Vasculitis Malaysia

Hypertension- Translating guidelines into Clinical Practice- Dr Chow Yok Wai

2010-07-16T21:27:00.000-07:00

Topic : Hypertension- Translating guidelines into Clinical Practice

Speaker: Dr Chow Yok Wai, Consultant Nephrologist and Physician,
Hospital Pantai Ayer Keroh, Melaka

Date: 16/7/2010 7.00pm

Venue: Ramada Renaissance Melaka

Who should attend?
General Practitioners/Primary care physicians
Physicians/Registrar/Medical Officers

Public Forum- Yong Peng by Dr Chow Yok Wai and Dr Pang Kim Keng

2010-06-06T21:10:00.000-07:00

Date : 26/6/2010 (Saturday)

Venue : Yong Peng High School

(Jalan Sekolah Cina, 83700 Yong Peng, Johor)

Time : 6.30pm - 10.00pm

Target of Pax : 150 pax

Speakers: Dr Pang Kim Keng, Consultant Urologist, Hospital Pantai Ayer Keroh, Melaka

Dr Chow Yok Wai, Consultant Nephrologist, Hospital Pantai Ayer Keroh, Melaka

Programme : 6.30pm - Registration

7.00pm - Talk by Dr Pang and Q&A (Common Urological Diseases)

8.00pm - Talk by Dr Chow and Q&A (Protect you kidneys, Control diabetes)

9.00pm - Blood Pressure and Blood Glucose Screening

Mycophenolate mofetil and intravenous cyclophosphamide are similar as induction therapy for class V lupus nephriti

2010-02-03T06:52:00.000-08:00

Original Article

Kidney International (2010) 77, 152–160; doi:10.1038/ki.2009.412; published online 4 November 2009
Mycophenolate mofetil and intravenous cyclophosphamide are similar as induction therapy for class V lupus nephritis

Jai Radhakrishnan1,6, Dimitrios-Anestis Moutzouris2,6, Ellen M Ginzler3, Neil Solomons4, Ilias I Siempos5 and Gerald B Appel1

Class V lupus nephritis (LN) occurs in one-fifth of biopsy-proven cases of systemic lupus erythematosus. To study the effectiveness of treatments in this group of patients, we pooled analysis of two large randomized controlled multicenter trials of patients with diverse ethnic and racial background who had pure class V disease. These patients received mycophenolate mofetil (MMF) or intravenous cyclophosphamide (IVC) as induction therapy for 24 weeks, with percentage change in proteinuria and serum creatinine as end points. Weighted mean differences, pooled odds ratios, and confidence intervals were calculated by using a random-effects model. A total of 84 patients with class V disease were divided into equal groups, each group had comparable entry variables but one received MMF and one received IVC. Within these groups, 33 patients on MMF and 32 patients on IVC completed 24 weeks of treatment. There were no differences between the groups in mean values for the measured end points. Similarly, no difference was found regarding the number of patients who did not complete the study or who died. In patients with nephrotic syndrome, no difference was noted between those treated with MMF and IVC regarding partial remission or change in urine protein. Hence we found that the response to MMF as induction treatment of patients with class V LN appears to be no different from that to IVC.

Protect your kidneys control diabetes- Health Forum 6th March 2010

2010-01-29T00:30:00.001-08:00

Dr Chow Yok Wai MRCP (UK) - Curriculum Vitae

2009-12-24T18:54:00.000-08:00

1. Education
Basic Medical Qualification
University Attended: University of Science Malaysia
Degree Obtained: Doctor of Medicine (MD)
Date Awarded: 25th July 1999

MRCP (Member of the Royal College of Physicians)
Date Awarded: 22nd July 2003

2. Working Experience and Present Appointment
July 1999-October 2000
• Commenced internship training in Sultanah Aminah Hospital Johor Bahru
(HSAJB), Malaysia
• Underwent 18 months training in General Medicine, General Surgery
(Including Plastic Surgery, Urology, Neurosurgery and Paediatric Surgery),
Obstetrics and Gynaecology, Orthopaedics and General Paediatrics.
November 2000-March 2001
• Joined the Department of Medicine HSAJB as a medical officer
• Commenced subspeciality rotation in Nephrology
April 2001-October 2001
• Haematology rotation
November 2001-March 2002
• Neurology rotation
April 2002-November 2002
• Infectious Disease and Intensive Medicine rotation
December 2002-July 2003
• Cardiology, Respiratory Medicine, Gastroenterology and Endocrinology
rotation

July 2003-December 2004
• Granted membership to the Royal College of Physicians of United Kingdom
• Assigned to a 35 bedded general medical ward as general physician in Sultanah Aminah Hospital Johor Bahru, Malaysia upon completion of MRCP
January 2005-June 2006
• Commencement of Nephrology Subspeciality Training
(Nephrology Followship Programme) in HSAJB
July 2006-December 2007
• Completion of Nephrology Subspeciality Training in Kuala Lumpur
Hospital, Malaysia
January 2008- February 2010
• Attached to the Nephrology Unit, Department of Medicine, HSAJB as Consultant Nephrologist and Physician.

February 2010 – to date
• Consultant Nephrologist and Physician,
Hospital Pantai Ayer Keroh
75450, Melaka

3. Registration as a Medical Practitioner
5th July 2000
• Full registration with the Malaysian Medical Council

12th November 2008
• Full registration with the General Medical Council of United Kingdom

4. Official Gazettement by the Government of Malaysia (Ministry of Health)
22nd January 2005
• Gazetted as a Internal Medicine specialist

1st January 2008
• Gazetted as a Nephrologist

5. Professional Activities
2003
Clinical attachment in the New Royal Infirmary of Edinburgh, Scotland
Department of Nephrology

Position: Visiting Registrar
Supervisor: Dr Robin Winnie
Consultant Nephrologist
New Royal Infirmary of Edinburgh
Edinburgh

2008
Attended the Malaysian Nephrology Board Examination on 9th May 2008
Conducted by the Nephrology Board Malaysia
(Malaysian Society of Nephrology, Ministry of Health Malaysia, Academy of
Medicine, Malaysia)
Obtained the highest marks in the 2008 cohort

2008-2009
Awarded a full scholarship by the Public Services Department of Malaysia to
pursue a one year advance nephrology training in the field of vasculitis in Addenbrooke’s Hospital Cambridge University Hospitals NHS Foundation Trust

Position: Honorary Clinical Research Fellow

Supervisor: 1) Dr David Jayne
Director of Lupus and Vasculitis,
Addenbrooke’s Hospital
Cambridge University Hospitals NHS Foundation Trust

2) Professor Kenneth Smith
Cambridge Institute of Medical Research,
University of Cambridge,
United Kingdom.

2008- 2010
Coordinator for National Glomerulonephritis Registry, National Renal Registry
(Johor Bahru)

6. Professional Bodies
National
Member of the Malaysian Medical Association
Member of the Malaysian Society of Nephrology
Member of the Postgraduate Renal Society, Malaysia
International
Member of the Royal College of Physicians of Edinburgh
Member of the International Society of Nephrology
Member of the IgA International Network

7. Research Activities
Publications:-
Acute renal failure in the same hospital 10 years apart; A comparison of two prospective studies in Sultanah Aminah Hospital, Johor Bahru
Medical Journal of Malaysia Vol 62 No1 March 2007

Lactic acidosis in HIV patients receiving highly active antiretroviral therapy- the Johor Bahru experience
Medical Journal of Malaysia Vol 62 No 1 March 2007

Quality improvement in Department of Nephrology, Kuala Lumpur Hospital- an audit on clinical performance indicators
Journal of Quality Improvement Vol 10 No 2 2007

Rituximab in Behcet’s Disease
Submitted to Annals of Rheumatic Diseases for publication (May 2009)

Participation in clinical studies:-
National
HDP study- Hospitalisation rate among Dialysis Patients study (2007)

International
GIANT study- Greatest International Antibiotic Trial study (2006)

A randomized double blind placebo controlled multicenter study to evaluate the efficacy and safety of two doses of Ocrelizumab in patients with WHO or ISN Class III or IV nephritis due to systemic lupus erythematosus (2008)

A phase III multicentre, double blind, double dummy, randomized flexible dose comparative study of MCI-196 versus Simvastatin for the treatment of dyslipidaemia in subjects with chronic kidney disease on dialysis (2009)

Evaluation of chronic kidney disease patterns through the global information database (GRID) (2009)

Biologic therapies in neuro-behcet’s disease, an international collaborative case-series- neurobehcet study group, International Society of Behcet’s Disease (2009)

CHiC-TRIAD (Cambridge Hinxton Centre for Translational Research In Autoimmune Disease) (2008-2009)

MY-CYC (A randomised clinical trial of mycophenolate mofetil versus cyclophosphamide for remission induction in ANCA associated vasculitis) (2008-2009)

Abstracts:-
National
OUTCOME OF A COMMUNITY BASED HEALTH SCREENING PROGRAMME DURING THE PUBLIC AWARENESS CAMPAIGN ON KIDNEY CARE IN JOHOR BAHRU, MALAYSIA
Oral Presentation- 22nd MSN Annual Seminar in Nephrology
Prevention of Chronic Kidney Disease

MYCOPHENOLATE MOFETIL IN RELAPSING LUPUS NEPHRITIS
23rd Malaysian Society of Nephrology Annual Seminar
Johor Bahru, Malaysia
11-13th April 2007

MYCOPHENOLATE MOFETIL IN MEMBRANOUS NEPHROPATHY
23rd Malaysian Society of Nephrology Annual Seminar
Johor Bahru, Malaysia
11-13th April 2007

International
ACUTE RENAL FAILURE IN THE SAME HOSPITAL 10 YEARS APART;
A COMPARISON OF TWO PROSPECTIVE STUDIES IN SULTANAH AMINAH
HOSPITAL, JOHOR BAHRU, MALAYSIA
Poster Presentation- 3rd World Congress of Nephrology
Post Congress Satellite Symposium
Acute Renal Failure: From Bench to Bedside
1st -3rd July 2005

LACTIC ACIDOSIS IN HIV PATIENTS RECEIVING HIGHLY ACTIVE ANTIRETROVIRAL THERAPY- THE JOHOR BAHRU EXPERIENCE
Oral Presentation
14th IUSTI Asia Pacific International Conference- 27th to 30th July, 2006

ADULT POLYCYSTIC KIDNEY DISEASE IN PATIENTS ON DIALYSIS IN MALAYSIA
11th Asian Pacific Congress of Nephrology 2008

ELDERLY PATIENTS INITIATING DIALYSIS IN MALAYSIA
11th Asian Pacific Congress of Nephrology 2008

MALIGNANCY POST RENAL TRANSPLANTATION: A 25 YEAR EXPERIENCE
11th Asian Pacific Congress of Nephrology 2008

LATE ACUTE ANTIBODY MEDIATED REJECTION ASSOCIATED WITH CALCINEURIN INHIBITOR MINIMISATION
11th Asian Pacific Congress of Nephrology 2008

CASTLEMAN’S DISEASE OF THE KIDNEY IN PATIENT WITH SLE
11th Asian Pacific Congress of Nephrology 2008

CLINICAL BENEFITS OF ICODEXTRIN: A SINGLE CENTRE EXPERIENCE
11th Asian Pacific Congress of Nephrology 2008

‘SUDOKU’ INCREASES COGNITIVE FUNCTION IN HAEMODIALYSIS PATIENTS- A PROSPECTIVE PILOT STUDY (Awarded Best Abstract)
11th Asian Pacific Congress of Nephrology 2008

SHORT TERM INFECTIOUS COMPLICATIONS POST RENAL TRANSPLANT
11th Asian Pacific Congress of Nephrology 2008

Neuro Behcet's Disease

2009-04-08T13:16:00.000-07:00

Types of NBD

NBD is sub-classified into two major forms: parenchymal and non-parenchymal. The two types rarely occur in the same individual and their pathogeneses are probably different (7).

A. Parenchymal NBD
The parenchymal form of NBD is characterized by focal or multifocal involvement of the brain parenchyma. It is the most common presentation reported in NBD (11), comprising 81% of cases in a study of 200 NBD patients (7). This form of NBD commonly presents with an attack of hemiparesis, cognitive changes, sphincteric troubles and possible fever in men in their third decade. The most common clinical findings are pyramidal tract and brain stem signs. An acute attack may be followed by a relapsing or progressive course (2, 7).

MRI findings in parenchymal NBD: MRI is the most sensitive imaging modality for assessing patients with NBD (18); it shows focal or multifocal CNS abnormalities in the clinically affected areas (17, 19, and 20). The distribution and the MRI signal behavior of parenchymal NBD lesions will be reviewed.

Distribution of parenchymal NBD: Lesions in parenchymal NBD are located in the brainstem, thalami and basal ganglia. However, it can also involve the cerebral hemispheres, cerebellum, spinal cord, and other sites.

Brainstem-thalamic-basal ganglia: Parenchymal NBD lesions have predilection to the brainstem-thalamic-basal ganglia region (Fig 1). They characteristically involve the junction of midbrain and thalamus (meso-diencephalic junction) (Fig 2). The reason for this predilection is unknown (10, 21, 22, and 23). Extensive large lesions are commonly seen in these regions during acute attacks (7).
In the brainstem, the midbrain is most commonly affected site (Fig 3), with the cerebral peduncles involved and the red nuclei characteristically spared (17). The next most common location is the ponto-bulbar region (7, 22), with lesions that usually involve the basis pontis (Fig 4) and occasionally extend to brachium pontis (17, 19, 20, and 21). Although the white matter is more often involved in NBD, lesions may also be seen within the grey matter structures, including brainstem nuclei, cortex and basal ganglia (21, 24). Figure 5 shows symmetrical involvement of midbrain colliculi in parenchymal NBD. The disease commonly involves the basal ganglia and internal capsule region unilaterally (Fig 6), or bilaterally in one third of the cases (7). The globus pallidus and adjacent internal capsule is the most common site affected in this region (Fig 7) (10, 22).
High linear signal intensity in T2 weighted images along the posterior limb of the internal capsule is highly suggestive of NBD (Fig 8). This sign can be seen unilaterally or bilaterally with variable symmetry and severity (7, 22, and 23).
Lesions located in the basal ganglia regions tend to extend caudally along the corticospinal tracts (Fig 9). High signal intensity changes may thus be seen in the fiber tracts of the brainstem and may further extend to the cervical cord (Fig 10). The reversibility of signal changes along this pathway in follow-up MR studies is suggestive that they may represent edema (22). However, in chronic NBD cases, non-reversible signal changes in this distribution can be attributed to wallerian degeneration of the tracts documented in pathological studies (16, 24).

Hemispheric lesions: In sub-acute NBD (a few months after an acute attack,: during either remission or progressive worsening of an acute attack), hemispheric lesions may be seen concomitant with brainstem-thalamic-basal ganglia lesions (7). Hemispheric lesions of NBD are usually subcortical rather than periventricular (Fig 11) (25). However, extensive periventricular white matter changes have been reported (Fig 12) (19, 20, and 26).

Other brain lesions:Cerebellar lesions are less common in parenchymal NBD (7). However, lesions in the cerebellar white matters are occasionally encountered in parenchymal NBD (Fig 13) (7, 22). Involvement of the cranial nerves, rootlet of the spinal nerves and peripheral nerves is exceedingly rare. Contrary to the common belief, isolated aseptic meningitis is distinctly uncommon in BD. Instead, diffuse meningoencephalitis is common (22).

Brainstem atrophy:In chronic stage of NBD, gliosis and atrophy may occur (19, 23, and 27) with striking involvement of the brainstem (Fig 14). Isolated atrophy of the brainstem with relative sparing of the cerebral cortex, though not very frequent, is characteristic of chronic NBD (26).

Spinal cord:The spinal cord is less commonly involved in NBD compared to brainstem-thalamic-basal ganglia or hemispheric regions. The spinal cord lesions tend to extend over two or more vertebral segments posterolaterally and may involve the cervico-medullary junction (Fig 15) (22).

MRI signal behavior of parenchymal NBD:The parenchymal lesions of NBD are hyper intense on T2-weighted images. High-signal intensity lesions represent demyelination, gliosis or transient inflammation and secondary edema supported by the resolution of MR abnormalities in response to methyl-prednisolone and other immunosuppressive therapies (28). Most lesions are somewhat visible on T1 weighted images, but can be very subtle (Fig 8) (7, 19, 22, 23, and 27). Hypointense lesions on T1-weighted images may be seen in chronic lesions (Fig 7) (17). Fluid attenuation inversion recovery (FLAIR) sequences increase MRI sensitivity for NBD lesions, especially juxta-cortical and periventricular (Fig 16) (19). Some lesions may demonstrate contrast enhancement during the acute or the sub-acute phase that resolves in remissions (22, 30). The area of contrast uptake can be nodular, linear, crescentic or irregular and is usually smaller than 5 mm (Fig 17) (18, 25). These changes likely reflect a breakdown in the blood-brain barrier (18).

Temporal course of MRI lesions in parenchymal NBD: Acute phase: There are extensive large parenchymal lesions with predominant involvement of the brainstem-thalamic-basal ganglia region that tend to enhance in contrast studies (Fig 1).
Sub-acute phase: There is marked regression in the appearance of the lesions seen during the acute phase, while there may be smaller scattered lesions in the cerebral white matters and brainstem-thalamic-basal ganglia regions (Fig 11).
Chronic phase: There is marked reduction in the size of the parenchymal lesions that corresponds to clinical remissions and possible appearance of brainstem atrophy (Fig 18) (7, 27, and 31).

Pathological findings in parenchymal NBD:Autopsy studies and biopsy specimens of parenchymal NBD reveal widespread meningoencephalitis with multifocal necrotic foci that tend to accumulate mostly in the brainstem and basal ganglion region (23, 24). A non-specific inflammatory reaction with peri-vascular neurtorphilic or lymphocytic cuffing is commonly seen (23, 24). Gliosis and inflammatory axonal injury are reported in chronic lesions (Fig 19) (32, 33).
The precise pathologic mechanism of parenchymal NBD lesions has not been established. It has been hypothesized that parenchymal NBD lesions could be venous infarcts (22). This hypothesis needs more pathological support as vasculitis cannot usually be demonstrated within the parenchymal NBD lesions (17, 31-33). The presence of abnormal T-cell lymphocyte function points to a possible aberrant immune response to antigenic components of infectious agents such as Streptococcus species (6, 32 and 33).

B. Non-parenchymal NBD:
In the non-parenchymal group, CNS dysfunction is due to involvement of major vessels (vascular NBD) or rarely aseptic meningitis (7). The vascular form of NBD is more often reported from the Middle East (14, 34) and France (35), and less often from other parts of the world (7, 17) with geographical and ethnic variations in disease expression and severity (36).
Vascular NBD usually affects major intracranial vessels with frequent involvement of the venous sinuses, cerebral veins and less commonly the intracranial arteries (7, 35, 37-39). The rare arterial involvements in NBD include thrombosis and aneurysms of the large cerebral arteries (37, 38). Venous sinus thrombosis is the most frequent vascular manifestation in NBD (35, 40) followed by thrombosed deep and cortical cerebral veins (41). The association of venous parenchymal infarcts with venous thrombosis depends on the efficacy of the collateral circulation within the cerebral venous system. The extensive collateral circulation usually allows for a significant degree of compensation in the early stages of sinus venous thrombosis (42).
Vascular NBD usually manifests with acute neurological attacks. The arterial involvement usually presents with stroke that evolves over several hours (7). Raised intracranial pressure is the main clinical manifestation of venous sinus thrombosis with a spectrum of clinical presentations such as headaches, papilloedema, focal neurological deficits, seizures and coma. However, the clinical diagnosis of acute dural sinus occlusion can be difficult to make and is frequently delayed (42).

MRI findings in vascular NBD: The most common MRI findings in vascular NBD are occlusion of the cerebral venous sinuses without or with venous infarcts (7). MRI in conjunction with MR venography (MRV) is highly sensitive in detecting such lesions (42-44).
Venous sinus thrombosis: MRI findings include:
Direct visualization of a thrombus within the vessel. The increased intensity thrombus, detected on T1- and T2- weighted images, may partially or totally replace the flow void of the normal venous channel (Fig 20) (42).
MR venography (MRV) shows lack or impaired flow in the occluded sinus and identifies venous collaterals (Fig 21) (45, 46).

Venous infarcts: Venous infarcts are characterized by their non-arterial distribution. They involve the white matter and/or the cortical-white matter junction, and are often associated with hemorrhage. Bilateral cerebral involvement can occur, including the superior cerebral white matter of the convexities from superior sagittal sinus thrombosis (Fig 22) or the basal ganglia and thalami from internal cerebral vein thrombosis (41, 42).

Parenchymal versus non-parenchymal NBD:
Differentiating between parenchymal and non-parenchymal NBD has significant diagnostic, pathologic, therapeutic, and prognostic implications. Vascular NBD due to isolated intracranial hypertension and dural venous sinus thrombosis have better prognosis if detected and treated early. Acute lesions of parenchymal NBD can be reversible with appropriate treatment such as corticosteroids (11).
The most common forms of NBD, parenchymal and vascular, are distinguished by their unique clinical presentations, characteristic changes in the cerebrospinal fluid (CSF), and MRI findings. Clinically, the parenchymal form manifests with signs and symptoms referable to the brainstem with pyramidal findings, cognitive impairment, ataxia and sphincter disturbance; while the vascular form usually causes raised intracranial pressure due to occlusion of the dural sinuses or very rarely an arterial stroke. CSF findings are different in both groups. In parenchymal NBD, CSF shows pleocytosis with predominance of polymorphonuclear cells, with or without elevated protein level and rarely positive oligoclonal bands. In vascular NBD, the CSF is usually normal except for elevated pressure (22). Conventional MRI can usually differentiate between parenchymal and vascular NBD. However, in some cases, the differentiation between vascular and parenchymal NBD may be difficult because of the presence of parenchymal lesions in the former or in rare instances the coexistence of the two forms. In such cases, special sequences of MRI, such as diffusion-weighted imaging (DWI) or magnetic resonance spectroscopy (MRS), can provide additional information.

2009-04-05T02:23:00.001-07:00

Focal Glomerulosclerosis

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2009-04-05T02:22:00.000-07:00

MPGN/MCGN

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2009-04-05T02:12:00.000-07:00

Membranous GN

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2009-04-05T02:11:00.003-07:00

Nodular GN

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2009-04-05T02:11:00.001-07:00

Tubulointerstitial diseases

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2009-04-05T02:10:00.002-07:00

Minimal Light Microscopic Changes

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2009-04-05T02:10:00.001-07:00

Crescentic GN

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2009-04-05T02:09:00.002-07:00

Diffuse Proliferative GN

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2009-04-05T02:09:00.001-07:00

Mesangial proliferative GN

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2009-04-05T02:08:00.001-07:00

Focalproliferative Glomerulonephritis

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Basement Membrane Abnormalities

2009-04-05T01:52:00.000-07:00

Basement Membrane Abnormalities

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EULAR recommendations- vasculitis (Medium and small vessel vasculitis)

2009-02-26T01:01:00.000-08:00

Medium and small vessel vasculitis- Wegener’s, Microscopic polyangiitis, Churg-strauss, PAN, Cryoglobulinaemia.

1. Management is to be in expert centres
• Its rarity makes management in normal settings suboptimal
• Specialised services may be required

2. ANCA must be done (both indirect IF and ELISA)

3. Positive biopsy in strongly supportive of vasculitis
• Fibrinoid necrosis
• Pauci-immune GN (segmental necrosis, extracapillary proliferation)
• Granuloma
• Biopsy esp helpful in ANCA negative pt

4. Clinic visits- structured assessment is required (ie checklist- clinical, urine, laboratory)
• To avoid missing the multi-organ involvement

5. Treatment is based on severity
• Localised
• Early systemic (any, without organ threatening or life threatening disease)
• Generalized (renal or other vital organ failure, serum creat >500umol/l)
• Severe (renal or other vital organ failure, serum creat >500umol/l)
• Refractory (unresponsive to steroids/cyclophos)

6. WG/MPA
• Induction:- Cyclophosphamide (oral 2mg/kg/d max 200mg/d) and prednisolone (1mg/kg/d max 60mg/d)
• Pulse IV Cyclophos- higher remission rate with lower A/E but higher rate of relapse
1. EUVAS regime- 15mg/kg (max 1.2g) 2 weekly IV cyclophosphamide for 3 pulses, then 3 weekly for 3-6 pulses. (dose adjusted for age and renal function)

7. PAN/CSS
• Induction:- Cyclophosphamide + steroids- better remission vs steroid alone.
• IV versus oral cyclophosphamide
• Lower A/E and equal efficacy in PAN pt

8. Cyclophosphamide therapy
• Mesna
• PCP prophylaxis (480mg dly or 960mg 3 times a week)

9. Non organ threatening or non life threatening ANCA associated vasculitis
• MTX (oral/IV) and steroids- acceptable less toxic alternative

10. Plasma exchange in pt with RPGN (with serum creatinine > 500umol/l)

11. Maintenance therapy- Steroids + Azathioprine, lefllunomide or MTX

12. Patients who failed remission or relapse despite on maximum doses of standard therapy –
• MMF
• Anti TNF (infliximab)
• RTX
• IVIG
• ATG

13. Cryoglobulinemia
• Mixed essential- treat as small vessel vasculitis
• RTX in hepatitis associated cryoglobulinemic vasculitis may be of benefit

14. HepC associated cryoglobulinaemic vasculitis- anti viral therapy

15. HepB associated PAN- antiviral therapy + steroids + plasma exchange

EULAR recommendations: Vasculitis (Large vessel vasculitis)

2009-02-26T00:51:00.000-08:00

Large vessel vasculitis (LVV)- Takayasu and GCA:-

1. Thorough clinical and imaging assessment of the arterial tree when a diagnosis of Takayasu is suspected-
• MRA/PET could assist in diagnosis and document of extent of involvement but has its limitations (not widely available, operator dependant).
• Conventional angiogram is gold standard

2. In Giant cell arteritis,
• Temporal artery biopsy should be performed
• 1cm tissue length is required
• skip lesions may lead to false negative HPE
• Don’t delay treatment while waiting for biopsy. Treat first.
• If CRP/ESR is not elevated, think of another diagnosis
• USG of the temporal artery looking for vessel wall oedema was 88% sensitive and 97% specific in diagnosing GCA

3. Start steroids early and at high dose for induction of remission of LVV
• Prednisolone- 1mg/kg (max 60mg) daily
• Maintain for 1 month then taper
• Taper should not be in the form of EOD therapy which is a/w higher relapse rate
• At 3 months, steroid dose should be at 10-15mg/d
• Steroid duration could be for several years
• Must give bone protection during this period

4. Immunosuppressive agents should be considered in LVV as adjunctive therapy
• Methotrexate (20-25mg weekly)
• Azathioprine (2mg/kg/d)
• Cyclophosphamide (in steroid resistant Takayasu)

5. Monitoring of LVV- clinical and inflammatory markers

6. Use low dose aspirin in all GCA pt

7. Reconstructive surgery for Takayasu should be performed during the quiescent phase at expert centres

Equivalent anti-inflammatory doses of different oral corticosteroids

2009-02-09T05:28:00.000-08:00

This table takes no account of mineralocorticoid effects, nor does it take account of variations in duration of action

Prednisolone 5mg

is equivalent to betamethasone 750 mcg

is equivalent to cortisone acetate 25 mg

is equivalent to dexamethasone 750 mcg

is equivalent to deflazacort 6mg

is equivalent to hydrocortisone 20mg

is equivalent to methylprednisolone 4mg

is equivalent to traimacinolone 4mg

Journal Club- 21/1/2009 CIMR

2009-01-21T08:00:00.000-08:00

The histone deacetylase HDAC11 regulates the expression of interleukin 10 and immune tolerance

Alejandro Villagra1, Fengdong Cheng1, Hong-Wei Wang1, Ildelfonso Suarez1,2, Michelle Glozak3,4, Michelle Maurin1, Danny Nguyen1, Kenneth L Wright1,4, Peter W Atadja5, Kapil Bhalla6, Javier Pinilla-Ibarz1,4, Edward Seto3,4 & Eduardo M Sotomayor1,3,4

Antigen-presenting cells (APCs) induce T cell activation as well as T cell tolerance. The molecular basis of the regulation of this critical ‘decision’ is not well understood. Here we show that HDAC11, a member of the HDAC histone deacetylase family with no prior defined physiological function, negatively regulated expression of the gene encoding interleukin 10 (IL-10) in APCs. Overexpression of HDAC11 inhibited IL-10 expression and induced inflammatory APCs that were able to prime naive T cells and restore the responsiveness of tolerant CD4+ T cells. Conversely, disruption of HDAC11 in APCs led to upregulation of expression of the gene encoding IL-10 and impairment of antigen-specific T cell responses. Thus, HDAC11 represents a molecular target that influences immune activation versus immune tolerance, a critical ‘decision’ with substantial implications in autoimmunity, transplantation and cancer immunotherapy.

Humoral Immunity

2009-01-15T03:00:00.000-08:00

Humoral Immunity

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Journal Club- 14th January 2009 (CIMR)

2009-01-14T06:41:00.000-08:00

Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease

Maria Florencia Delgado1, Silvina Coviello1, A Clara Monsalvo1, Guillermina A Melendi1,2, Johanna Zea Hernandez1,2, Juan P Batalle1, Leandro Diaz1, Alfonsina Trento3, Herng-Yu Chang4, Wayne Mitzner4, Jeffrey Ravetch5, Jose ́ A Melero3, Pablo M Irusta1,6 & Fernando P Polack1,2,7,8

Abstract:

Respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants. A formalin-inactivated RSV vaccine was used to immunize children and elicited nonprotective, pathogenic antibody. Immunized infants experienced increased morbidity after subsequent RSV exposure. No vaccine has been licensed since that time. A widely accepted hypothesis attributed the vaccine failure to formalin disruption of protective antigens. Here we show that the lack of protection was not due to alterations caused by formalin but instead to low antibody avidity for protective epitopes. Lack of antibody affinity maturation followed poor Toll-like receptor (TLR) stimulation. This study explains why the inactivated RSV vaccine did not protect the children and consequently led to severe disease, hampering vaccine development for 42 years. It also suggests that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR agonists in their formulation, and it identifies affinity maturation as a key factor for the safe immunization of infants.

Renal pathology- An introduction

2009-01-12T06:52:00.000-08:00

Introduction to Renal Pathology

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Immunology for the non immunologist

2009-01-08T13:16:00.000-08:00

Intro Immune

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IVIG Protocol- Addenbrooke's Hospital (For use in Vasculitis and Lupus)

2008-12-11T11:33:00.000-08:00

Dose:
0.67 g/kg for 3 days (ie Total Dose is 2g/kg).

0.4g/kg for 5 days for patients with history of thrombosis

0.4g/kg for 5 days for patients > 60y/o

0.4g/kg for 5 days for Serum Creatinine > 150umol/l

Give 1st 5g bottle over 1 hour.
Then subsequent bottles at 1/2 hour each.

Side Effects:
IVIG is potentially nephrotoxic!
(ARF can occur in pt with pre-existing renal impairment. Renal recovery is usually complete within 10 days)
(But I have seen 1 doctor's dad who went into ESRD following IVIG infusions. Please be careful)
Common side effects: fever, chills, backache, malaise.
Headache/Backache and chills usu respond to slowing of the infusion rate
Rarely, allergic reactions occur.
Patients with autoimmune disease often experience 'flu-like' symptoms and headaches for 7-10days after IVIG infusion.
Rarely, they may experience rashes and other inflammatory phenomena.

Haemodialysis patients:
Dose: 0.67g/kg given after 3 consecutive dialysis sessions (Total dose 2g/kg)
IVIG can be administered through the venous return limb of the extracorporeal circuit.
IVIG may be started during the HD session or given entirely at the end of the HD session via the venous limb of the fistula.
Following completion of the infusion, it is safe for the patient ot go home 1 hour after initial treatment.

CAPD patients:
As in normal patients but beware of fluid overload.

C57BL/6 lab mouse

2008-12-10T10:48:00.000-08:00

C57BL/6, often referred to as "C57 black 6" or just "black 6" is a common inbred strain of lab mouse. It is probably the most widely used "genetic background" for genetically modifed mice for use as models of human disease. They are the most widely used lab mouse strain, due to the availability of congenic strains, easy breeding, robustness, and their relationship to GM models, making them ideal controls.

Dark brown, nearly black, coat. Easily irritable temperament. They have a tendency to bite, and cannot be handled like a typical pet mouse or even more docile laboratory strains such as BALB/c.

C57BL/6 mice which are group-housed also display barbering behavior, in which the dominant mouse in a cage selectively removes hair from its subordinate cage mates. Mice that have been barbered have large bald patches on their bodies, commonly around the head, snout, and shoulders, although barbering may appear anywhere on the body. Both hair and vibrissae may be removed. Barbering is more frequently seen in female mice; male mice are more likely to display dominance through fighting.[1]

C57BL/6 as a "Th1 responder"

C57BL/6 has certain immunophenotypes that distinguish it from other inbred strains like BALB/c. For example the immunological response to the same pathogen in C57BL/6 mice is often of an opposite spectrum compared to BALBb/c mice, namely C57BL/6 shows Th1 and BALB/c shows Th2 response in response to intracellular pathogen Leishmania major, where a Th1 response results in a resistant ie healer phenotype (since the pathogen is intracellular), whereas a Th2 response results in a susceptible (nonhealer) phenotype.[citation needed]

Though this trait had been observed in these two strains since 1988, in an article published in 2000 by Mills et al.[2] these observations were systematized and generalized to other strains of mice. Even without biasing towards Th1 or Th2 by priming through infection, the strains differ in their macrophages' ability to be activated, as measured from their arginine metabolic programs when stimulated by Interferon gamma or LPS or both:

* M-1 macrophages from typical Th1 responders: C57BL/6 or B10.D2 mice, preferentially produce NO by action of iNOS
* M-2 macrophages from typical Th2 responders: DBA or BALB/c mice, preferentially produce ornithine and urea by action of arginase.

Rules and Guidelines for Nomenclature of Mouse and Rat Strains

2008-12-10T10:38:00.000-08:00

Revised: July 2007
International Committee on Standardized Genetic Nomenclature for Mice
Chairperson: Dr. Janan T. Eppig
(e-mail: jte@informatics.jax.org)
Rat Genome and Nomenclature Committee
Chairperson: Dr. Goran Levan
(e-mail: Goran.Levan@gen.gu.se)

In 2001, the International Committee on Standardized Nomenclature for Mice and the Rat Genome and Nomenclature Committee agreed to establish a joint set of rules for strain nomenclature, applicable to strains of both species. These rewritten guidelines reflect this collaboration, in addition to documenting new and revised rules for the naming of strains. This document is updated annually by the international nomenclature committees for mouse and rat.

Reference to former versions of the rules for mouse strain nomenclature can be found in Snell (1941), Committee for Standardized Genetic Nomenclature in Mice (1952, 1960, 1976, 1981, 1989, 1996), Festing (1979, 1993), Staats (1986), Maltais et al. (1997). The November 2006 revision is available here. Reference to former rules for rat strain nomenclature can be found in Committee on Rat Nomenclature (1992).

Current nomenclature rules for naming genes are available online at:

For mouse: http://www.informatics.jax.org/mgihome/nomen/gene.shtml#genenom
For rat: http://rgd.mcw.edu/nomen_rules.html

Table of Contents

1. Introduction
1.1 Mice
1.2 Rats
2. Laboratory codes
3. Inbred Strains and Hybrids
3.1 Definition
3.2 Nomenclature of Inbred Strains
3.3 Indication of Inbreeding
3.4 Substrains
3.5 Hybrids
4. Strains Made from Multiple Inbred Strains
4.1 Recombinant Inbred Strains
4.2 Mixed Inbred Strains
4.3 Recombinant Congenic Strains
4.4 Advanced Intercross Lines
5. Coisogenic, Congenic, and Segregating Inbred Strains
5.1 Coisogenic Strains
5.2 Congenic Strains
5.3 Consomic Strains
5.4 Segregating Inbred Strains
5.5 Conplastic Strains
6. Outbreds and Closed Colonies
6.1 Outbreds
6.2 Closed Colonies 7. References

1. Introduction

Mice and rats used in the laboratory derive from a variety of sources. Production of inbred strains means that these backgrounds can be defined and thus require nomenclature conventions. It should be borne in mind that genetic drift means that there may still be unknown genetic differences between individuals within strains.
1.1 Mice

Most laboratory mice have contributions from both Mus musculus musculus and Mus musculus domesticus. There is evidence that smaller contributions also may have come from Mus musculus molossinus and Mus musculus castaneus. Therefore, they should not be referred to by species name, but rather as laboratory mice or by use of a specific strain or stock name. (In addition, some recently developed laboratory mouse strains are derived wholly from other Mus species or other subspecies, such as M. spretus).

Mouse strain names should be registered through the Mouse Genome Database (MGD) at http://www.informatics.jax.org/mgihome/submissions/submissions_menu.shtml.
1.2 Rats

Laboratory rat strains derive from the Rattus norvegicus species. Another species, Rattus rattus, also is used as an experimental model, but has not contributed to the common laboratory rat strains.

Rat strain names should be registered through the Rat Genome Database (RGD) at http://rgd.mcw.edu.
2. Laboratory codes

A key feature of mouse and rat nomenclature is the Laboratory Registration Code or Laboratory code, which is a code of usually three to four letters (first letter uppercase, followed by all lowercase) that identifies a particular institute, laboratory, or investigator that produced, and may hold stocks of, a mouse or rat strain. Substrains should be identified by Laboratory codes, as should congenic and other strains where several different forms exist that are not otherwise distinguishable. Laboratory codes are assigned by the Institute of Laboratory Animal Research (ILAR) (http://dels.nas.edu/ilar_n/ilarhome/search_lc.shtml).

Examples of Laboratory codes are:

J The Jackson Laboratory
Rl W.L. and L.B. Russell
Jr John Rapp
Mcw Medical College of Wisconsin
Kyo Kyoto University

3. Inbred Strains and Hybrids
3.1 Definition

Strains can be termed inbred if they have been mated brother x sister for 20 or more consecutive generations, and individuals of the strain can be traced to a single ancestral pair at the 20th or subsequent generation. At this point the individuals' genomes will on average have only 0.01 residual heterozygosity (excluding any genetic drift) and can be regarded for most purposes as genetically identical. Inbred strains must be continuously mated brother x sister (or equivalent) thereafter

Other breeding schemes can be used to produce inbred strains; consecutive parent x offspring mating may be used, provided that the younger of the parents is always used (i.e., the offspring that is mated to parent is subsequently mated to its offspring). Other breeding schemes are acceptable provided that the inbreeding is equivalent to 20 successive generations of sib mating (Green 1981).
3.2 Nomenclature of Inbred Strains

An inbred strain should be designated by a unique brief symbol made up of uppercase, roman, letters, or a combination of letters and numbers beginning with a letter. (Note that some pre-existing strains do not follow this convention; e.g., mouse strain 129P1/J)

Care should be taken that mouse and rat strains do not overlap in strain designations. (Note: a few historical examples exist of similar mouse and rat strain designations and these are allowed to stand, with their substrain designations identifying them as unique).

Inbred strains that have a common origin, but are separated before F20 are related inbred strains, and symbols should reflect this relationship.

Examples:

Mouse strains: NZB, NZC, NZO
Rat strains: SR, SS

3.3 Indication of Inbreeding

The number of brother x sister inbreeding generations can be indicated, if necessary, by addition in parentheses of F followed by the number of generations.

Example:

Rat strain: ACI/N (F159)

If there is not information as to the total number of generations, but a minimum number of recent inbreeding generations is known, this can be shown by a question mark + the known number of subsequent generations of inbreeding.

Example:

Mouse strain: C3H/HeJ-ruf (F?+25)

3.4 Substrains

Established inbred strains may genetically diverge with time into substrains, due to a number of circumstances:

* If two branches are separated after 20 but before 40 generations of inbreeding there still will be enough residual heterozygosity that two genetically different substrains will result (Green 1981).

* If branches are separated for more than 20 generations from a common ancestor, it is likely that genetic variation between the branches will have occurred by mutation and genetic drift.

* If genetic differences are proven by genetic analysis to have occurred between branches.

Substrains are given the root symbol of the original strain, followed by a forward slash and a substrain designation. The designation is usually the Laboratory code of the individual or laboratory originating the strain.

Examples:

IS/Kyo Substrain of IS rat strain originating at Kyoto University.
A/He Substrain of A mouse strain originating from Walter Heston.

If a laboratory originates more than one substrain, serial numbers should be added to the Laboratory code.

Example:

Mouse strains: FL/1Re, FL/2Re

(Note that historical exceptions to this rule exist; for example, in mouse, BALB/c is not a substrain, DBA/1 and DBA/2 are separate strains and are not substrains.)

Substrains may give rise to further substrains by continued maintenance by a different investigator or through establishment of a new colony. In addition, substrains arise if demonstrable genetic differences from the original substrain are discovered. In either case, further substrain designations are added, without the addition of another slash.

Examples:

C3H/HeH Mouse substrain derived at Harwell (H) from the Heston (He) substrain of C3H.
SR/JrIpcv Rat substrain derived at Institute of Physiology, Czech Academy of Sciences (Ipcv) from the John Rapp (Jr) substrain of SR

Laboratory codes should be accumulated because genetic differences will accumulate with time, the rate depending to some extent on varying levels of quality control at the facilities that have housed and bred the strain or substrain. Organizations distributing mice and rats should include the number of generations the strain has been separated from the parent strain in the information they provide regarding the strain. Strain names can be abbreviated in publications after the first mention of the full proper designation.
3.5 Hybrids

Mice or rats that are the progeny of two inbred strains, crossed in the same direction, are genetically identical, and can be designated using uppercase abbreviations of the two parents (maternal strain listed first), followed by F1. Note that reciprocal F1 hybrids are not genetically identical, and their designations are, therefore, different.
Examples:

D2B6F1 Mouse that is the offspring of a DBA/2 mother and C57BL6/J father. A full F1 designation is (DBA/2N x C57BL/6J)F1.
B6D2F1 Mouse that is the offspring of the reciprocal cross. A full F1 designation is (C57BL/6J x DBA/2N)F1.
CB1BD22F1 Mouse that is the offspring of two recombinant inbred strains, a CXB1 mother and BXD22 father; full F1 designation is (CXB1/ByJ x BXD22/TyJ)F1.

Further crosses produce offspring that are no longer genetically identical, but it may still be appropriate to give them designations reflecting their parentage, similar to those for F1 hybrids.

Examples:

D2B6F2 are offspring of a D2B6F1 intercross.
B6(D2AKRF1) are offspring of a (DBA/2 x AKR/J)F1 male backcrossed to a C57BL/6J female.

In all the above cases, for clarity, the full strain symbols should be given in any publication when the hybrids or crosses are first referred to. If a hybrid is constructed using a substrain known to differ from the "standard" strain genetically and/or phenotypically, the substrain should be indicated in the hybrid symbol; e.g., BALB/cBy = CBy, C3H/HeSn = C3Sn.

Approved abbreviations for common mouse strains are listed below:

129 129 strains (may include subtype, e.g., 129S6 for strain 129S6/SvEvTac)
A A strains
AK AKR strains
B C57BL
B6 C57BL/6 strains
B10 C57BL/10 strains
BR C57BR/CD
C BALB/c strains
C3 C3H strains
CB CBA
D1 DBA/1 strains
D2 DBA/2 strains
HR HRS/J
L C57L/J
R3 RIIIS/J
J SJL
SW SWR

4. Strains Made from Multiple Inbred Strains

Mice or rats can be produced that have a defined genetic background, derived from two or more inbred strains, and that may or may not be genetically identical. Such animals should be designated appropriately, according to the breeding scheme that produced them.
4.1 Recombinant Inbred Strains

Recombinant inbred (RI) strains contain unique, approximately equal proportions of genetic contributions from two original progenitor inbred strains. Traditionally, recombinant inbred (RI) strains are formed by crossing animals of two inbred strains, followed by 20 or more consecutive generations of brother x sister matings (Bailey 1971, Taylor 1978). Alternate breeding schemes can be used, such as creating RI strain sets from Advanced Intercross Lines, where F2 animals are nonsib mated for several generations, followed ultimately by 20 or more consecutive generations of brother x sister matings. Note that if backcrossing to one of the parental strains is involved, this will create recombinant congenic strains and should be named accordingly. RI strains should be designated by uppercase one- or two-letter abbreviations of both parental strain names, with the female strain written first, and separated by an uppercase letter X with no intervening spaces. All members of RI sets involving the same two strains will be serially numbered regardless of whether they were created in one or more laboratories. Sequential numbers may be obtained from MGD (email to: nomen@informatics.jax.org).

Examples:

CXB Recombinant inbred mouse strain derived from a cross of BALB/c x C57BL/6J.

Multiple RI are given serial numbers.

Examples:

BXD1, BXD2, BXD3 Members of the BXD set of mouse RI strains derived from a cross of C57BL/6 x DBA/2.
HXB1, HXB2, HXB3 Members of the HXB set of rat RI strains derived from a cross of SHR/OlaIpcv x BN-Lx/Cub.

If the second strain abbreviation ends in a number (e.g., CX8 RI strains), a hyphen should be used to separate it from the serial number (e.g., CX8-1).

Recombinant inbred strains may be intercrossed for mapping complex traits. Such F1s are called recombinant inbred intercrosses (RIX) and are symbolized the same as F1s between other inbred strains

Example:

(BXD1/Ty X AXB19/Pgn)F1 An F1 between a female BXD1/Ty and a male AXB19/Pgn.

4.2 Mixed Inbred Strains

Incipient inbred stocks or inbred strains that are derived from only two parental strains (one of which could be a gene-targeted ES cell line) can be designated using uppercase abbreviations for the two strains, separated by a semicolon. The strain designation preceding the semicolon should be the host and the strain following the semicolon the donor, specifically for targeted mutations where the donor is the ES cell line. When the two progenitor strains do not have a donor/host relationship, the convention followed is the same as when constructing a F1 hybrid designation; that is, the abbreviation of the strain from which the female originated in the first cross is given before the semicolon. Laboratory codes and serial numbers should be used to distinguish strains produced in different laboratories, or multiple strains from the same laboratory. Because these designations may be used for a mixed stock before it is fully inbred, these stocks should not be assumed to be inbred unless accompanied by an inbreeding generation number (e.g., > F20).

Example:

B6;129-Acvr2tm1Zuk A mixed strain derived from C57BL/6J and a 129 ES cell line carrying a targeted knockout of the Acvr2 gene.

A mutant strain, incipient or inbred, derived from more than two progenitor strains or having genetic contribution from an unknown source is considered a "mixed" inbred and may be designated as STOCK followed by a space (i.e., no hyphen) and the mutation(s) or chromosome anomaly it carries.

Example:

STOCK Rb(16.17)5Bnr An inbred strain of unknown or complex background carrying the Robertsonian translocation Rb(16.17)5Bnr.

Once such a mutant stock achieves inbred status, it should be given the appropriate strain designation. It may be designated using the symbols for the genetic mutations it carries in all uppercase, provided the symbols are short. Because the change in strain name is optional, though strongly recommended, some strains designated as STOCK may be inbred.

Example:

JIGR/Dn An inbred strain developed from a mixed background stock carrying the mutations gr(grizzled) and ji (jittery).

When a mutant allele or chromosomal aberration is maintained by crossing animals bearing the mutation to an F1 hybrid at every generation or at alternate generation(s), the stock is designated by the symbol that would be used for that F1, but without the "F1" suffix, and followed by the appropriate allele or chromosome anomaly symbol.

Examples:

B6C3Fe a/a-Dh The Dh (dominant hemimelia) mutation is maintained by crossing to a (B6C3Fe a/a)F1 at each generation, but the stock itself is not an F1.

4.3 Recombinant Congenic Strains

Recombinant Congenic (RC) Strains are formed by crossing two inbred strains, followed by a few (usually two) backcrosses of the hybrids to one of the parental strains (the "recipient" strain), with subsequent inbreeding without selection for any specific markers (Demant and Hart, 1986). Such inbred strains will consist of the background recipient strain genome interspersed with homozygous segments of the donor (the amount of donor strain genome depending on the number of original backcrosses, 2 backcrosses will give on average 12.5%).

RC Strains should be regarded as fully inbred when the theoretical coefficient of inbreeding approximates that of a standard inbred strain. For this purpose, one generation of backcrossing will be regarded as being equivalent to two generations of brother x sister mating. Thus, a strain produced by two backcrosses (N3, equivalent to F6) followed by 14 generations of brother x sister mating (F14) would be fully inbred.

RC strains should be designated by an uppercase abbreviation of the two strains, recipient strain listed first, separated by lowercase "c."

Example:

CcS Recombinant congenic strain between BALB/c recipient and STS donor.

Multiple RC are given serial numbers.

Example:

CcS1, CcS2, CcS3, etc.

If the second strain abbreviation ends in a number (e.g., 129P2), a hyphen should be used to separate it from the serial number.
4.4 Advanced Intercross Lines

Advanced intercross lines (AIL) are made by producing an F2 generation between two inbred strains and then intercrossing in each subsequent generation, but avoiding sibling matings (Darvasi and Soller, 1995). The purpose is to increase the possibility of tightly linked genes recombining.

The symbols should contain the Laboratory code of the laboratory that has produced the line, followed by a colon, the two inbred strain abbreviations, separated by a comma, with the generation number included in the symbol following a hyphen. Generations are designated G3, G4, etc. beginning with the first non-sib cross after the F2 generation.

Example:

Pri:B6,D2-G# This is an AIL stock created at Princeton from the inbred strains C57BL/6 x DBA/2. The G number will increase with each generation.

5 Coisogenic, Congenic, and Segregating Inbred Strains

There are several ways in which inbred strains may differ at only a small part of the genome.
5.1 Coisogenic Strains

Coisogenic strains are inbred strains that differ at only a single locus through mutation occurring in that strain. Strains containing targeted mutations in ES cells that are then crossed to, and maintained, on the same inbred substrain from which the ES cells were derived can be regarded as coisogenic, but the possibility of mutations elsewhere should be considered. Similarly, chemically or radiation induced mutants on an inbred background can be considered coisogenic, although other genomic alterations could be present. A coisogenic strain may accumulate genetic differences over time by genetic drift unless periodically backcrossed to the parental strain.

Coisogenic strains should be designated by the strain symbol (and where appropriate the substrain symbol) followed by a hyphen and the gene symbol of the differential allele, in italics.

Example:

129S7/SvEvBrd-Fyntm1Sor A targeted mutation of the Fyn gene was produced using the AB1 ES cell line derived from 129S7/SvEvBrd. Chimeric animals were mated to 129S7/SvEvBrd and the allele subsequently maintained on this coisogenic strain.

Example:

C57BL/6JEi-tth The tumor with tilted head mutation in the C57BL/6JEi strain.

In some cases, such mutations will be maintained in heterozygous condition. It should be noted that this means that the strain designation does not reflect the breeding system, nor indicate the specific genotype of a given mouse or rat.

Example:

C57BL/6J-cph The congenital progressive hydronephrosis mutation arose on the C57BL/6J strain. It is a coisogenic strain, but because homozygotes are generally juvenile lethal, the strain is maintained by breeding heterozygotes +/cph x +/cph.

If the number of generations of inbreeding since the mutation arose in a coisogenic strain is to be shown, it can be indicated by adding the number of generations since the mutation to the number before:

Example:

F110 + F23 indicates 23 generations of brother x sister matings since the occurrence of a mutation at F110 in an inbred strain.

5.2 Congenic Strains

Congenic strains are produced by repeated backcrosses to an inbred (background) strain, with selection for a particular marker from the donor strain (Snell 1978, Flaherty 1981). Congenic lines that differ at a histocompatibility locus and therefore resist each other's grafts are called congenic resistant (CR) lines.

A strain developed by this method is regarded as congenic when a minimum of 10 backcross generations to the background strain have been made, counting the first hybrid or F1 generation as generation 1. At this point the residual amount of unlinked donor genome in the strain is likely to be less than 0.01. (Note that the amount of donor genome linked to the selected gene or marker is reduced at a much slower rate, approximately equivalent to 200/N, where N is the number of backcross generations for N>5 (Flaherty 1981; Silver 1995).

Marker assisted breeding or marker assisted selection breeding, also known as "speed congenics" permits the production of congenic strains equivalent to 10 backcross generations in as few as 5 generations. (Markel et al., 1997; Wakeland et al., 1997). Provided that the appropriate marker selection has been used, these are termed congenic strains if the donor strain contribution unlinked to the selected locus or chromosomal region is less than 0.01. Ideally, descriptions of speed congenic strains in first publications thereof should include the number and genomic spacing of markers used to define the congenicity of the strain. Because speed congenics depend upon thorough marker analysis and can vary by particular experimental protocol, the inbred status of speed congenics should be regarded with caution.

Congenic strains are designated by a symbol consisting of three parts. The full or abbreviated symbol of the recipient strain is separated by a period from an abbreviated symbol of the donor strain, this being the strain in which the allele or mutation originated, which may or may not be its immediate source in constructing the congenic strain. (In cases where the chromosome on which the mutation arose is unknown, e.g., the donor is not inbred or is complex or an F1 hybrid, the symbol Cg should be used to denote congenic. The use of the donor strain symbol or Cg is essential to distinguish congenic from coisogenic strains. Cg is also used to designate a strain constructed by crossing together two congenic strains that have been backcrossed separately to the same host background, but where their respective donor strains differ. Cg is also applied where alleles originate from a single donor strain, but the congenic strain also carries other coisogenic alleles. In each case, the use of Cg indicates that alleles in the strain name came from more than one source). A hyphen then separates the strain name from the symbol (in italics) of the differential allele(s) introgressed from the donor strain.

Examples:

B6.AKR-H2k A mouse strain with the genetic background of C57BL/6 but which differs from that strain by the introduction of a differential allele (H2k) derived from strain AKR/J.
LEW.BN-RT1n A rat strain with the genetic background of LEW but which differs from that strain by the introduction of a differential segment (RT1n) derived from strain BN.
DA.F344-Cia5 A rat strain with the genetic background of DA onto which a segment from the F344 strain containing the Cia5 QTL has been transferred.
B6.Cg- KitW-44J Gpi1a A mouse strain with the genetic background of C57BL/6, but where the donor strain is mixed, the Kit allele originating from C3H/HeJ and the Gpi1 allele originating from CAST/Ei.

If several lines derived from the same host background and donor strains and carrying the same differential allele(s) are available, the individual lines should be distinguished by adding a forward slash followed by serial numbers and Laboratory codes.

Examples:

C.B10-H2b/1Sn
C.B10-H2b/2Sn

Parentheses may be used to show that an inbred, incipient congenic or congenic inbred strain may have a minor contribution from one other strain.

Examples:

B6(C)-mut A mutation originates on an inbred (e.g., C57BL/6J), is crossed out to or onto another background (e.g., BALB/c) and then is crossed back onto the original background.
C.129P(B6)-Il2tm1Hor A targeted mutation created in a 129 ES cell line and transferred from a B6;129P mixed background to BALB/c.
B6(C)-mut A mutation arises on a congenic strain carrying another mutation (e.g., B6.C-m) and the original mutation is bred out of the new strain. Note if the original mutation is not proved to be removed from the strain, the symbol would be B6.C m-mut.
B6(C)-mut A mutation arises on a hybrid or mixed background stock (e.g., B6CF1) and is backcrossed onto one of the original inbred strains (e.g., C57BL/6J).
B6.C(Cg)-mut A mutation has been backcrossed from one strain (e.g., BALB/c) onto another (e.g., C57BL/6J) but arose on a mixed background or has had a varied history and the origin of the chromosomal segment is unknown.

If the chromosomal segment that has been transferred is defined by several genes or multiple DNA loci, the segment can be defined in the symbol by listing the most proximal and the most distal markers demonstrated to be in the segment in parentheses, separated by a hyphen.

Examples:

B6.Cg-(D4Mit25-D4Mit80)/Lt A congenic strain made by introducing into C57BL/6J a segment of chromosome 4 from an outbred or mixed strain (=Cg), extending between the two defined markers.
B6.CBA-(D4Mit25-D4Mit80)/Lt A similar congenic strain in which the donor chromosomal segment comes from the CBA/J strain.

Note that the markers defining the segment only describe the most proximal and distal markers tested, and this does not imply that there are not other untested markers further proximal or distal. If several lines are made, in the same or different labs that contain the same segment and would be otherwise indistinguishable, then a forward slash, serial number and Laboratory code should be appended.

If necessary, the number of backcross generations should be indicated by N and the number in parentheses following the strain name; generations should not be incorporated into the strain name. Incipient congenics may be given congenic nomenclature at N5, as long as the number of generations of backcrossing is clearly documented in information accompanying the strain. In cases where it is necessary to use more complex mating systems, the generations should be expressed as N equivalents (NE) and the strain regarded as congenic at a minimum of NE10. For example, when backcrossing a recessive gene onto an inbred background, after 10 rounds of backcrossing and intercrossing to recover a homozygote for the next backcross (20 generations), the strain would be at NE10. When a congenic strain is maintained by brother x sister matings after backcrossing, the number of brother x sister generations follows the number of backcross generations, e.g., (N10F6), 10 generations of backcrossing followed by 6 generations of brother x sister inbreeding; (NE12F17), a complex system of backcrosses and intercrosses genetically equivalent to 12 backcrosses, followed by 17 generations of brother x sister matings.

When generating speed congenics N will be less than 10 initially, nevertheless the actual number should be given in parentheses following the N, e.g., N(6), and the details of breeding system and markers used detailed elsewhere in a publication or database.
5.3 Consomic Strains

Consomic strains (also called chromosome substitution strains, Nadeau et al., 2000) are produced by repeated backcrossing of a whole chromosome onto an inbred strain. As with congenic strains, a minimum of 10 backcross generations is required, counting the F1 generation as generation 1. For autosomes it is necessary to genotype progeny to ensure that the selected donor chromosome has not recombined with the corresponding recipient chromosome. The generic designation for consomic strains is HOST STRAIN-Chr #DONOR STRAIN.

Examples:

SHR-Chr YBN In this consomic rat strain, the Y chromosome from BN has been backcrossed onto SHR.
C56BL/6J-Chr 19SPR In this consomic mouse strain, a M.spretus Chromosome 19 has been backcrossed onto C57BL/6J.
C56BL/6J-Chr 1A/J Chr 3DBA/2J In this consomic mouse strain, Chromosome 1 from the A/J strain and Chromosome 3 from the DBA/2J strain have been backcrossed onto C57BL/6J.

Experience shows that on occasion it is impossible to transfer an entire chromosome from one strain to another due to lethal effects on a particular chromosome. For example, a consomic set on which PWD/Ph individual chromosomes were transferred to C57BL/6J revealed that Chr 11 and Chr X cannot be transferred intact. To designate "sections" of transferred chromosomes that contribute to a consomic set, regions can be indicated as a decimal 1, 2, 3, etc.

Thus, a part of Chr 11 of this consomic set would be: C57BL/6J-Chr 11.1PWD/Ph/ForeJ

Although consomic strains are similar in concept and development to congenic strains, in consomic nomenclature the name of the host strain is not abbreviated, and no period followed by the donor strain is required because the strain of origin is shown in the superscript. Capitalization of all letters in the superscript and non-italicization of the chromosome letter/number and of the superscript distinguish a chromosome identifier from an allele symbol.
5.4 Segregating Inbred Strains

Segregating inbred strains are inbred stains in which a particular allele or mutation is maintained in heterozygous state. They are developed by inbreeding (usually brother x sister mating) but with heterozygosity selected at each generation. They are designated like other inbred strains since the segregating locus is part of the standard genotype of the strain (see Section 5.1 Coisogenic Strains). When segregating coat color alleles are part of the inbred strain's normal phenotype, they need not be included in the strain name (see examples below). Details of inbred strain genotypes are available in publications and databases.

Examples:

129P3/J This mouse strain segregates for the tyrosinase alleles albino (Tyrc) and chinchilla (Tyr c-ch)
WB/Re This mouse strain segregates for the dominant white spotting allele of the kit oncogene (KitW).

Strains that carry linked alleles in coupling or repulsion should be designated so that it is clear that the alleles are linked and the phase of the linked genes is specified.

Examples:

B6.Cg-m Leprdb/+ + In this strain the m andLeprdb alleles are carried on one chromosome (in coupling) and the wild type alleles on the other.
B6.Cg-m +/+ Leprdb In this strain the m and Leprdb alleles are carried on different homologs of the chromosome (in repulsion); this is also called a balanced strain.

5.5 Conplastic Strains

Conplastic strains are strains in which the nuclear genome from one strain has been crossed onto the cytoplasm of another, i.e., the mitochondrial donor is always the female parent during the backcrossing program. The designation is NUCLEAR GENOME-mtCYTOPLASMIC GENOME.

Example:

C57BL/6J-mt BALB/c A strain with the nuclear genome of C57BL/6J and the cytoplasmic (mitochondrial) genome of BALB/c.

Such a strain is developed by crossing male C57BL/6J mice with BALB/c females, followed by repeated backcrossing of female offspring to male C57BL/6J. As with congenic strains, a minimum of 10 backcross generations is required, counting the F1 generation as generation 1.
6. Outbreds and Closed Colonies
6.1 Outbreds

Outbred stocks are genetically undefined; that is, no two individuals from an outbred stock are the same. Outbreds are intentionally not bred with siblings or close relatives, as the purpose of an outbred stock is to maintain maximum heterozygosity. One advantage of using outbred stocks is lower cost, because outbreds have relatively long lifespan, are resistant to disease, and have high fecundity. They are useful for experimentation where genotype is unimportant and where a random genetic population is desired. For outbreds, the common strain root is preceded by the Laboratory Code of the institution holding the stock.

Examples:

Tac:ICR The ICR outbred stock maintained by Taconic Farms, Inc.
Hsd:NIH Swiss The NIH Swiss outbred stock maintained by Harlan Sprague Dawley, Inc.

6.2 Closed Colonies

A closed colony contains limited genetic diversity, and is maintained neither by sib-mating (inbred), nor by selective mating to maximize heterozygosity (outbred). All matings occur within the colony members, but breeders need not be selected from specific parentage. No animals are introduced into the colony from outside the stock from generation to generation.

Closed colonies may be established as a way to more readily maintain a difficult mutation, where the desire is to maintain a reasonably uniform background, but poor mating performance prohibits use of sib-mating schemes. Note that closed colonies describe a permanent mating system and this does not apply, for example, if an inbred strain is out-crossed to a near relative in a single generation because of a temporary breeding crisis.

Closed colony designations consist of the strain of origin and appropriately designated mutations (if applicable), followed by [cc] to indicate closed colony.

Example:

C57BL/6Tac-Bmp4tm1Blh[cc] A closed colony of mice originating from the C57BL/6Tac inbred strain and carrying the Bmp4tm1Blh targeted mutation.

7. References

Bailey, D.W. 1971. Recombinant inbred strains, an aid to finding identity, linkage, and function of histocompatibility and other genes. Transplantation 11:325-327

Committee on Rat Nomenclature. 1992. Definition, nomenclature, and conservation of rat strains. ILAR News 34: S1-S26

Committee on Standardized Genetic Nomenclature for Mice. 1952. Standardized nomenclature for inbred strains of mice. Cancer Res. 12:602-613.

Committee on Standardized Genetic Nomenclature for Mice. 1960. Standardized nomenclature for inbred strains of mice, second listing. Cancer Res. 20:145-169.

Committee on Standardized Genetic Nomenclature for Mice. 1976. Nomenclature for inbred strains of mice preserved by freezing. Mouse News Lett 54:2-3.

Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. Rules for nomenclature of inbred strains, pp. 368-372. In: Genetic Variants and Strains of the Laboratory Mouse, Green, M.C. (ed.), First Edition, Gustav Fischer Verlag, Stuttgart, 1981.

Committee on Standardized Genetic Nomenclature for Mice, Chair: Lyon, M.F. Rules for nomenclature of inbred strains, pp. 632-635. In: Genetic Variants and Strains of the Laboratory Mouse, Lyon, M.F., A.G. Searle (eds.), Second Edition, Oxford University Press, Oxford, 1989.

Committee on Standardized Genetic Nomenclature for Mice, Chair: Davisson, M.T. Rules for nomenclature of inbred strains, pp. 1532-1536. In: Genetic Variants and Strains of the Laboratory Mouse, Lyon MF, Rastan S, Brown SDM (eds.), Third Edition, Oxford University Press, Oxford, 1996

Darvasi A, Soller M. 1995. Advanced intercross lines, an experimental population for fine genetic mapping. Genetics 141: 1199-1207

Demant, P. and Hart, A.A.M. 1986. Recombinant congenic strains- a new tool for analyzing genetic traits determined by more than one gene. Immunogenetics 24:416-422.

Eppig JT. 2006. Mouse Strain and Genetic Nomenclature: an Abbreviated Guide. In: The Mouse in Biomedical Research, Volume 1, Second Edition. Fox J, Barthold S, Davisson M, Newcomer C, Quimby F, Smith A, eds. Academic Press. pp 79-98.

Festing, M.F.W. 1979. Inbred strains in biomedical research, Macmillan Press, London: Oxford University Press, New York.

Festing, M.F.W. 1993. Origins and characteristics of inbred strains of mice, 11th listing. Mouse Genome 91:393-550.

Flaherty L. 1981. Congenic strains. In The Mouse in Biomedical Research, Vol. 1, Foster HL, Small JD, Fox JG (eds.), Academic Press, New York, pp. 215-222.

Green EL. 1981. Genetics and Probability in Animal Breeding Experiments. Oxford University Press, New York

Maltais LJ, Blake JA, Eppig JT, Davisson MT 1997. Rules and guidelines for mouse gene nomenclature: a condensed version. International Committee on Standardized Genetic Nomenclature for Mice. Genomics 45: 471-476

Markel P, Shu P, Ebeling C, Carlson GA, Nagle DL, Smutko JS, Moore KJ. 1997. Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains. Nat Genet. 17:280-284.

Nadeau JH, Singer JB, Matin A, Lander ES. 2000. Analyzing complex genetic traits with chromosome substitution strains. Nat Genet. 24: 221-225.

Silver LM. 1995. Mouse Genetics: Concepts and Applications. Oxford University Press, Oxford.

Snell GD. 1941. Biology of the Laboratory Mouse, 1st Edition, McGraw-Hill, New York

Snell GD. 1978. Congenic resistant strains of mice. In Origins of Inbred Mice, Morse HC (ed.), Academic Press, New York, pp. 1-31.

Staats J. 1985. Standardized Nomenclature for Inbred Strains of Mice: eighth listing. Cancer Res. 45: 945-977

Taylor B.A. 1978. Recombinant inbred strains: use in gene mapping. In Origins of Inbred Mice, Morse, HC. (eds.), Academic Press, New York, pp. 423-438.

Wakeland E, Morel L, Achey K, Yui M, Longmate J. 1997. Speed congenics: a classic technique in the fast lane (relatively speaking). Immunol Today 18: 472-477.

Campath (Alemtuzumab)

2008-12-04T10:43:00.000-08:00

DNA derived humanized monoclonal antibodyy.
Targeted against CD52.
CD52 is epressed on surface of normal and malignant B and T lymphocytes, NK cells, monocytes, macrophages, tissues of the male reproductive system.
Erythrocytes or hematopoetic stem cells do not have CD52.
Mature sperms have CD52.
Skin and male reproductive tract hace CD52.

Mechanism of action: antibody dependent lysis of leukemic cells following cell surface binding.

Lab monitoring: FBC weekly. CD4 counts.

About 2% risk of antibodies towards Campth (may cross react with other monoclonals)

No long term studies on Carcinogenesis, Mutagenesis, Impairment of fertility

Contraception should be used in childbearing women and men during Campth use and 6 months following Campath therapy

Side Effects:-
Infusion related A/E: 6% discontinue Campath d/t infusion related A/E
Rigors, drug fever, nausea, vomiting, hypotension, rash, fatigue, urticaria,
Dyspnea, pruritus, headache, diarrhoea.

Infection: 43% risk despite PCP/Herpes prophylaxis (1/3 of pt Grade ¾ severity)

Hematological: Pancytopenia, marrow hypoplasia, Anemia, cytopenias, ITP

Dosing:
Campath therapy should be initiated at a dose of 3 mg administered as a 2 hour IV infusion daily.

When the Campath 3 mg daily dose is tolerated (e.g.infusion-related toxicities are ≤ Grade 2), the daily dose should be escalated to 10 mg and continued until tolerated.

When the 10 mg dose is tolerated, the maintenance dose of Campath 30 mg may be initiated.

The maintenance dose of Campath is 30 mg/day administered three 3 times per week on alternate days (i.e., Monday, Wednesday, and Friday) for up to 12 weeks.

In most patients, escalation to 30 mg can be accomplished in 3 - 7 days.

Dose escalation to the recommended maintenance dose of 30 mg administered three times per week is required.

Single doses of Campath greater than 30 mg or cumulative weekly doses of greater than 90 mg should not be administered since higher doses are associated with an increased incidence of pancytopenia.

Campath should be administered intravenously only.
The infusion should be administered over a 2 hour period.
DO NOT ADMINISTER AS AN INTRAVENOUS PUSH OR BOLUS.

Recommended Concomitant Medications:
Diphenhydramine 50 mg and acetaminophen 650 mg administered 30 minutes prior to Campath infusion.
In cases where severe infusion-related events occur, treatment with hydrocortisone 200 mg was used in decreasing the infusion-related events.

Patients should receive anti-infective prophylaxis to minimize the risks of serious opportunistic infections.
trimethoprim/sulfamethoxazole DS twice daily (BID) three times per week and famciclovir or 348 equivalent 250 mg twice a day (BID) upon initiation of Campath therapy.
Prophylaxis should be continued for 2 months after completion of Campath therapy or until the CD4+ count is ≥ 350

Campath therapy should be discontinued during serious infection, serious hematologic toxicity, or other serious toxicity until the event resolves.
Campath therapy should be permanently discontinued if evidence of autoimmune anemia or thrombocytopenia appears.

Dose is adjusted to Figure above

Preparation:
Aseptic technique should be used.
Withdraw the necessary amount of Campath from the ampoule into a syringe.
Filter with a sterile, low-protein binding, non-fiber releasing 5 μm filter prior to dilution.
Inject into 100 mL sterile 0.9% Sodium Chloride USP or 5% Dextrose in Water USP. Gently invert the bag to mix the solution.
Campath should be used within 8 hours after dilution.
Campath solutions may be stored at room temperature (15-30°C) or refrigerated.
Campath solutions should be protected from light.

MYCYC- A RCT of MMF vs Cyclophosphamide in remission induction of ANCA associated vasculitis

2008-12-04T10:17:00.000-08:00

CD 27, CD28

2008-12-01T13:00:00.001-08:00

CD27

Also called tumor necrosis factor receptor superfamily member 7

Binds to CD70

Marker of T cell activation; also regulates B cell activation and immunoglobulin synthesis

Uses: help differentiate memory-type CD8+ T cells (CD27+) from effector-type CD8+ T cells (important against pathogens, CD27-); memory B cells (CD27+) from naïve B cells (CD27-)

Positive staining (normal): T cells, memory B cells, NK cells, plasma cells, medullary thymocytes

Positive staining (disease): myelomas (64%, Br J Haematol 2006;132:168)

Negative staining: hairy cell leukemia (Haematologica 2005;90:266)

References: OMIM 186711

CD28

T cells require 2 signals for full activation - the first by binding of the antigen/MHC complex on antigen presenting cell to the T cell receptor; the second is delivered by the interaction of CD28 with its ligands CD80 (B7-1) or CD86 (B7-2), found on activated B cells, and is called a costimulation signal (diagram of 2 signals)

However, “superantagonistic” anti-CD28 antibodies, awaiting clinical trials for autoimmune diseases, activate mature T cells with only one signal (Ann Rheum Dis 2005;64 Suppl 4:iv91)

The costimulatory signal induces T cell activation and survival, interleukin-2 production, T-helper type 2 development and clonal expansion

CD28 is a constitutive, high abundance, low affinity receptor; its binding also increases expression of CTLA4 (CD152), a structurally related cell surface receptor on T cells which has the same ligands but opposite effects (J Clin Immunol 2002;22:1, Curr Pharm Des 2006;12:149); CTLA4 competes with CD28 for the same ligands (diagram)

Imbalance in CTLA-4/CD28 expression at the maternal-fetal interface may confer susceptibility to unexplained pregnancy loss (Int J Gynaecol Obstet 2006;93:123)

CD8+, CD28+ T cells: antigen specific cytotoxic T cells (class I restricted) (90% of CD8+ T cells)

CD8+, CD28- T cells: suppressor T cells; increased in various infectious diseases and autoimmune diseases and associated with aging Loss of T cell CD28 expression is associated with aging, and frequency of CD28(null) T cells predicts immune incompetence in elderly; these T cells are functionally active and long-lived, but have no/limited proliferative capacity (Immunol Rev 2005;205:158)

Uses: no significant clinical use by pathologists

Positive staining (normal): CD4+ T cells (95%), CD8+ T cells (50%); activated B cells, plasma cells (some)

Positive staining (disease): myeloma (95%)

References: OMIM 186760, Blood 2005;105:13

IV pulse cyclophosphamide for vasculitis- Addenbrooke's Hospital

2008-11-30T04:29:00.000-08:00

CYC is given as intravenous pulses at weeks 0, 2, 4 and then every 3 weeks until the remission is reached at 3 - 6 months from start of therapy (max 10 doses min 6).

a) CYC may be stopped from 3 months onward provided patient in remission (BVAS 0 for 2 consecutive study assessments). After completion of CYC, AZA to be commenced.

b) Reductions for renal function and age according to table above.

c) Maximum CYC pulse is 1.2g.

d) Upon completion of CYC course AZA to be commenced.

e) Dissolve CYC in water for injection, then dilute in saline 0.9% 500 ml and administer as IV infusion over one hour.

f) Mesna is optional and will be administered orally in the same dose in mg as CYC in mg either from IV vials or in the form of tablets on days when CYC is administered. (If it has to be administered IV reduce mesna dose to 60% of the CYC dose).

g) Prevention of emesis: the choice of antiemetic drugs to cover the CYC pulses should follow local practice. Ondansetron is suitable for this indication.

h)Check FBC on day of pulse or previous day.
If WBC prior to pulse < 4 x 109/L, then postpone pulse until WBC > 4 x 109/L, while checking WBC at least weekly. Reduce dose of pulse by 25%. With any further episodes of leucopenia, make equivalent dose reduction.

i) Check FBC between days 10 and 14 after a pulse. If the leucocyte nadir (i.e. the lowest leucocyte count between two CYC pulses) is < 3 x 109/L, even if the WBC just previous to the next pulse is > 4 x 109/L, then reduce the dose of the next pulse by:
j) leucocyte nadir 1 - 2 x 109/L reduce CYC dose of last pulse by 40 % of previous dose.
k) leucocyte nadir 2 - 3 x 109/L reduce CYC dose of last pulse by 20 % of previous dose.

l) If in remission by three months, or between three and six months, may switch to AZA 2mg/kg/day.

Anti-inflammatory cytokines

2008-11-29T04:08:00.000-08:00

A general term for those immunoregulatory cytokines that counteract various aspects of inflammation, for example cell activation or the production of pro-inflammatory cytokines, and thus contribute to the control of the magnitude of the inflammatory responses in vivo.
These mediators act mainly by the inhibition of the production of pro-inflammatory cytokines or by counteracting many biological effects of pro-inflammatory mediators in different ways.
The major anti-inflammatory cytokines are:-
IL4,
IL10, and
IL13.

Other anti-inflammatory mediators include
IL16,
IFN-alpha,
TGF-beta,
IL1ra,
G-CSF,
soluble receptors for TNF or IL6.

Proinflammatory Cytokines

2008-11-29T04:06:00.000-08:00

A general term for those immunoregulatory cytokines that favour inflammation.
The major pro-inflammatory cytokines that are responsible for early responses are:-
IL1-alpha, IL1-beta,
IL6, and
TNF-alpha.

Other pro-inflammatory mediators include:-
LIF,
IFN-gamma,
OSM,
CNTF,
TGF-beta,
GM-CSF,
IL11,
IL12,
IL17,
IL18,
IL8 and a variety of other chemokines that chemoattract inflammatory cells.

These cytokines either act as endogenous pyrogens (IL1, IL6, TNF-alpha), up-regulate the synthesis of secondary mediators and pro-inflammatory cytokines by both macrophages and mesenchymal cells (including fibroblasts, epithelial and endothelial cells), stimulate the production of acute phase proteins, or attract inflammatory cells.

The net effect of an inflammatory response is determined by the balance between pro-inflammatory and anti-inflammatory cytokines. It should be noted that the common and clear-cut classification of cytokines as either pro anti-inflammatory or pro-inflammatory may be misleading. The type, duration, and also the extent of cellular activities induced by one particular cytokine can be influenced considerably by the nature of the target cells, the micro-environment of a cell, depending, for example, on the growth and activation state of the cells, the type of neighboring cells, cytokine concentrations, the presence of other cytokines, and even on the temporal sequence of several cytokines acting on the same cell.

Elispot

2008-11-29T02:37:00.000-08:00

The Enzyme-linked immunosorbent spot (ELISPOT) assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983.[1]

The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. ELISPOT assays were originally developed to enumerate B cells secreting antigen-specific antibodies, and have subsequently been adapted for various tasks, especially the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.

By virtue of exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses) which were not possible before are now relatively easy. This exceptional sensitivity is in part because the product is rapidly captured around the secreting cell: before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This makes ELISPOT assays much more sensitive than conventional ELISA measurements. Limits of detection are below 1/100,000 rendering the assay uniquely useful for monitoring antigen-specific responses, applicable to a wide range of areas of immunology research, including cancer, transplantation, infectious disease, and vaccine development. The assay has gained a recent increase in popularity, especially as a surrogate measure for CTL responses, in large part because it is both reliable and highly sensitive.

While ELISPOT assay techniques have existed for more than two decades now advancements are still being made in the assay. Modern ELISPOT analysis is typically performed using ELISPOT readers, which employ computer vision techniques to enumerate the actively producing cells. This allows much of the analysis process to be automated, and permits a greater level of accuracy than what can be achieved using manual inspection.

Etanercept- from uptodate

2008-11-29T01:00:00.000-08:00

Etanercept: Drug information

SPECIAL ALERTS
Tumor Necrosis Factor (TNF) Blockers and Malignancy Risk - June 5, 2008

The U.S. Food and Drug Administration (FDA) issued an Early Communication to healthcare professionals regarding a possible association between TNF blocker (adalimumab, certolizumb pegol, etanercept, and infliximab) use and the development of malignancies in children and young adults. Over the last 10 years, the FDA has received ~30 reports of cancer in children or young adults who had been treated with TNF blockers prior to the age of 18 years. TNF blockers were given for the treatment of Juvenile Idiopathic Arthritis (JIA [formerly termed Juvenile Rheumatoid Arthritis]), Crohn's disease, or other indications in combination with other immunosuppressive medications (eg, azathioprine, 6-mercaptopurine or methotrexate). Approximately half of the reported cancers were lymphomas (Hodgkin's and non-Hodgkin's), which are cancers involving the cells of the immune system.

TNF blockers work by suppressing the immune system. The prescribing information for each TNF blocker contains warnings regarding the possible association of malignancy development with use. Malignancies may not be detected in short-term studies; long-term studies are necessary to identify the impact of TNF blocker therapy on malignancy development. The manufacturers of the four TNF blockers available in the U.S. are being asked by the FDA to provide information regarding all cases of cancer reported in children taking TNF blockers. The FDA is expected to report its findings in approximately 6 months, after completing a safety review and evaluation.

Additional information is available at http://www.fda.gov/medwatch/safety/2008/safety08.htm#TNF

Etanercept (Enbrel®): Revised Prescribing Information With the Addition of a Boxed Warning Concerning the Risk of Infection, Including Tuberculosis - May 2008

These product labeling changes have previously been incorporated into the etanercept Lexi-Comp monograph.

The FDA MedWatch alert can be found at: http://www.fda.gov/medwatch/safety/2008/safety08.htm#Enbrel

U.S. BRAND NAMES — Enbrel®

PHARMACOLOGIC CATEGORY
Antirheumatic, Disease Modifying
Tumor Necrosis Factor (TNF) Blocking Agent

DOSING: ADULTS
Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis: SubQ:
Once-weekly dosing: 50 mg once weekly
Twice-weekly dosing: 25 mg given twice weekly (individual doses should be separated by 72-96 hours)

Plaque psoriasis:
Initial: 50 mg twice weekly, 3-4 days apart (starting doses of 25 or 50 mg once weekly have also been used successfully); maintain initial dose for 3 months
Maintenance dose: 50 mg once weekly

DOSING: PEDIATRIC — Juvenile idiopathic arthritis: Children 2-17 years: SubQ:

(For additional information see "Etanercept: Pediatric drug information")

Once-weekly dosing: 0.8 mg/kg (maximum: 50 mg/dose) once weekly

Twice-weekly dosing: 0.4 mg/kg (maximum: 25 mg/dose) twice weekly (individual doses should be separated by 72-96 hours)

DOSING: ELDERLY — SubQ: Refer to adult dosing. Although greater sensitivity of some elderly patients cannot be ruled out, no overall differences in safety or effectiveness were observed.

DOSAGE FORMS — Excipient information presented when available (limited, particularly for generics); consult specific product labeling.

Injection, powder for reconstitution:
Enbrel®: 25 mg [contains sucrose 10 mg; diluent contains benzyl alcohol]

Injection, solution [preservative free]:
Enbrel®: 50 mg/mL (0.51 mL, 0.98 mL) [contains sucrose 1%; natural rubber/natural latex in packaging]

DOSAGE FORMS: CONCISE
Injection, powder for reconstitution:
Enbrel®: 25 mg

Injection, solution [preservative free]:
Enbrel®: 50 mg/mL (0.51 mL, 0.98 mL)

GENERIC EQUIVALENT AVAILABLE — No

ADMINISTRATION — Administer subcutaneously. Rotate injection sites. New injections should be given at least one inch from an old site and never into areas where the skin is tender, bruised, red, or hard. Note: If the physician determines that it is appropriate, patients may self-inject after proper training in injection technique.

Powder for reconstitution: Follow package instructions carefully for reconstitution. The maximum amount injected at any single site should not exceed 25 mg.

Solution for injection: May be allowed to reach room temperature prior to injection.

USE — Treatment of moderately- to severely-active rheumatoid arthritis (RA); moderately- to severely-active polyarticular juvenile idiopathic arthritis (JIA); psoriatic arthritis; active ankylosing spondylitis (AS); moderate-to-severe chronic plaque psoriasis

ADVERSE REACTIONS SIGNIFICANT — Percentages reported for adults except where specified.

>10%:
Central nervous system: Headache (17%; children 19%)
Gastrointestinal: Abdominal pain (5%; children 19%), vomiting (3%; children 13%)
Local: Injection site reaction (14% to 37%; erythema, itching, pain or swelling)
Respiratory: Respiratory tract infection (upper; 12% to 29%), rhinitis (12% to 16%)
Miscellaneous: Infection (35%; children 63%), positive ANA (11%), positive antidouble-stranded DNA antibodies (15% by RIA, 3% by Crithidia luciliae assay)

≥3% to 10%:
Cardiovascular: Edema (2% to 8%)
Central nervous system: Dizziness (7%)
Dermatologic: Rash (5%)
Gastrointestinal: Dyspepsia (4%), nausea (children 9%)
Neuromuscular & skeletal: Weakness (5%)
Respiratory: Pharyngitis (7%), respiratory disorder (5%), sinusitis (3%), cough (6%)

<3%, postmarketing, and/or case reports: Abscess, adenopathy, allergic reactions, alopecia, anemia, angioedema, anorexia, aplastic anemia, appendicitis, aseptic meningitis, bursitis, cerebral ischemia, chest pain, cholecystitis, coagulopathy; demyelinating CNS disorders (suggestive of multiple sclerosis, transverse myelitis, or optic neuritis); deep vein thrombosis, depression, diarrhea, dyspnea, erythema multiforme, fatigue, fever, flushing, flu-like syndrome, gastrointestinal hemorrhage, heart failure, hepatitis (autoimmune), hydrocephalus (with normal pressure), hyper-/hypotension; infections (bacterial, fungal, protozoal, viral); interstitial lung disease, intestinal perforation, joint pain, leukopenia, lupus-like syndrome, lymphadenopathy, malignancies (including lymphoma), membranous glomerulopathy, MI, mouth ulcer, multiple sclerosis, myocardial ischemia, neutropenia, ocular inflammation, optic neuritis, pancytopenia, pancreatitis, paresthesia, polymyositis, pruritus, psoriasis exacerbation, pulmonary disease, pulmonary embolism, renal calculus, sarcoidosis, seizure, stroke, Stevens-Johnson syndrome, subcutaneous nodules, taste disturbances, thrombocytopenia, thrombophlebitis, toxic epidermal necrolysis, transaminases increased, tuberculosis, tuberculous arthritis, urinary tract infection, urticaria, vasculitis (cutaneous), weight gain, xerophthalmia, xerostomia

CONTRAINDICATIONS — Hypersensitivity to etanercept or any component of the formulation; patients with sepsis (mortality may be increased); active infections (including chronic or local infection)

WARNINGS / PRECAUTIONS
Boxed warnings:

* Infections: See "Concerns related to adverse effects" below.

* Tuberculosis: See "Concerns related to adverse effects" below.

Concerns related to adverse effects:

* Anaphylaxis/hypersensitivity reactions: Allergic reactions may occur, but anaphylaxis has not been observed. If an anaphylactic reaction or other serious allergic reaction occurs, administration should be discontinued immediately and appropriate therapy initiated.
* Autoimmune disorder: Positive antinuclear antibody titers have been detected in patients (with negative baselines). Rare cases of autoimmune disorder, including lupus-like syndrome or autoimmune hepatitis, have been reported; monitor and discontinue if symptoms develop.
* Hepatitis B: Rare reactivation of hepatitis B has occurred in chronic carriers of the virus; evaluate prior to initiation and during treatment in patients at risk for hepatitis B infection.
* Infections: [U.S. Boxed Warning]: Serious and potentially fatal infections have been reported including bacterial sepsis and tuberculosis. Discontinue administration if patient develops a serious infection or sepsis. Caution should be exercised when considering the use in patients with chronic infection, history of recurrent infection, or predisposition to infection (such as poorly-controlled diabetes). Do not give to patients with an active chronic or localized infection. Patients should be educated about the symptoms of infection and closely monitored for signs and symptoms while undergoing treatment.
* Malignancy: Use may affect defenses against malignancies; impact on the development and course of malignancies is not fully defined. As compared to the general population, an increased risk of lymphoma has been noted in clinical trials; however, rheumatoid arthritis has been previously associated with an increased rate of lymphoma.
* Tuberculosis: [U.S. Boxed Warning]: Tuberculosis (disseminated or extrapulmonary) has been reported in patients receiving etanercept; both reactivation of latent infection and new infections have been reported. Patients should be evaluated for latent tuberculosis infection with a tuberculin skin test prior to starting therapy. Treatment of latent tuberculosis should be initiated before therapy is used. Some patients who tested negative prior to therapy have developed active infection; monitor for signs and symptoms of tuberculosis in all patients.

Disease-related concerns:

* Demyelinating CNS disease: Use with caution in patients with pre-existing or recent onset CNS demyelinating disorders; rare cases of new onset or exacerbation of CNS demyelinating disorders have occurred; may present with mental status changes and some may be associated with permanent disability. Optic neuritis, transverse myelitis, multiple sclerosis, and new onset or exacerbation of seizures have been reported.
* Heart failure: Use with caution in patients with heart failure or decreased left ventricular function; worsening and new-onset heart failure has been reported.
* Hematologic disorders: Use with caution in patients with a history of significant hematologic abnormalities; has been associated with pancytopenia and aplastic anemia (rare cases in postmarketing experience). Patients must be advised to seek medical attention if they develop signs and symptoms suggestive of blood dyscrasias; discontinue if significant hematologic abnormalities are confirmed.
* Wegener's granulomatosis: Use is not recommended for use in patients with Wegener's granulomatosis who are receiving immunosuppressive therapy due to higher incidence of noncutaneous solid malignancies.

Concurrent drug therapy issues:

* Anakinra: Due to higher incidence of serious infections, should not be used in combination with anakinra unless no satisfactory alternatives exist, and then only with extreme caution.

Special populations:

* Pediatrics: Safety and efficacy have not been established in children <2 years of age.
* Varicella virus exposure: Patients with a significant exposure to varicella virus should temporarily discontinue therapy; treatment with varicella zoster immune globulin should be considered.

Dosage form specific issues:

* Latex: Some dosage forms may contain dry natural rubber (latex).

Other warnings/precautions:

* Immunizations: Patients should be brought up to date with all immunizations before initiating therapy. Live vaccines should not be given concurrently; there is no data available concerning secondary transmission of live vaccines in patients receiving therapy.

RESTRICTIONS
An FDA-approved patient medication guide is available and must be distributed when dispensing an outpatient prescription (new or refill) where this medication is to be used without direct supervision of a healthcare provider. Medication guides are available at http://www.fda.gov/cder/Offices/ODS/medication_guides.htm.

DRUG INTERACTIONS
Abatacept: Anti-TNF Agents may enhance the adverse/toxic effect of Abatacept. An increased risk of serious infection during concomitant use has been reported. Risk D: Consider therapy modification

(For additional information: Launch Lexi-Interact™ Drug Interactions Program )

Anakinra: Anti-TNF Agents may enhance the adverse/toxic effect of Anakinra. An increased risk of serious infection during concomitant use has been reported. Risk X: Avoid combination

Cyclophosphamide: Etanercept may enhance the adverse/toxic effect of Cyclophosphamide. An increased risk of solid cancer development may be present. Risk D: Consider therapy modification

Echinacea: May diminish the therapeutic effect of Immunosuppressants. Risk D: Consider therapy modification

Natalizumab: Immunosuppressants may enhance the adverse/toxic effect of Natalizumab. Specifically, the risk of concurrent infection may be increased. Risk X: Avoid combination

Rilonacept: Anti-TNF Agents may enhance the adverse/toxic effect of Rilonacept. Risk X: Avoid combination

Trastuzumab: May enhance the neutropenic effect of Immunosuppressants. Risk C: Monitor therapy

Vaccines (Inactivated): Immunosuppressants may diminish the therapeutic effect of Vaccines (Inactivated). Risk C: Monitor therapy

Vaccines (Live): Immunosuppressants may enhance the adverse/toxic effect of Vaccines (Live). Vaccinal infections may develop. Immunosuppressants may also decrease therapeutic response to vaccines. Risk X: Avoid combination

ETHANOL / NUTRITION / HERB INTERACTIONS — Herb/Nutraceutical: Echinacea may decrease the therapeutic effects of etanercept (avoid concurrent use).

PREGNANCY RISK FACTOR — B (show table)

PREGNANCY IMPLICATIONS — Developmental toxicity studies performed in animals have revealed no evidence of harm to the fetus. There are no studies in pregnant women; this drug should be used during pregnancy only if clearly needed. A pregnancy registry has been established to monitor outcomes of women exposed to etanercept during pregnancy (877-311-8972).

LACTATION — Excretion in breast milk unknown/not recommended

BREAST-FEEDING CONSIDERATIONS — It is not known whether etanercept is excreted in human milk. Because many immunoglobulins are excreted in human milk and the potential for serious adverse reactions exists, a decision should be made whether to discontinue nursing or to discontinue the drug, taking into account the importance of the drug to the mother.

PRICING — (data from drugstore.com)
Kit (Enbrel)
25 mg (4): $748.92

Solution (Enbrel)
50 mg/mL (3.92): $1471.69

Solution (Enbrel SureClick)
50 mg/mL (3.92): $1541.98

MONITORING PARAMETERS
Signs and symptoms of infection (prior to and during therapy); latent TB screening prior to therapy initiation

CANADIAN BRAND NAMES — Enbrel®

INTERNATIONAL BRAND NAMES — Enbrel (AR, AT, AU, BE, BG, BR, CH, CN, CO, CR, CZ, DE, DK, ES, FI, FR, GB, GR, GT, HK, HN, IE, IL, IN, IT, KP, MX, MY, NI, NL, NO, PA, PE, PH, PL, PT, RU, SE, SG, SV, TH, TR, VE)

MECHANISM OF ACTION — Etanercept is a recombinant DNA-derived protein composed of tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. Etanercept binds tumor necrosis factor (TNF) and blocks its interaction with cell surface receptors. TNF plays an important role in the inflammatory processes and the resulting joint pathology of rheumatoid arthritis (RA), polyarticular-course juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), and plaque psoriasis.

PHARMACODYNAMICS / KINETICS
Onset of action: ~2-3 weeks; RA: 1-2 weeks

Half-life elimination: RA: SubQ: 72-132 hours

Time to peak: RA: SubQ: 35-103 hours

Excretion: Clearance: Children: 45.9 mL/hour/m2; Adults: 89 mL/hour (52 mL/hour/m2)

PATIENT INFORMATION — If self-injecting, follow instructions for injection and disposal of needles exactly. If redness, swelling, or irritation appears at the injection site, contact prescriber. Do not have any vaccinations while using this medication without consulting prescriber first. You may experience headache or dizziness (use caution when driving or engaging in tasks requiring alertness until response to drug is known). If stomach pain or cramping, unusual bleeding or bruising, persistent fever, paleness, blood in vomitus, stool, or urine occurs, stop taking medication and contact prescriber immediately. Also immediately report skin rash, unusual muscle or bone weakness, or signs of respiratory flu or other infection (eg, chills, fever, sore throat, easy bruising or bleeding, mouth sores, unhealed sores).

Infliximab administration protocol

2008-11-29T00:43:00.000-08:00

Protocol for Infliximab Infusion (Adults)

Indications and Dosage:
Rheumatoid arthritis, used in combination with methotrexate, where response to at least two DMARDS has been inadequate (NICE guidance).
• 3mg/kg IV infusion, followed by additional 3mg/kg at weeks 0, 2 and 6 weeks, then every 8 weeks depending on response/need. Clinical response is usually achieved within 12 weeks. Continued therapy should be reconsidered in patients who show no evidence of therapeutic benefit within this time.

Severe, active Crohn’s disease – treatment of severe, active disease in patients who have not responded despite a full and adequate course of therapy with a corticosteroid or immunosuppressant; or who are intolerant or have medical contraindications for such therapies.
• 5mg/kg IV infusion at 0, 2 and 6 weeks. In patients who respond alternative strategies for continued treatment are: • Maintenance: repeat infusions of 5mg/kg every 8 weeks depending on response/need or • Readministration: 5mg/kg infusion if signs and symptoms recur within 16 weeks of last infusion

Fistulising, active Crohn’s disease - treatment of fistulising, active Crohn’s disease, in patients who have not responded, despite full and adequate course of conventional treatment.
• initial 5mg/kg IV infusion, followed with additional 5mg/kg infusion doses at 2 and 6 weeks after the first infusion. If a patient does not respond after 3 doses, no additional treatment with infliximab should be given. In patients who respond alternative strategies for continued treatment are: • Additional infusions of 5mg/kg every 8 weeks or • Readministration if signs and symptoms of the disease recur within 16 weeks of last infusion followed by infusions of 5mg/kg every 8 weeks.

Ulcerative colitis - treatment of moderately to severely active ulcerative colitis in patients who have had an inadequate response to conventional therapy including corticosteroids and 6-mercaptopurine or azathioprine, or who are intolerant to or have medical contraindications for such therapies.
• 5 mg/kg given as an intravenous infusion over a 2 hour period followed by additional 5 mg/kg infusion doses at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter. • Clinical response is usually achieved within 14 weeks of treatment, i.e. three doses. • Continued therapy should be carefully reconsidered in patients who show no evidence of therapeutic benefit within this time period.

Ankylosing spondylitis – in patients who have severe axial symptoms, elevated serological markers of inflammatory activity and who have responded inadequately to conventional therapy.
• 5mg/kg IV infusion followed by additional 5mg/kg infusion at 2 and 6 weeks after the first infusion, then every 6-8 weeks depending on response/need thereafter. If a patient does not respond by 6 weeks (ie after 2 doses), no additional treatment with infliximab should be given.

Psoriatic arthritis, in combination with methotrexate, for treatment of active and progressive psoriatic arthritis in patients who have responded inadequately to DMARDs. • 5mg/kg IV infusion followed by 5mg/kg infusion at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter depending on response/need.

Pretreatment:

Patients should be supplied with the package leaflet and special Alert card.

Contraindications:

• patients with TB or other severe infections such as sepsis, abcesses and opportunistic infections
• patients with moderate of severe heart failure
• hypersensitivity to infliximab, other murine proteins or any of the excipients

Special Warnings and Precautions for Use:
Infusion reactions • Associated with acute infusion-related reactions (eg wheezing, hypotension, pallor, nausea, anaphylactic shock and delayed hypersensitivity) which may develop within seconds or within a few hours following infusion. If this occurs then stop infusion immediately and refer to doctor.
• For severe reactions patients may require treatment with IV hydrocortisone, nebulised salbutamol and chlorphenamine.
• For mild reactions (including: drop in systolic BP < 40mmHg normal blood pressure) the infusion should be temporarily stopped until symptoms subside. Following discussion with a doctor the infusion may be restarted at a slower rate. Some patients may be rechallenged after several weeks.
• Hydrocortisone and/or paracetamol 30 mins before infliximab can prevent mild and transient effects. Infections
• Monitor closely for infections, including TB, before, during and up to 6 months after treatment

When to Withold Treatment

• Presentation of signs and symptoms of intercurrent infection. This includes upper respiratory tract infection and skin ulcer.
• Worsening of coexisting illness such as CCF or diabetes
• Suspected malignancy
• Persistent hypotension (systolic < 100mmHg)
• Impending Surgery (4 week gap for standard procedures; 8 week gap for high sepsis- risk surgery) – restart after wound healing

Exclude Infection Prior to Each Administration:

• Take a complete history and examination
• Monitor: • temperature, • urinalysis – refer to doctor if positive for blood or protein
• Send for the following bloodtests: FBC, ESR, U&Es, LFTs, CRP
• Additional tests for RA patients: Rheumatoid factor, ANA-dsDNA

Administration and Monitoring:

• Infliximab is obtained from the Aseptic Dispensing Unit (ADU), Pharmacy.
24 hour notice is required.
• IV Hydrocortisone 100-200mg stat prior to infliximab - not always necessary, there is evidence to suggest that this reduces the incidence of infliximab antibodies. Check with the prescriber.
• Insert venflon and flush with normal saline.
Monitor blood pressure, pulse and temperature every 30 minutes during the infusion and observation periods.
Patients should be observed for at least 1-2 hours post infusion for acute infusion related reactions.
• Infliximab is given in 250ml of sodium chloride 0.9%, and should be infused over a 2 hour period.
Occasionally there are patient specific deviations from this regimen.
The drug can be given through a normal giving set, via a 1.2-micron low-protein binding filter.
Filters can be ordered from supplies: "1.2micron filter lipid solutions priming volume 0.2cc male/female connectors" Medex code MX1483 Supplier = NHS Logistics, code FTC192 Rheumatoid arthritis patients – for carefully selected patients who have received 3 initial infusions over a 2 hour period, subsequent infusions can be infused over a period of not less than 1 hour, and patients observed for 1 hour afterwards.

Side Effects and Toxicity:

Recognised side effects of treatment include flu-like symptoms, headache, transient fever, gastrointestinal upset and skin rashes. Please consult the infliximab data sheet available on-line at www.medicines.org.uk for a complete list. Rarely patients have become ANA positive and developed lupus-like syndromes while receiving treatment. Adverse reactions should be reported to the CSM using the “Yellow Cards” in the usual manner.

Plasmapheresis Protocol- Hospital Kuala Lumpur

2008-11-29T00:36:00.000-08:00

1-2 plasma volume/session
PV= (1-Hct)(b+cW)
Hct= Hematocrit
b=1530 for males, 864 for females
c=41 for males, 47.2 for females
W= lean body weight

Rough estimation: at Hct 45.
Plasma Volume=40ml/kg (BW)
Usually 5-7 sessions of plasmapheresis is required exchanges is usu done 24 hours apart
Replacement flluid- 5% human albumin
Monitor VS every 15 min
Measure PT,PTT and pre&post procedure RP, LFT, FBC

Anticoagulation: Heparin 50u/kg stat than 1000u/h
target ACT 1.5-2.0X normal
adjust dose accordingly
stop heparin 30min prior to end of procedure

To avoid hemolysis, PE must be done at
TMP 50-60mmHg
Qb 100-150mls/min

Complications of PE
Hypotension- bld loss, decrease oncotic pressure
Bleeding d/t reduction in coagulation factors
Edema- reduction of intravascular oncotic pressure
Loss of cellular elements
Ethylene Oxide hypersensitivity

Related to anticoagulation
Bleeding- heparin related
Hypocalcemia leading to arrhythmias/ hypotension/ numbness/ tingling sensation

CD86

2008-11-28T11:37:00.000-08:00

Also called B7-2

T cells need two signals for activation - the first signal is antigen peptide presented on MHC class II through the T cell receptor

The second (costimulatory) signal is delivered by CD80 or CD86, expressed on surface of antigen presenting cells, which interact with either CD28 or CD152 (CTLA-4)

CD80 and CD86 appear to have opposing functions on regulatory T cells (J Immunol 2004;172:2778)

Polymorphisms are associated with liver transplant acceptance (Transpl Immunol 2005;15:69) and systemic sclerosis (Int J Immunogenet 2006;33:155)

Increased expression may cause excessive antigen presentation in fulminant hepatic failure as an early step in its pathogenesis before the onset of tissue damage (Am J Pathol 1999;154:1711)

High circulating soluble levels are poor prognostic factor in myeloma (Br J Haematol 2006;133:165); are associated with severe asthma (Thorax 2004;59:870)

Receptor for some adenovirus species (Virus Res 2006;122:144)

Associated with H. pylori dependent early stage high grade MALT lymphoma of stomach (World J Gastroenterol 2005;11:4357)

Uses: no significant clinical use by pathologists

Diagram: costimulatory signal

Micro images: inflamed skin; fulminant hepatic failure; chronic HBV infection of liver; liver sinusoidal endothelial cells; thyroid carcinoma (E-H); normal esophagus; esophageal carcinoma

early stage high grade gastric MALT lymphoma - H. pylori dependent case (CD86+); H. pylori independent case (CD86-)

Positive staining (normal): interdigitating dendritic cells in T zones of secondary lymphoid organs, Langerhans cells, peripheral blood dendritic cells, memory B cells, germinal center B cells, monocytes, endothelial cells, activated T cells

Positive staining (disease): AML (29%, Clin Cancer Res 2005;11:5708), ulcerative colitis (100%, Dig Dis Sci 2004;49:1738)

Negative staining: immature dendritic cells

References: OMIM 601020

CD43

2008-11-28T11:36:00.000-08:00

Also called leukosialin, sialophorin

Appears to be important for immune function and may be part of a physiologic ligand-receptor complex involved in T-cell activation

Serves as a ligand for E-selectin on T cells and may regulate T cell trafficking (Blood 2006;107:1421, J Immunol 2005;175:8042)

Defective expression in T cells of males with Wiskott-Aldrich syndrome (OMIM 301000)

Uses: pan T cell marker, diagnosis of granulocytic sarcoma (J Clin Pathol 2005;58:211), classify T cell and low grade B cell lymphoma subtype, differentiate pulmonary MALT lymphoma (CD20+ CD43+) from lymphoid hyperplasia (CD43 neg, AJSP 2002;26:76)

Micro images: anaplastic large B cell lymphoma #1 of breast (figure c); #2-post transplant tumor of stomach (figure D); #3-AIDS associated; blastic NK lymphoma; #1 of breast (figure 6); atypical SLL/CLL

Positive staining (normal): most T cells, activated B cells, B cells in terminal ileum (Appl Immunohistochem Mol Morphol 2005;13:138), plasma cells (Scand J Immunol. 1991 Feb;33(2):211-8.), NK cells, granulocytes, monocytes, megakaryocytes, erythroid cells, hematogones (Br J Haematol 2005;128:820), Langerhans cells, brain, thymocytes, some macrophages, platelets (weak)

Positive staining (disease): T/NK cell lymphomas - anaplastic large cell (variable), blastic NK, hepatosplenic gamma-delta T cell, lymphoplasmacytic (variable), NK/T cell-nasal type (96%, Hum Path 2004;35:86), peripheral T cell, subcutaneous panniculitis-like, CD4+ CD56+ lineage negative malignancies (AJSP 2005;29:1274)

B cell lymphomas - ALL (most), Burkitt’s (almost all), Burkitt’s-like (almost all, AJSP 2005;29:1652), lymphoblastic (variable), mantle cell (100%, AJCP 2003;119:218), marginal zone (variable), nodal marginal zone (variable, AJSP 2003;27:762), plasmablastic, SLL/CLL (AJCP 1999;112:319)

other - AML, granulocytic sarcoma, hemangioma, Langerhans cell histiocytosis, mast cell disease (AJSP 2000;24:703), plasmacytoma; early colonic adenoma (Oncol Rep 2004;11:327)

Negative staining: colonic epithelium, follicular lymphoma, Hodgkin’s lymphoma, lymphoepithelioma-like thymic carcinoma, primary effusion lymphoma, splenic marginal zone lymphoma

References: OMIM 182160

CD23

2008-11-28T11:35:00.000-08:00

Also known as low affinity IgE receptor, Fc fragment of IgE receptor, FCER

A type C lectin that can be secreted

After physiologic germinal cell development, the follicular dendritic cell meshwork expands and follicular dendritic cells in the light zone of the germinal center become CD23 positive

CD23 acts as a B cell growth and activation factor, promoting differentiation into plasma cells

Regulates IgE synthesis through CD21 and IgE binding (J Exp Med 2005;202:751), and mediates IgE related immune responses (Clin Rev Allergy Immunol 2005;29:61)

Shows variability in flow cytometry expression between specimens from same patient (AJCP 2002;117:615)

CD21, CD23 and CD35 are dendritic cell markers

High expression on B cells in peripheral blood is associated with bullous pemphigoid (J Dermatol Sci 2004;35:53)

CD23 antibodies may decrease adherence of Plasmodium falciparum-infected erythrocytes (Cell Microbiol 2004;6:839)

Mantle cell lymphoma: usually CD23 negative, but rarely/often has CD23 present with dim intensity by flow cytometry, AJCP 2003;120:760 / AJCP 2001;116:893; CD23+ mantle cell cases have high cyclin D1 levels, AJCP 2002;117:237); rarely has CD23+ cells in peripheral blood (AJCP 2002;118:758)

Uses: differentiate SLL/CLL (CD23+) vs. mantle cell lymphoma or MALT lymphoma (CD23-); B cell marker, particularly for SLL/CLL, mediastinal large B cell lymphoma and lymphoplasmacytic lymphoma; distinguish nodal mantle cell lymphoma from follicular lymphoma by identifying a disrupted follicular dendritic cell pattern (Int J Surg Pathol 2005;13:73), may identify prognostically favorable cases of diffuse large B cell lymphoma (Clin Cancer Res 2003;9:722), high soluble CD23 is associated with aggressive disease and poorer prognosis in CLL (Leuk Lymphoma 2002;43:549, Clin Lab Haematol 2006;28:30)

Micro images: angioimmunoblastic T cell lymphoma (figure F: CD23 highlights extrafollicular meshworks of follicular dendritic cells); CD23 negative MALT lymphoma with CD23+ follicular centers; CD23 negative mantle cell lymphoma with CD23+ follicular center

contributed by Leica Microsystems, Biosystems Division: normal tonsil; follicular lymphoma

Positive staining (normal): activated mature B cells expressing IgM or IgD (particularly mantle cells), activated monocytes / macrophages, T cell subsets, platelets, eosinophils, Langerhans cells, follicular dendritic cells, intestinal epithelium (encodes IgE receptor, Gastroenterology 2005;129:928)

Positive staining (disease): B-cell CLL/SLL (almost all cases; high levels, Leuk Res 2002;26:809; atypical cases may have higher levels, AJCP 2001;116:655); follicular dendritic cell tumors (including inflammatory pseudotumor type- AJSP 2001;25:721), mediastinal large B cell lymphoma (70%, Histopathology 2004;45:619), lymphoplasmacytic lymphoma (61%, usually dim intensity by flow cytometry, AJCP 2005;124:414, Clin Lymphoma 2005;5:246), hairy cell leukemia (17%, AJCP 2006;125:251), diffuse large B cell lymphoma (16%),

Negative staining: other B cell lymphomas including Burkitt’s lymphoma, Burkitt-like lymphoma (AJSP 2005;29:1652), follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma (AJSP 1999;23:59, Mod Path 1998;11:967); also most T cell lymphomas, inflammatory fibroid polyps of the gastrointestinal tract (although of dendritic cell origin, AJSP 2004;28:107), follicular dendritic cell tumor, interdigitating dendritic cell tumor

References: OMIM 151445, Hum Path 1999;30:648 (early study)

CD21

2008-11-28T11:34:00.000-08:00

Also called CR2, C3d receptor, EBV receptor

Binds to Epstein Barr virus (EBV) and HHV8 (J Virol 2005;79:4651), breakdown products of complement component C3 (C3d), CD23 (plays a role in IgE synthesis) and possibly gamma interferon

Follicular dendritic cells produce a different isoform of CD21 than B cells (J Exp Med 1997;185:165)

CD21, CD23 and CD35 are dendritic cell markers

Hodgkin’s lymphoma demonstrates disruption of follicular dendritic cell-germinal cell clusters (evaluated by CD21 and CD23)

Note: although CD21 is the receptor for EBV, it is not necessarily expressed in EBV+ tumors

Shows variability in flow cytometry expression between specimens from same patient (AJCP 2002;117:615)

Uses: diagnose follicular dendritic cell sarcomas (AJSP 1996;20:944); assess follicular dendritic cell meshwork infrastructure (AJCP 2005;124:182, AJSP 2001;25:388), distinguish cutaneous or nodal mantle cell lymphoma from follicular lymphoma (AJSP 2001;25:732, Int J Surg Pathol 2005;13:73), distinguish splenic littoral cell angioma (CD21+ lining cells) from splenic hamartomas (CD21-, AJSP 1997;21:827), confirm that atypical cells have follicular dendritic origin in fine needle aspirates of hyaline-vascular Castleman’s disease (Diagn Cytopathol 2000;22:230)

Micro images: normal germinal center

follicular dendritic cell sarcoma - (1) liver (figure 2B); (2) figure 1: liver tumor (inset: splenic tumor); 2a/b: H&E; 2c: CD21; 2d: CD35; (3) stomach (figure 4 is CD21/CD35 cocktail)

follicular lymphoma - well organized clusters of dendritic cells in follicular lymphoma #1 (testicular)); #2 (head and neck); #3 (head and neck); peripheral T cell lymphoma resembling follicular lymphoma

Positive staining (normal): mature B cells (particularly marginal and mantle cells), follicular dendritic cells, pharyngeal and cervical epithelial cells, some thymocytes, some T cells

Positive staining (tumors): follicular dendritic cell sarcoma (AJSP 2004;28:988, AJSP 2001;25:721), hairy cell leukemia, B cell lymphomas (particularly mantle and marginal zone), hyaline vascular variant of Castleman's disease (AJSP 2002;26:662), splenic littoral cell angiomas (lining cells are CD21+, AJSP 1997;21:827), some T-ALL

Negative staining: dendritic cell neurofibroma with pseudorosettes (AJSP 2001;25:587), interdigitating dendritic cell sarcoma (AJCP 2001;115:589), histiocytic sarcoma (AJSP 2004;28:1133), inflammatory fibroid polyps of GI tract (AJSP 2004;28:107), splenic hamartomas, plasma cells

References: AJSP 2001;25:721, Mod Path 2002;15:50, OMIM 120650

CD19, CD20

2008-11-28T11:32:00.000-08:00

CD19

Coreceptor with CD21

Earliest B cell antigen in fetal tissue

Regulates B cell development, activation and differentiation (J Immunol 2003;171:5921)

May define intrinsic and antigen receptor-induced signaling thresholds critical for clonal expansion of the B cell pool and humoral immunity (Curr Dir Autoimmun 2005;8:55)

More common in plasma cells in steroid resistant ulcerative colitis than Crohn’s disease (Virchows Arch 2006;448:412); presence of CD19+ cells in intestinal mucosa may predict long remission after infliximab (anti-TNF alpha) therapy in Crohn’s disease (Hepatogastroenterology 2005;52:1128)

Uses: diagnosis of B cells and B cell disorders; may be more sensitive than CD20 to detect B cell acute leukemias (Zhongguo Shi Yan Xue Ye Xue Za Zhi 2005;13:943), to differentiate follicular lymphoma (dimmer CD19 in CD10+ B cells by flow cytometry) from reactive hyperplasia (AJCP 2005;124:576)

Flow cytometry images: follicular lymphoma with IgG light chain restriction; biphenotypic acute leukemia with CD19 and myeloperoxidase coexpression (figure B); hairy cell leukemia variant: A-CD20 (bright) and CD22+; B-CD11c+ and CD22+; C-CD103+ and CD25 negative; D-CD19+ and FMC7+; E-kappa+; F-lambda negative

Positive staining (normal): Pre B cells, B cells (considered a pan B cell antigen); first B cell antigen after HLA-DR, follicular dendritic cells

Positive staining (disease): B cell lymphomas and leukemias but often weak/negative in follicular lymphoma or diffuse large B cell lymphoma (Histopathology 2006;48:239, Cytometry B Clin Cytom 2005;63:28), occasional myeloid leukemias (AML-AJCP 1998;109:211; AML-M0-AJCP 2001;115:876; CML blast phase-AJCP 2004;121:836), occasional anaplastic large cell lymphoma by flow cytometry (AJCP 2003;119:205)

Negative staining: plasma cells, myeloma (AJCP 2004;121:482), most T cell lymphomas, often L&H cells in lymphocyte predominant Hodgkin’s lymphoma, often post-transplant B cell lymphoproliferative disorder

References: OMIM 107265

CD20

Also called L26, membrane spanning 4 domains (MS4A1)

33kd phosphoprotein with 3 hydrophobic regions that traverse the cell membrane, creating a structure similar to an ion channel that allows for the influx of calcium required for cell activation

Initially expressed on B cells after CD19/CD10 expression and before CD21/CD22 and surface immunoglobulin expression; retained on mature B cells until plasma cell development

Delivers early signal in B cell activation, allowing resting B cells to respond to later antigens

Closely related to FMC7, which recognizes a CD20 epitope (Leukemia 2003;17:1384), particularly if there is strong CD20 expression (AJCP 2003;120:754)

Rituximab is a chimeric murine-human anti-CD20 antibody used to treat B cell lymphomas; treatment may cause selection of CD20 negative (but CD79a+) tumor subclones (AJSP 2005;29:1399)

Rituximab is also used to treat autoimmune disorders (Clin Immunol 2005;117:207), TTP/HUS (Acta Cytol 2005;19:423), ABO incompatible transplantation (Transplant Proc 2005;37:1205) and transplant rejection (Clin Transplant 2005;19:137)

Anti CD20-antibody attached to radioisotopes is also used to treat B cell lymphomas (Clin Exp Med 2006;6:1)

Case reports: CD20+ T cell lymphomas (Am J Hematol 2002;71:331, AJCP 1994;102:483, Mod Path 2001;14:105, Mod Path 2000;13:1244); rarely stains nucleoli of malignant T cells (Acta Cytol 2005;49:365), but see J Clin Pathol 2004;57:442

Uses: commonly used marker for B cells

Micro images: normal lymph node #1; #2

angioimmunoblastic T cell lymphoma (figure d)

diffuse large B cell lymphoma - bone; brain-#1; #4 (figure A); #5 (intravascular); #6 (intravascular); liver; nasal cavity (figure 3A); ovary CD20 (fig 3), CD3 (fig 4); sclerosing; skin (figure 1D); small intestine; unknown site #1-intravascular; #2-sclerosing subtype

other leukemia/lymphoma - follicular lymphoma #1; #2 (childhood); hairy cell leukemia #1; #2-variant type (figure D); Hodgkin’s lymphoma #1-lymphocyte predominant (figures C&D); #2 (figure C); #3-mostly negative Reed-Sternberg cells); lymphoplasmacytic lymphoma/ Waldenström macroglobulinemia #1 (brain-figure D); MALT lymphoma #1 of bladder; #2 of liver; #3 of lung;

SLL of colon (figure C)

post-transplantation lymphoproliferative disorder - #1; #2 (polymorphic subtype-figure C); #3 of liver: H&E, CD20, EBV

other - ectopic hamartomatous thymoma (figure C); lymphocytic mastitis: CD20+/CD3- lymphocytes (B, not T cells); immunoblastic myofibroblastic tumor (figure 2d-reactive B cells)

Additional images: intravascular large cell lymphoma (figure 2a); post-transplant lymphoproliferative disorder

Virtual slides: diffuse large B cell lymphoma

Flow cytometry images: hairy cell leukemia variant - A-CD20 (bright) and CD22+; B-CD11c+ and CD22+; C-CD103+ and CD25 negative; D-CD19+ and FMC7+; E-kappa+; F-lambda negative

Positive staining (normal): most B cells (considered a pan B cell antigen), also follicular dendritic cells

Positive staining (disease): 90% of B cell lymphomas; also B-CLL, hairy cell leukemia, spindle cell thymomas (AJSP 1992;16:988), 40% of pre B ALL/LBL; 80% of nodular lymphocyte predominant Hodgkin’s lymphoma, 20% of classic Hodgkin’s lymphoma (may be an adverse prognostic factor, Br J Haematol 2004;125:701); dimly expressed in T cells (benign and neoplastic, particularly in bone marrow, AJCP 1996;106:78, AJCP 1994;102:483), some myelomas (Mod Path 2004;17:1217, Blood 2003;102:1070)

Negative staining: non-hematopoietic cells, most T cells, plasma cells, mastocytosis

Note: staining does not work well with Bouin’s fixative

Flow cytometry: brighter expression in follicular lymphomas than normal B cells (AJCP 2005;124:576)

References: OMIM 112210, J Biol Chem 2004;279:19893 (presence in lipid rafts)

CD16

2008-11-28T11:31:00.000-08:00

CD16a

Also known as Fc gamma receptor III A, low affinity immunoglobulin gamma Fc region receptor III-A

Receptor for the Fc portion of IgG; binds various IgG molecules, including rheumatoid factor

Mediates antibody dependent cytotoxicity of foreign cells, phagocytosis and other antibody-dependent responses; also platelet satellitism (AJCP 1995;103:740)

Affinity to ligand is regulated by glycosylation (Immunology 2003;110:335)

CD14+ CD16+ monocytes have increased capacity to produce proinflammatory cytokines such as TNF-alpha, and are elevated in various inflammatory diseases, including coronary artery disease (Thromb Haemost 2004;92:419)

Polymorphisms influence: (a) the severity but not the incidence of IgA nephropathy in Japanese patients (Nephrol Dial Transplant 2005;20:2439); (b) pathogenesis of coronary artery disease (Atherosclerosis 2005;180:277), (c) clinical response to rituximab (Cancer Res 2004;64:4664)

If target cell has class I MHC, then NK cell's killer cell inhibitory receptor (KIR) inhibits cytolysis

Note: preincubation with CD16/CD32 antibodies is commonly used to prevent nonspecific binding

Uses: NK cell and macrophage marker; to subtype leukemia/lymphoma

Diagrams: NK cell mediated cytotoxicity [CD16 / FcgammaRIII is activating receptor on NK cell]

Positive staining (normal): NK cells (10-20%), granulocytes, macrophages, T cells (reactive), immature thymocytes, placental trophoblast

Positive staining (disease): NK proliferative disorders, T cell large granular lymphocyte leukemia, hepatosplenic gamma-delta T cell lymphoma

References: OMIM 146740

CD16b

Also known as Fc gamma receptor III B, low affinity immunoglobulin gamma Fc region receptor III-B

Highly homologous to CD16a

The most common receptor for the Fc domain of IgG on leukocytes

The only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor

Bears allotypes that define the human neutrophil antigen-1 (HNA-1 and NA) system involved in major post-transfusional reactions (Tissue Antigens 2004;64:119)

Low copy number is associated with glomerulonephritis in systemic lupus erythematosus (Nature 2006;439:851)

CD16+ eosinophils are upregulated in allergic conditions (J Allergy Clin Immunol 2002;109:463)

Affinity to ligand is regulated by glycosylation (Immunology 2003;110:335)

Uses: no significant clinical use by pathologists

Positive staining: neutrophils

CD8

2008-11-28T11:30:00.000-08:00

Also called T cell suppressor/cytotoxic cells, OKT8

Cell surface glycoprotein, member of immunoglobulin superfamily; at 2p12

Heterodimer of an alpha and a beta chain linked by two disulfide bonds; heterodimer on thymocytes and homodimer on peripheral blood T cells

MHC class I restricted receptor; binds to nonpolymorphic region of class I molecules; may increase avidity of interactions between cytotoxic T cell and target cell during antigen-specific activation

Can kill target cells by recognizing peptide-MHC complexes on them or by secreting cytokines capable of signaling through death receptors on target cell surface

CD8 alpha cells promote survival and differentiation of activated T cells into memory CD8+ T cells, which may become clonal (but not malignant) in the elderly (Immunol Rev 2005;205:170)

Contribute to initiation, progression and regulation of autoimmune responses (Curr Opin Immunol 2005;17:624)

Associated with lymphoepithelioma-like carcinoma of lung (AJSP 2002;26:715); low CD8:CD3 ratio in epidermotrophic component of lymphoid infiltrate is suggestive of mycosis fungoides (Mod Path 2003;16:857)

Uses: cytotoxic T cell marker

Drawings: (1) CD8+ T cell interaction with antigen presenting cell #1; #2; (3) destruction of viral infected cell

Micro images: (4) infiltrating lymphocytes in lymphoepithelioma-like carcinoma of cervix-figure 3; (5) T cell lymphoma (type not specified); (6) sinus lining cells in splenic hamartomas #1 are CD8+; #2 (figure 3A); (8) nodal cytotoxic T cell lymphoma; (9) mycosis fungoides; (10) intraepithelial lymphocytes in duodenum (figure 1c); (11) increased intraepithelial lymphocytes at villous tip (figure 2e)

Positive staining (normal): cortical thymocytes (70-80%), T cells (25-35% of mature peripheral T cells, mostly cytotoxic T cells); NK cells (30%, which are also CD3 negative)

Positive staining (disease): epidermotrophic lymphocytes in mycosis fungoides (AJSP 2002;26:450), subcutaneous panniculitis-like T cell lymphoma, indolent T cell lymphoblastic proliferation, sinus lining cells in splenic hemartoma, heterotopic ovarian splenoma, NK/T cell lymphoma (variable), some post-thymic T cell lymphomas, rarely mantle cell lymphoma (AJCP 1998;109:689), rarely CLL (AJCP 1994;102:212, Archives 2000;124:1361), T cell infiltrate in 30% of cases of nodular regenerative hyperplasia of liver (Hum Path 2004;35:1241)

Negative stains: adult T cell leukemia/lymphoma, littoral cell hemangioma of spleen

References: Mod Path 2002;15:1131; OMIM 186910 (alpha chain), OMIM 186730 (beta chain)

CD4

2008-11-28T11:28:00.000-08:00

Also called OKT4

Nonpolymorphous glycoproteins belonging to immunoglobulin superfamily

Expressed on surface of T helper cells; serves as coreceptor in MHC class II-restricted antigen induced T cell activation

CD4+ CD25+ T cells maintain peripheral tolerance and prevent autoimmunity (Curr Top Microbiol Immunol 2005;293:115)

Serves as HIV receptor on T cells, macrophages, brain

Downregulated by HIV Nef protein during AIDS progression (J Virol 2003;77:11536, J Biol Chem 2003;278:33912)

Normally CD4 > CD8; in HIV patients, CD4/CD8 ratio is inverted (i.e. CD4 < CD8) and patients are at risk for opportunistic infections

Homologous to CD223

Uses: classify lymphomas and inflammatory conditions; serum levels are marker of HIV disease progression and response to therapy (CD4+ cells are killed by HIV); serum levels also increased by transient stress (AJCP 2002;117:819)

Drawings: (1) CD4+ T cell and antigen presenting cell; (2) HIV entry into T cells

Positive staining (normal): thymocytes (80-90%), T helper cells, macrophages, Langerhans cells, dendritic cells, granulocytes

Positive staining (disease): many post-thymic T cell leukemia/lymphomas, indolent T cell lymphoblastic proliferation, pityriasis lichenoides, CD4+ CD56+ hematodermic malignancies (blastic NK lymphoma), histiocytic lymphoma / sarcoma, acute myeloid leukemia (AJCP 1995;104:204), some pyothorax associated lymphomas, cutaneous lymphomatoid granulomatosis (AJSP 2001;25:1111), lymphomatoid papulosis (variable), florid antiviral inflammatory response (Mod Path 2003;16:166)

Negative staining: NK cells, T cell lymphoma with cytotoxic phenotype, hepatosplenic alpha/beta and gamma/delta lymphoma, enteropathy associated T cell lymphoma, B cell lymphoma (usually), Hodgkin’s lymphoma (usually), nonhematopoietic neoplasms

References: Cell 1985;42:93 (early article), OMIM 186940

Gating in FACS

2008-11-28T11:20:00.000-08:00

Gating will allow you to view cells of interest by any combination of criteria that you choose. Gating does not change the intensity value assigned to an event as is the case for changes in voltage or compensation.

It simply lets you decide which data to view and which data to ignore or discard.

It is important to check that small changes in your gates don't have significant effects on your results or else your data will be prone to artifact.

When you create or format a data plot (i.e. on the Data menu select "Format Histogram" or "Format Dot Plot") you can select any of the gates that you have created.

This will filter the data and plot will only display those events which meet the gate criteria.

Gating will not discard data unless you have requested this under "Acquisiton and Storage". Gating can subsequently be changed when you analyze your data without any loss of information.

To set up a gate you first draw a "Region" using one of the tools on the tool palette (there are four geometric shapes to choose from outlined with dotted lines). Note that the "Marker bar" (designated with an M) and the quadrant maker tool next do not define regions. They are used for statistical analysis only and can't be used to filter data like the regions/gates.

Regions which are commonly employed include: PI for DNA content: FL2 area vs FL2 width. This window is useful for gaiting out apoptotic cells (lower left quadrant) and doublets (a separate cloud with increased FL2 width). FSC vs SSC: This is useful for gating out RBC, myeloid cells etc, from blood or marrow. Each region that is setup automatically defines a gate (e.g. G1 = R1). To delete regions select "Region List" under the Gates menu. To combine regions into more complex gating criteria use the "Gate List" under the Gates menu. (e.g. G5 = (R1 or R2) and R3).

What are Northern, Southern, Western, Southwestern Blots?

2008-11-28T10:35:00.000-08:00

Southern blotting was the original of the four.
It got its name from the developer, Edwin Southern.
Western blotting was named as a sort of joke. (North, South, East, West).
The rest arose based on these first two.

Southern blotting uses gel electrophoresis for the detection of a specific DNA sequence in a sample of DNA.

Western blotting also uses gel electrophoresis but it is to detect proteins and separate them based on size and shape.

Northern blotting is for the detection of RNA sequences, and so is geared towards detecting gene expression (the technique is very similar to Southern blotting only formaldehyde is used to denature the RNA).

There is no Eastern blot, but there is a southwestern one. It is used to find where proteins (DNA binding proteins) bind to specific sequences of DNA.

Far-western blotting is a molecular biological method which is based on the technique of western blotting. While usual western blotting uses an antibody to detect a protein of interest, far-western blotting uses a non-antibody protein, which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is rather employed to detect protein:protein interactions.

By the way, they all use gel electrophoresis.

Far-Eastern blotting is different from the above. It is a technique developed in the 1990s by T. Taki and colleagues at the Cellular Technology Institute of Otsuka Pharmaceutical Co., Japan for the analysis of lipids separated by high-performance thin layer chromatography (HPTLC). The lipids are transferred from the HPTLC plate to a PVDF membrane for further analysis, for example by enzymatic or ligand binding assays[1] or mass spectrometry[2].

Cholesterol, glycerophospholipids and sphingolipids are major constituents of the cell membrane and in certain cases function as second messengers in cell proliferation, apoptosis and cell adhesion in inflammation and tumor metastasis. Far-eastern blotting was established as a method for transferring lipids from an HPTLC plate to a polyvinyledene difluoride (PVDF) membrane within a minute. Applications of this with other methods have been studied. Far-eastern blotting allows for the following techniques:

* Purification of glycosphingolipids and phospholipids.
* Structural analysis of lipids in conjunction with direct mass spectrometry.
* Binding study using various ligands such as antibodies, lectins, bacterium, viruses, and toxins, and
* Enzyme reaction on membranes.

Not only analysis of lipids but also metabolites of drugs and natural compounds from plants, and environmental hormones are possible by this method.

Alum

2008-11-28T00:07:00.000-08:00

Alum, (IPA: /ˈæləm/) refers to a specific chemical compound and a class of chemical compounds. The specific compound is the hydrated aluminum potassium sulfate with the formula KAl(SO4)2.12H2O. The wider class of compounds known as alums have the related stoichiometry, AB(SO4)2.12H2O.

Alum is used in vaccines as an adjuvant to enhance the body's response to immunogens.

Thalidomide

2008-11-27T23:59:00.000-08:00

WARNING

SEVERE, LIFE-THREATENING HUMAN BIRTH DEFECTS.

IF THALIDOMIDE IS TAKEN DURING PREGNANCY, IT CAN CAUSE SEVERE BIRTH DEFECTS OR DEATH TO AN UNBORN BABY. THALIDOMIDE SHOULD NEVER BE USED BY WOMEN WHO ARE PREGNANT OR WHO COULD BECOME PREGNANT WHILE TAKING THE DRUG. EVEN A SINGLE DOSE [1 CAPSULE (REGARDLESS OF STRENGTH)] TAKEN BY A PREGNANT WOMAN DURING HER PREGNANCY CAN CAUSE SEVERE BIRTH DEFECTS.

BECAUSE OF THIS TOXICITY AND IN AN EFFORT TO MAKE THE CHANCE OF FETAL EXPOSURE TO THALOMID® (thalidomide) AS NEGLIGIBLE AS POSSIBLE, THALOMID® (thalidomide) IS APPROVED FOR MARKETING ONLY UNDER A SPECIAL RESTRICTED DISTRIBUTION PROGRAM APPROVED BY THE FOOD AND DRUG ADMINISTRATION. THIS PROGRAM IS CALLED THE "SYSTEM FOR THALIDOMIDE EDUCATION AND PRESCRIBING SAFETY (S.T.E.P.S.® )".

UNDER THIS RESTRICTED DISTRIBUTION PROGRAM, ONLY PRESCRIBERS AND PHARMACISTS REGISTERED WITH THE PROGRAM ARE ALLOWED TO PRESCRIBE AND DISPENSE THE PRODUCT. IN ADDITION, PATIENTS MUST BE ADVISED OF, AGREE TO, AND COMPLY WITH THE REQUIREMENTS OF THE S.T.E.P.S.® PROGRAM IN ORDER TO RECEIVE PRODUCT.

PLEASE SEE THE FOLLOWING BOXED WARNINGS CONTAINING SPECIAL INFORMATION FOR PRESCRIBERS, FEMALE PATIENTS, AND MALE PATIENTS ABOUT THIS RESTRICTED DISTRIBUTION PROGRAM.

PRESCRIBERS

THALOMID® (thalidomide) may be prescribed only by licensed prescribers who are registered in the S.T.E.P.S.® program and understand the risk of teratogenicity if thalidomide is used during pregnancy.

Major human fetal abnormalities related to thalidomide administration during pregnancy have been documented: amelia (absence of limbs), phocomelia (short limbs), hypoplasticity of the bones, absence of bones, external ear abnormalities (including anotia, micro pinna, small or absent external auditory canals), facial palsy, eye abnormalities (anophthalmos, microphthalmos), and congenital heart defects. Alimentary tract, urinary tract, and genital malformations have also been documented.1 Mortality at or shortly after birth has been reported at about 40%.2

Effective contraception (see CONTRAINDICATIONS) must be used for at least 4 weeks before beginning thalidomide therapy, during thalidomide therapy, and for 4 weeks following discontinuation of thalidomide therapy. Reliable contraception is indicated even where there has been a history of infertility, unless due to hysterectomy or because the patient has been postmenopausal for at least 24 months. Two reliable forms of contraception must be used simultaneously unless continuous abstinence from heterosexual sexual contact is the chosen method. Women of childbearing potential should be referred to a qualified provider of contraceptive methods, if needed. Sexually mature women who have not undergone a hysterectomy or who have not been postmenopausal for at least 24 consecutive months (i.e., who have had menses at some time in the preceding 24 consecutive months) are considered to be women of childbearing potential.

Before starting treatment, women of childbearing potential should have a pregnancy test (sensitivity of at least 50 mIU/mL). The test should be performed within the 24 hours prior to beginning thalidomide therapy. A prescription for thalidomide for a woman of childbearing potential must not be issued by the prescriber until a written report of a negative pregnancy test has been obtained by the prescriber.

Male Patients: Because thalidomide is present in the semen of patients receiving the drug, males receiving thalidomide must always use a latex condom during any sexual contact with women of childbearing potential even if he has undergone a successful vasectomy.

Once treatment has started, pregnancy testing should occur weekly during the first 4 weeks of use, then pregnancy testing should be repeated at 4 weeks in women with regular menstrual cycles. If menstrual cycles are irregular, the pregnancy testing should occur every 2 weeks. Pregnancy testing and counseling should be performed if a patient misses her period or if there is any abnormality in menstrual bleeding.

If pregnancy does occur during thalidomide treatment, thalidomide must be discontinued immediately.

Any suspected fetal exposure to THALOMID® (thalidomide) must be reported immediately to the FDA via the MedWatch number at 1-800-FDA-1088 and also to Celgene Corporation at 1-888-423-5436. The patient should be referred to an obstetrician/gynecologist experienced in reproductive toxicity for further evaluation and counseling.

FEMALE PATIENTS

Thalidomide is contraindicated in WOMEN of childbearing potential unless alternative therapies are considered inappropriate AND the patient MEETS ALL OF THE FOLLOWING CONDITIONS (i.e., she is essentially unable to become pregnant while on thalidomide therapy):

* she understands and can reliably carry out instructions.
* she is capable of complying with the mandatory contraceptive measures, pregnancy testing, patient registration, and patient survey as described in the System for Thalidomide Education and Prescribing Safety (S.T.E.P.S.® ) program.
* she has received both oral and written warnings of the hazards of taking thalidomide during pregnancy and of exposing a fetus to the drug.
* she has received both oral and written warnings of the risk of possible contraception failure and of the need to use two reliable forms of contraception simultaneously (see CONTRAINDICATIONS), unless continuous abstinence from heterosexual sexual contact is the chosen method. Sexually mature women who have not undergone a hysterectomy or who have not been postmenopausal for at least 24 consecutive months (i.e., who have had menses at some time in the preceding 24 consecutive months) are considered to be women of childbearing potential.
* she acknowledges, in writing, her understanding of these warnings and of the need for using two reliable methods of contraception for 4 weeks prior to beginning thalidomide therapy, during thalidomide therapy, and for 4 weeks after discontinuation of thalidomide therapy.
* she has had a negative pregnancy test with a sensitivity of at least 50 mIU/mL, within the 24 hours prior to beginning therapy. (See PRECAUTIONS, CONTRAINDICATIONS)
* if the patient is between 12 and 18 years of age, her parent or legal guardian must have read this material and agreed to ensure compliance with the above.

MALE PATIENTS

Thalidomide is contraindicated in sexually mature MALES unless the PATIENT MEETS ALL OF THE FOLLOWING CONDITIONS:

* he understands and can reliably carry out instructions.
* he is capable of complying with the mandatory contraceptive measures that are appropriate for men, patient registration, and patient survey as described in the S.T.E.P.S.® program.
* he has received both oral and written warnings of the hazards of taking thalidomide and exposing a fetus to the drug.
* he has received both oral and written warnings of the risk of possible contraception failure and of the presence of thalidomide in semen. He has been instructed that he must always use a latex condom during any sexual contact with women of childbearing potential, even if he has undergone a successful vasectomy.
* he acknowledges, in writing, his understanding of these warnings and of the need to use a latex condom during any sexual contact with women of childbearing potential, even if he has undergone a successful vasectomy. Sexually mature women who have not undergone a hysterectomy or who have not been postmenopausal for at least 24 consecutive months (i.e., who have had menses at any time in the preceding 24 consecutive months) are considered to be women of childbearing potential.
* if the patient is between 12 and 18 years of age, his parent or legal guardian must have read this material and agreed to ensure compliance with the above.

VENOUS THROMBOEMBOLIC EVENTS

The use of THALOMID® (thalidomide) in multiple myeloma results in an increased risk of venous thromboembolic events, such as deep venous thrombosis and pulmonary embolus. This risk increases significantly when thalidomide is used in combination with standard chemotherapeutic agents including dexamethasone. In one controlled trial, the rate of venous thromboembolic events was 22.5% in patients receiving thalidomide in combination with dexamethasone compared to 4.9% in patients receiving dexamethasone alone (p = 0.002). Patients and physicians are advised to be observant for the signs and symptoms of thromboembolism. Patients should be instructed to seek medical care if they develop symptoms such as shortness of breath, chest pain, or arm or leg swelling. Preliminary data suggest that patients who are appropriate candidates may benefit from concurrent prophylactic anticoagulation or aspirin treatment.
DRUG DESCRIPTION

THALOMID® (thalidomide), α-(N-phthalimido) glutarimide, is an immunomodulatory agent. The empirical formula for thalidomide is C13H10N2O4 and the gram molecular weight is 258.2. The CAS number of thalidomide is 50-35-1.

Thalidomide is an off-white to white, odorless, crystalline powder that is soluble at 25°C in dimethyl sulfoxide and sparingly soluble in water and ethanol. The glutarimide moiety contains a single asymmetric center and, therefore, may exist in either of two optically active forms designated S-(-) or R-(+). THALOMID® (thalidomide) is an equal mixture of the S-(-) and R-(+) forms and, therefore, has a net optical rotation of zero.

THALOMID® (thalidomide) is available in 50 mg, 100 mg, 150 mg and 200 mg capsules for oral administration. Active ingredient: thalidomide. Inactive ingredients: pregelatinized starch and magnesium stearate. The 50 mg capsule shell contains gelatin, titanium dioxide, and black ink. The 100 mg capsule shell contains black iron oxide, yellow iron oxide, titanium dioxide, gelatin, and black ink. The 150 mg capsule shell contains FD&C blue #2, black iron oxide, yellow iron oxide, titanium dioxide, gelatin, and black and white ink. The 200 mg capsule shell contains FD&C blue #2, titanium dioxide, gelatin, and white ink.

Other common side effects

Thalidomide is associated with:-
drowsiness/somnolence,
peripheral neuropathy,
dizziness/orthostatic hypotension,
neutro-penia, and
HIV viral load increase.

Hypersensitivity to THALOMID® (thalidomide) and bradycardia in patients treated with thalidomide have been reported.

Somnolence, dizziness, and rash are the most commonly observed adverse events associated with the use of thalidomide.

Plasmapheresis Protocol- Addenbrooke's Hospital

2008-11-27T13:12:00.000-08:00

Dose:-

7 sessions over 14 days
60mls/kg
5% Albumin
Use of FFP at end of procedure- at discretion of treating doctor

Other aspects of plasma-exchange applies.

Anticoagulation: Heparin 50u/kg stat than 1000u/h
target ACT 1.5-2.0X normal
adjust dose accordingly
stop heparin 30min prior to end of procedure

To avoid hemolysis, PEx must be done at
TMP 50-60mmHg
Qb 100-150mls/min

Complications of PEx
Hypotension- bld loss, decrease oncotic pressure
Bleeding d/t reduction in coagulation factors
Edema- reduction of intravascular oncotic pressure
Loss of cellular elements
Ethylene Oxide hypersensitivity

Related to anticoagulation
Bleeding- heparin related
Hypocalcemia leading to arrhythmias/ hypotension/ numbness/ tingling sensation

RITUXIMAB-Mechanism of action in NHL

2008-11-27T12:13:00.000-08:00

Azathioprine

2008-11-27T12:11:00.001-08:00

After hepatic conversion to 6-mercaptopurine, the cytotoxic effects of azathioprine are mediated by the impairment of purine synthesis, incorporation of purines into DNA, and impairment of the endonuclease repair activity of DNA polymerase (20). The drug is well-absorbed after oral administration and elimination requires hepatic metabolism by xanthine oxidase; an important drug interaction is with xanthine oxidase inhibitors, such as allopurinol. Lymphocyte function is reduced, B-cells more than T-cells, and there is suppression of the cellular component of the inflammatory response. The major adverse effects are nausea and vomiting, dose-dependent myelosuppression and reversible, cholestatic, hepatic toxicity. An increased incidence of malignancies, particularly lymphomas and skin cancers, has been observed with prolonged administration after organ transplantation

Cyclophosphamide

2008-11-27T12:09:00.000-08:00

Cyclophosphamide is an inactive pro-drug, converted by the mixed function oxidase system in the liver to the alkylating agents 4-hydroxy-cyclophosphamide and phosphoramide mustard, which alkylate guanine nucleotides, thus blocking cell division (20). Bioavailability after oral administration is greater than 75%, but there are large variations between individuals in the rate of production of active metabolites. A phenotypic variation in carboxylator activity affects the production of the inactive metabolite carboxyphosphoramide from 4-hydroxy-cyclophosphamide, which may influence efficacy and toxicity. The relation of renal and hepatic failure to the production and elimination of active metabolites has not been fully determined. Bladder toxicity is caused by renal excretion of the metabolite acrolein which can cause haemorrhagic cystitis and a markedly increased risk of bladder cancer (20). Other adverse effects include nausea and vomiting, myelosuppression with neutropenia, infections due to immunosuppression (4), alopecia and infertility. Permanent ovarian failure occurs in over 50% of women after one year’s exposure and is age-related; male infertility has been less well studied. The incidence of leukaemia and /or lymphoma is increased tenfold; less common adverse effects include pulmonary fibrosis, hepatitis and the syndrome of inappropriate ADH secretion.

RITUXIMAB

2008-11-27T11:57:00.000-08:00

Administration

* DO NOT ADMINISTER AS AN INTRAVENOUS PUSH OR BOLUS

* Premedicate before each infusion.

Administer only as an intravenous infusion
* Interrupt the infusion or slow the infusion rate for infusion reactions (see BOXED WARNING and WARNINGS AND PRECAUTIONS)
* First infusion: Initiate infusion at a rate of 50 mg/hr. In the absence of infusion toxicity, increase infusion rate by 50 mg/hr increments every 30 minutes, to a maximum of 400 mg/hr
* Subsequent infusions: Initiate infusion at a rate of 100 mg/hr. In the absence of infusion toxicity, increase rate by 100 mg/hr increments at 30-minute intervals, to a maximum of 400 mg/hr

Preparation

* RITUXAN is supplied as 100 mg/10 mL (NDC 50242-051-21) and 500 mg/50 mL (NDC 50242-053-06) solution in a single-use vial

Stability and storage

* RITUXAN vials are stable at 2°C–8°C (36°F–46°F). Do not use beyond expiration date stamped on carton. RITUXAN vials should be protected from direct sunlight. Do not freeze or shake
* RITUXAN solutions for infusion may be stored at 2°C–8°C (36°F–46°F) for 24 hours. RITUXAN solutions for infusion have been shown to be stable for an additional 24 hours at room temperature. However, since RITUXAN solutions do not contain a preservative, diluted solutions should be stored refrigerated (2°C–8°C). No incompatibilities between RITUXAN and polyvinylchloride or polyethylene bags have been observed.

WARNING: FATAL INFUSION REACTIONS, TUMOR LYSIS SYNDROME (TLS), SEVERE MUCOCUTANEOUS REACTIONS, and PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY (PML)

Infusion Reactions
Rituxan administration can result in serious, including fatal infusion reactions. Deaths within 24 hours of Rituxan infusion have occurred. Approximately 80% of fatal infusion reactions occurred in association with the first infusion. Carefully monitor patients during infusions. Discontinue Rituxan infusion and provide medical treatment for Grade 3 or 4 infusion reactions.

Tumor Lysis Syndrome (TLS)
Acute renal failure requiring dialysis with instances of fatal outcome can occur in the setting of TLS following treatment of non-Hodgkin's lymphoma (NHL) patients with Rituxan.

Severe Mucocutaneous Reactions
Severe, including fatal, mucocutaneous reactions can occur in patients receiving Rituxan.

Progressive Multifocal Leukoencephalopathy (PML)
JC virus infection resulting in PML and death can occur in patients receiving Rituxan.

Premedication:-

We give the following:-
PO Paracetamol 1g
IV Chlorpheniramine 10mg
+/- IV Methylprednisolone

We have used the following Rituximab dosing protocol for our vasculitis patients in Addenbrooke's Hospital, Cambridge.

Rituximab 1g X 2 doses 2 weeks apart
Then 1g 6 monthly for 2 years.

RITUXVAS

2008-11-27T11:38:00.000-08:00

Background

1.1.The diseases

Wegener's granulomatosis (WG) and microscopic polyangiitis (MP) are syndromes of primary systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA) (appendix 1)(15,16,19). WG and MP share many histological features, including a necrotising glomerulonephritis which often leads to rapidly progressive renal failure. Isolated pauci-immune necrotising, cresentic glomerulonephritis, has many features to suggest that it represents a renal-limited form of vasculitis (RLV), including the presence of circulating anti-MPO or anti-Pr3 antibodies. Common histological and serological features, and a similar response to treatment, have justified a common approach to the treatment of WG, MP and RLV. These diseases are sub grouped according to severity. ‘Generalised’ vasculitis refers to systemic disease with renal involvement (creatinine up to 500) and or other imminent vital organ failure. Patients with ‘generalised’ vasculitis and those with more severe renal vasculitis (i.e. creatinine greater than 500) will be included in this trial.

1.2.Their treatment

Untreated generalised WG and MP follow a progressive course with fatal outcome due to vital organ failure (17). With the empirical introduction of corticosteroids and cytotoxic agents, five year survival has increased from under 20% to over 60% (18). Over the last decade the treatment of AASV has been standardised following the results of randomised trials. Cyclophosphamide (either daily oral or intravenous (IV) pulse) and prednisolone are used for remission induction (3 to 6 months) with longer therapy of azathioprine or methotrexate and low dose steroids to prevent disease relapse. Early systemic vasculitis may be treated with methotrexate in place of cyclophosphamide. The addition of plasma exchange improves renal recovery rates in severe renal vasculitis. Adverse events with current therapy are frequent and are the major cause of mortality. The frequency of severe adverse events (10-45%) is influenced by age and the severity of renal involvement. There is a clear need for a new safer, effective therapy for AASV (1,2,3,6). In order to address the questions of drug toxicity and frequent disease relapses, newer therapies with better safety profiles have been investigated in AASV. One such agent is rituximab, a murine/human chimeric anti CD 20 monoclonal antibody. Its use results in B cell depletion for 6-18 months (7). B cells are key factors in autoimmune diseases with roles in autoantibody production, cytokine release and antigen presentation to T-cells. The sequence of immune events that trigger AASV are not fully understood, however evidence suggests that ANCA are pathogenic (21). Thus interruption of the cell line leading to ANCA production may be beneficial in disease suppression. Rituximab is an established treatment in non-Hodgkin’s lymphoma with a good safety track record (7). Even though the individual is rendered B cell deplete, immunoglobulin levels are maintained, and infection rates are low. Recently its use in autoimmune diseases has been realised with excellent results in rheumatoid arthritis (8). Rituximab has been shown to be effective in AASV, inducing remission in patients intolerant of cyclophosphamide (5,10-12,22,26-28). Given its good safety profile and efficacy its use a first line agent in AASV needs to be investigated.

In RITUXVAS, a rituximab based regimen will be compared with a standard cyclophosphamide/azathioprine based regimen in the treatment of AASV with glomerulonephritis. In order to achieve results applicable to clinical practice, all cases from mild to severe renal failure will be included. Standard practice includes the use of oral steroids which will be applied similarly in both treatment groups. It is recognised that necrotising cresentic glomerulonephritis progresses rapidly and immediate immune suppression is required to reverse existing and spare further organ damage. The therapeutic effect of rituximab is not immediate. Therefore two initial doses of cyclophosphamide will be given along side rituximab, thus allowing early disease control. When dialysis dependant renal failure or pulmonary haemorrhage occur, plasma exchange or intravenous steroids are standard additional therapies. These will be allowed according to local practice, and randomisation will occur after plasma exchange. Patients in the cyclophosphamide limb will receive azathioprine as remission maintenance therapy. Patients receiving rituximab will not receive any additional maintenance therapy.

1.3. Rituximab

Rituximab is an anti CD 20 chimeric mouse/human monoclonal antibody, with human IgG1 constant regions and murine light/heavy chain variable regions (7). CD 20 is a ligand which exists on developing B cells, excluding stem cells and plasma cells. The role of the CD 20 ligand in nature is not fully understood, although mediation of apoptosis has been suggested. The mechanisms by which rituximab causes B cell depletion may involve complement induced B cell lysis, Fc receptor mediated cytotoxic cell killing or the direct induction of B cell apoptosis by rituximab. Rituximab therapy correlates positively with B cell depletion and rituximab levels. Different dosing regimens have been trialled. In patients with systemic lupus erythematosus (SLE), the efficacy of rituximab is dependant upon B cell depletion. 4 infusions of 375mg/m2 rituximab infusions (one per week for four weeks) were required for this to occur (13,14). In 3 published reports 375mg/m2 has resulted in B cell depletion and clinical response in patients with refractory vasculitis (10-13). On the basis of these results a dose of 4 x 375mg/m2 infusions will be used in RITUXVAS. In RITUXVAS, 2 doses cyclophosphamide will still be administered with the 1st and 3rd rituximab infusions, for two reasons. Firstly, the necrotising cresentic glomerulonephritis associated with AASV progresses rapidly and the therapeutic effect of rituximab is delayed. The use of cyclophosphamide/high dose steroid, which are standard components of induction therapy, will allow adequate immunosuppression in the early crucial treatment of AASV. Secondly, human antichimeric antibody (HACA) formation has been reported in NHL, SLE, and RA with varying frequencies, (4.3% of patients in RA (8)). The long term implications of these antibodies are not known. However, the possibility of anaphylactic reactions and rituximab resistance with further treatments exists. Co-administration of an immunosuppressant effective in the treatment of vasculitis is thus a logical approach to minimise HACA development. Rituximab has now been used to treat 300,000 patients with NHL. Long-term safety regarding carcinogenicity and fertility has yet to be established. However, no major long-term adverse sequelae have been reported (1). Up to 50% of patients receiving rituximab for an indication will develop infusion reactions with symptoms including; fevers, chills, rigors, flushing, throat irritation RITUXVAS EUVAS Trial 9
EUDRACT 2005-003610-15 rash rhinitis fatigue headache, nausea, vomiting, urticaria, angioedema, bronchospasm, myalgia, arthralgia, hypotension, hypertension and exacerbation of angina or congestive cardiac failure. Later reactions include diarrhoea, leucopenia, neutropenia, thrombocytopenia, anaemia, and infections in 30%, which may or may not be drug related (investigators brochure).

1.4. Aims of RITUXVAS:

1 To assess the rates of preliminary response and sustained remission of AASV following rituximab (on the basis of former studies, 86% sustained remission expected with rituximab compared to 75% in control group). 2 To assess safety of a rituximab regimen in terms of severe adverse events (in patients receiving standard therapies, adverse advent rate is 45% at 2 years, at least 50% of which are infection relapted. In comparison rituximab use is only rarely associated with infections, therefore 22.5% adverse event rate expected with rituximab compared to 45% at 2 years in control group).

2. Hypothesis: Rituximab leads to a higher rate of sustained remission compared to standard therapies (cyclophosphamide/azathioprine) with a lower rate of severe adverse events and reduced cyclophosphamide exposure.

3. Trial Design

International, randomised, controlled, prospective, open trial comparing a rituximab based regimen with a standard cyclophosphamide/azathioprine regimen in the treatment of active ‘generalised’ AASV. Informed consent and ethics committee approval will be obtained.

3.1. Inclusion Criteria (all four must be present)

1. A new diagnosis of WG, MP or renal-limited vasculitis (RLV)(appendix 1) 2. Renal involvement attributable to active WG, MP or RLV with at least one of the following: a) Biopsy demonstrating necrotizing glomerulonephritis. b) Red cell casts on urine microscopy or ≥ ++ haematuria 3. ANCA positivity ANCA positivity requires either (a) or (b) (a) PR3-ANCA by ELISA or a typical cANCA pattern by indirect immunofluorescence (IIF), or both. (b) MPO-ANCA by ELISA. A positive pANCA by IIF requires confirmation by MPO-ANCA ELISA.

3.2. Exclusion Criteria

1. Previous cyclophosphamide, (greater than 2 weeks of an oral or IV pulse cyclophosphamide regimen). 2. Co-existence of another multisystem autoimmune disease, e.g. SLE, Churg Strauss Syndrome, Henoch Schonlein Purpura, rheumatoid vasculitis, essential mixed cryoglobulinaemia, anti-glomerular basement membrane antibody positivity 3. Hepatitis B e antigen positive or Hepatitis C antibody positive. 4. Known HIV positive (HIV testing will not be a requirement for this trial). 5. Previous malignancy (usually exclude unless agreed with trial co-ordinator). 6. Pregnancy, breast feeding or inadequate contraception if female. 7 Allergy to a study medication 8 Live Vaccine within last 4 weeks

3.3. Randomisation

1 Variables known to influence primary end points will be balanced between groups by minimisation i Age ii Disease WG or MP/RLV iii Creatinine 2 Randomisation form (see TMF) sent by e-mail rbjones@doctors.org.uk or fax to 01223 586506 (clinical trials office).

3.4. Endpoints Primary end points will be assessed upon trial completion at 2 years. However interim analyses will be performed when 1 30 patients have completed 6 weeks, to assess efficacy (treatment response) and safety (severe adverse events). 2 40 patients have completed 6 months to assess efficacy (remission rates) and safety (severe adverse events).

i Primary

1 Sustained remission (BVAS = 0 at 6 months and sustained for 6 months). 2 Severe adverse events (CTCAE grade ≥ 3) at 2 years.

ii Secondary 1 Efficacy

Response rate at 6 weeks (BVAS < 50% baseline) Remission at 6 months (BVAS=0 for 2 months by 6 months) Time to remission (BVAS=0) Relapses (all relapses and major/minor) BVAS area under the curve Change in GFR Change in SF-36 Change in VDI

2. Safety Severe adverse events (CTCAE grade ≥ 3) at 6 weeks and 6 months

All adverse events Death Prednisolone cumulative dose Cyclophosphamide cumulative dose

iii Tertiary

Human anti-chimeric antibody testing Correlation of B cells with disease activity Change in ANCA and disease activity Histopathology predictors of outcome

3.5. Interventions

I Drug regimens a) Rituximab Regimen: Rituximab, 375mg/m2 IV once a week for 4 weeks (i.e. 4 doses total), with 2 doses of cyclophosphamide 15mg/kg, 2 weeks apart given with the 1st and 3rd rituximab dose (appendix 3).

b) Control (cyclophosphamide/azathioprine) Regimen; Cyclophosphamide 15mg/kg for 3-6 months (6-10 doses total) to be given IV according to protocol (appendix 3) for remission induction. Cyclophosphamide should be converted to azathioprine for remission maintenance. c) Steroids. All patients will receive 1g IV methylprednisolone, then same daily oral corticosteroid regimen (appendix 3). d) Plasma exchange or IV methylprednisolone will be allowed according to local practice for patients with organ threatening disease. NB randomisation should not occur until completion of plasma exchange to avoid loss of rituximab during plasma exchange. The first dose of cyclophosphamide can be given prior to completion of plasma exchange. e) Progressive disease Within the first 6 months disease progression defined as a persistence of nephritic sediment or activity on a renal biopsy and a failure to improve GFR≥ 10 mls/min, if GFR at diagnosis is < 50 mls/min (calculated by cockcroft gault formula, see TMF for formula) OR persistence or new occurrence of a major non-renal BVAS item at 6 weeks then additional treatment should occur: i) Rituximab limb: 3rd dose of cyclophosphamide (15mg/kg) ii) Cyclophosphamide limb: Plasma exchange or IV methylprednisolone (according to local practice). f) Relapse Therapy. Relapses will be categorised as major or minor.

i) Rituximab limb: Rituximab with steroid will be used for major and minor relapse. Additional cyclophosphamide may also be used for major relapse.

ii) Control limb: Increased azathioprine and steroid for minor relapse and cyclophosphamide and steroid for major relapse.

II Evaluations Study assessments (including history and examination) will be performed at entry, 1.5, 3, 6, 9, 12, 15, 18, 21 and 24 months and at the time of relapse (see appendix 5, and TMF for assessment schedule). At each assessment the following should be performed: a) Blood for routine testing. b) 10 mls serum saved for centralised immunological analysis (including ANCA) and HACA. c) BVAS for each assessment and at relapse;

Every 6 months SF-36 and VDI should be performed (23-25)(see TMF).

Initial Renal histology will be reviewed by a EUVAS panel of histopathologists, based in Milan.

3.6. Withdrawal and treatment failure

1 At patient's or physician's request. Reason for withdrawal is to be recorded in the CRF. 2 Patients not achieving remission within 6 months to be withdrawn from trial drug regimen and will be treated according to local practice. They will remain under trial follow up.

3.7. Adverse events

1 Adverse effects will be actively sought and recorded in CRFs at each evaluation (see section 4.5 and TMF). 2 Unexpected, severe adverse events attributable to study medication must be reported within 24 hours to trial management committee. (see TMF and section 4.5 for full details).

3.8. Statistical analysis

Primary end points will be assessed upon trial completion at 2 years. However interim analyses will be performed: (a) When 30 patients have completed 6 weeks trial participation to assess (1) treatment response (BVAS fall of > 50% baseline). (2) safety (adverse events CTCAE grade 3 or greater) and (b) When 40 patients have completed 6 months trial participation to assess (1) remission (BVAS 0 for 2 months by 6 months) (2) safety (adverse events CTCAE grade 3 or greater)Primary end point confidence Intervals. A total of 30 patients will receive rituximab. The expected sustained remission rate for rituximab is 86%, based on the results of former studies. The 95% confidence interval for a sustained remission rate of 86% is 70.3%-94.7% when the sample size is 30. The expected response rate of 86% is considered clinically significant as patients with active newly diagnosed generalised AASV without 3-6 months of cyclophosphamide are conventionally expected to experience disease progression (worsening of clinical signs and symptoms). The expected sustained remission rate in the control group is 75%. Analyses on the 40 patients should be considered exploratory.

3.9. Duration: 3 years: 6 months recruitment, two year follow-up per patient and 6 month analyses.

Visit the EUVAS website for details.