2HistoryPt is a 70 yo man originally presented to the ER on 12/20/07 c/o SOB x 3 days with increasing LE edema. Pt has a h/o CKD, asthma, HTN, CHF, CAD, AS w/porcine AVR 2001, HCV, chronic normocytic anemia thought 2/2 HCV and CKD.Pt has been admitted several times over the past year for similar reasons, including to the ICU in 11/07 for CHF which required a lasix drip for diuresis.Pt reported non-compliance w/meds 2/2 diarrhea for 4 days PTA. Non bloody, yellow-brown well formed diarrhea x 4 days. No fevers/chills, n/v, melena or brbpr. Denies use of NSAIDs.Pt was diuresed in the ICU, given blood transfusions, and started on vanco for a resistant staph epidermis UTI and was transferred to the floor 12/22.On 12/26/07, renal consulted for patient’s worsening CKD (crt from 1.5 baseline to 2.8) with nephrotic range proteinuria. Renal bx on 1/8/08.
8LM MICRO -13 glomeruli, one globally sclerotic -all glomeruli are enlarged and hypercellular and have a lobulated appearance-hypercellularity is mostly due to proliferation of endothelial cells andmesangial cells and occasional marginating inflammatory cells-occasional double contours are also noted-podocytes are acutely injured and reveal diffuse hypertrophy-tubulointerstitial compartment is remarkable for extensive interstitialfibrosis, accompanied by patchy dense inflammatory infiltrate of lymphocyte,monocytes and rare plasma cells-the few non-atrophic tubules are mildly hypertrophic and occasionally reveal aflattened epithelium and contain hyaline casts.Arterioles are generally unremarkableTrichrome: blue represent collagen = fibrosis / sclerotic glomeruliSclerotic glomeruli and atropic tubules
9IFIF--renal cortex containinf our glomeruli, two of which are gloabbbly sclerotic-there is 1-2+ diffuse background for IgG, IgM shows 3+ segmental semilinearstaining in the glomerular basement membranes-numberous intrtubular casts are noted within IgA, and kappa and lambda lightchains-C3 shows similar pattern of positive stain in the glomeruli with 3+ segmentalsemilinear staining in the glomerular basement membranes.-fibrinogen is diffusely positive in interstitial capillaries-C1q and albumin are negative
10EM EM -tissue with renal cortex containing a single glomeruli -GBM are remarkable for the presence of double contours due to interposition ofmesangial cell cytoplasm-podocytes are acutely injured and reveal extensive foot process effacementaccompanied by microvillous transformation-numerous electron dense deposits are noted in the mesangial areas as well as inthe capillary walls, within the double contours-no tubuloreticular inclusions are identified although endothelial cells appearoccasionally swollen.Electron dense material is also noted inlumina of interstitial capillaries
11EMElectron dense material is also noted inlumina of interstitial capillaries
13Hepatitis C virus-related cryoglobulinemia and glomerulonephritis pathogenesis and therapeutic strategies
14Introduction HCV HCV related disease: cryoglobulinemia and MPGN Treatment for our patientStandard antiviral (IFN-alpha and Ribavirin)?IFN-alpha?CG targeted treatment?
15HCV virus HCV is an RNA virus of the flaviviridae family 170 million persons infected worldwideThe natural targets of HCV are hepatocytes and, possibly, B lymphocyte
16The HCV Genome and Expressed Polyprotein Figure 1. The HCV Genome and Expressed Polyprotein.Two regions in E2, called hypervariable regions 1 and 2 (HVR 1 and HVR 2), show extreme sequence variability, which is thought to be the result of selective pressure by virus-specific antibodies.HCV encodes a single polyprotein of 3011 amino acids, which is then processed into 10 mature structural and regulatory proteins (Fig. 1).a capside protein, two envelope proteins (E1 and E2) and seven non-structural proteins involved in the viral life cycleThe region of the envelope protein E2 contains the binding site for CD81, a tetraspanin expressed on hepatocytes and B lymphocytes that is thought to function as a cell receptor or co-receptor of the virus. As the natural targets of HCV are hepatocytes and B lymphocytes, the identification of CD81 provides a mechanism by which these cells are infected by HCV.Viral replication, which occurs through a direct RNA-RNA mechanism without a DNA intermediate and genomic integration, is highly error-prone, resulting in the generation of different but related variants known as quasispecies.This genetic variability confers to the virus an important survival advantage against host immune surveillance, and may contribute to the persistence of HCV infection.N Engl J Med, Vol. 345, No. 1 July 5, 2001
17Genotypes There are at least six major genotypes 75% The different genotypes have different geographic distributions.Genotypes 1a and 1b are the most common in the United States (about 75 percent of cases).Genotypes 2 and 3 are present in only 10 to 20 percent of patients.There is little difference in the severity of disease or outcome of patients infected with different genotypes. Knowledge of the genotype is important because it has predictive value in terms of theresponse to antivrial therapy, with better responses associated with genotypes 2 and 3 than with genotype 1.There are at least six major genotypes
18HCV-associated Mixed Cryoglobulinemia (MC) Mixed cryoglobulins (MCs) are proteins that reversibly precipitate at ≤ 37°C and consist of a mixture of monoclonal or polyclonal IgM that have antiglobulin (rheumatoid factor-RF) activity and bind to polyclonal IgG.MCs are categorized asType I monoclonal Igs (IgG, IgM, and sometimes IgA)2/2 MM or Waldenström's macroglobulinemiaType II if the IgM RF is monoclonal2/2 persistent viral infection: HCV, HIVType III if polyclonal IgM RF is present2/2 connective tissue diseaseHCV involved in the pathogenesis of MCCharacterized by nonneoplastic proliferation of rheumatoid factor positive B-cell clones => CG productionp/w triad of purpura, asthenia (feeling of weakness), and arthalgiaMost common organs invovled are: skin (purpura, leg ulcers), nerves (peripheral neuropathy) and kidneys (GN)Vessel wall deposition of circulating CG leads to complement activation, and is responsible for the vasculitic lesions and organ damage
19Serum protein electrophoresis LABORATORY DETECTIONCryoglobulin precipitate in a cryocrit tubeThe cryocrit is approximately 25% (left panel)Serum protein electrophoresis (ELP) of the cryoprecipitate reveals both a homogeneous band and a smear pattern in the gamma zone,indicating a cryoglobulin composed of monoclonal and polyclonal gamma globulins.Immunofixation with specific antisera to the IgG, IgA, IgM, κ, and λ chainsdemonstrates type II cryoglobulinemia formed bymonoclonal IgM-κ (homogenous bands in the IgM heavy chain and κ light chain lanes) andpolyclonal IgG (a smear pattern in the IgG heavy chain and κ and λ light chain lanes).25%Serum protein electrophoresisCryoglobulin precipitate in a cryocrit tube
20Sequential steps for managing and treating patients with chronic HCV infection, genotype 1 In principle, all patients with chronic HCV infection are candidates for antiviral therapy.However, the risks and benefits of treatment must be assessed individually, particularly given the typically slow course of natural infection.Only a subgroup of infected persons will have a clear indication for therapy.persistently elevated alanine aminotransferase (ALT) levels and a liver biopsy showing fibrosis or at least moderate necrosis and inflammation.These persons have a high risk of disease progression, and treatment is strongly recommended in the absence of contraindications.Persons with elevated alanine aminotransferase levels and only minimal or mild necrotic and inflammatory changes may also be treated.However, the risk of progressive liver disease is lower, and follow-up by means of serial measurements of alanine aminotransferase and a biopsy at three to five years is considered a reasonable alternativePersons with persistently normal alanine aminotransferase levels and no histologic evidence of necroticand inflammatory changes or only minimal changes have an excellent prognosis without therapy. In fact,in some such persons alanine aminotransferase levels become elevated after treatment with interferon alfaAmerican Association for the Study of Liver Diseases. Hepatology 2004; 39:1147
21Sustained virologic response rates with peginterferon alfa-2a (pegIFN) or interferon alfa-2b (IFN) and ribavirin (RBV) according to genotype
22Contraindications to Treatment with Iterferon Alfa and Ribavirin Side Effects of Treatmetn with Interferon Alfa and RibavirinRenal Insufficiency (CrCl ~50)
23What treatment options are available? HCV related cryoglobulinemia and MPGNTreatment for our patientStandard antiviral (IFN-alpha and Ribavirin)?IFN-alpha?CG targeted treatment?
24Proposed Mechanisms of Action of Interferon Alfa against HCV Interferon alfa is a cytokine that has an important function in the innate antiviral immune response.It acts by attaching to cell-surface receptorsthat signal through the system of Janus-activated kinase and signal transducers and activators of transcription,leading to induction of multiple interferon stimulated genes (Fig. 1).These genes include double-stranded RNases, inhibitors of viral protein translation, and proteins that destabilize viral messenger RNA. Interferon alfa also induces the expression of genes involved in the immune response, resulting in activation of natural killer cells, maturation of dendritic cells, proliferation of memory T cells, and prevention of T-cell apoptosisInterferon alfa engages receptors on the hepatocyte cell-surface membrane,causing them to dimerize and to activate Janus-activated and tyrosine kinasesthat phosporylate the cytoplasmic signal transducers and activators of transcription (STAT) proteins.STAT1 and STAT2 dimerize and bind interferon regulatory factor 9 (IRF9), creating a large complex (interferon-stimulated gene factor 3, or ISGF3) that is translocated into the nucleus, where it binds to interferon-stimulated response elements (ISREs) on DNA.This engagement causes transcription of multiple (>100) interferon-stimulated gene (ISG) mRNAs, which exit the nucleus and encode proteins that alter cell metabolism and interfere with virus replication, protein synthesis, and assembly.Major ISGs thought to be important in inhibiting HCV replication include 2',5' oligoadenylate synthetase, which activates antiviral RNases; RNA-specific adenosine deaminase, which edits viral RNA; and protein kinase R, which inactivates protein translation from viral mRNA. The HCV replicative complex is associated with the cytoplasmic membranes of hepatocytes and comprises RNA replicative intermediates, viral mRNA, structural and nonstructural viral proteins, and assembling virions.
252nd phase (not randomized) Influence of Antiviral Therapy in Hepatitis C Virus–Associated Cryoglobulinemic MPGN (Alric, AJKD, 2004)Patients (n=25) with nephrotoic-range proteinuria, mixed CG, MPGN by biopsy, with HCVInitial phaseAll treated for nephrotic proteinuria with lasix, acei, plasma exchanges, and steroid2nd phase (not randomized)Group 1, (n=18) after 4-12 weeks of initial treatment receive antiviral treatment for minimal 6 mosGroup 2, (n=7) maintenance with low dose lasixFollow upInitial eval, end of antiviral tx, and 6 mos after discontinuation
26Influence of Antiviral Therapy in Hepatitis C Virus–Associated Cryoglobulinemic MPGN (Alric, AJKD, 2004)No difference was found between groups for the parameters studiedAt diagnosis of nephrotic-range proteinuria, the duration of HCV infection established on the history of patients for factors associated with HCV infection was and months for patients from groups 1 and 2, respectively.Patients from group 1 were undistinguishable from those from group 2 concerningserum alanine aminotransferases levels, urinary protein excretion, or serum albumin or creatinine levelsIn most patients, renal failure was mild,with mean serum creatinine concentrations of mg/dL ( mol/L) in group 1mg/dL ( mol/L) in group 2Only 6 of 18 patients from group 1 and 3 of 7 patients from group 2 had more severe renal disease, with a serum creatinine level greater than 1.7 mg/dL (150 mol/L).Type II cryoglobulinemia was found in16 of 18 patients from group 1 and in all 7 patients from group 2.All patients had a polyclonal immunoglobulin G (IgG) and an IgM monoclonal component. Polyclonal IgM and IgG type III cryoglobulins were found in only 2 of 18 patients from group 1. Cryoglobulin levels were variable, with means of g/L (range, 0.02 to 6.8 g/L) and g/L (range, 0.02 to 2.79 g/L) in groups 1 and 2, respectively.The most frequent HCV genotype was genotype 1 (11 of 18 patients, group 1; 4 of 7 patients, group 2). According to the Knodell index, liver disease was mild in both groups
27Influence of Antiviral Therapy in Hepatitis C Virus–Associated Cryoglobulinemic MPGN (Alric, AJKD, 2004)All 6 nonresponders were genotype 1TABLE 3: comparison to values before antiviral therapySignificant decrease in daily proteinuria in sustained virological respondersIn addition, a marked increase in serum albumin levels was observed in sustained responders at the end of combination therapy and end of follow-up compared with pretreatment valuesMixed cryoglobulinemia levels at the end of tx and end of fu were decreased compared to pretreatmentMoreover, CG disappeared in 5/12 viral responders
28Conclusion Promising but not appropriate for our patient Anemia requiring frequent transfusions prohibits the use of RibavirinAs per GI: ½ dose PEG-IFNResponse seen is genotype dependent:For full dose PEG-IFN: 1b ~20% vs 2b ~40%Interferon Alfa-2a Therapy in Cryoglobulinemia Associated with Hepatitis C Virus (Misiani, NEJM, 1994)
29prospective randomized, controlled trial Interferon Alfa-2a Therapy in Cryoglobulinemia Associated with Hepatitis C Virus (Misiani, NEJM, 1994)prospective randomized, controlled trial53 patients with HCV-associated type II cryoglobulinemia.27 patients received recombinant interferon alfa-2athrice weekly at a dose of 1.5 million units for a week and then 3 million units thrice weekly for the following 23 weeks.26 control patients did not receive anything apart from previously prescribed treatmentsAll patients were then followed for an additional 24 to 48 weeks.
30Interferon Alfa-2a Therapy in Cryoglobulinemia Associated with Hepatitis C Virus (Misiani, NEJM, 1994)Out of 27 who started, 2 stopped (Afib and depression)After the treatment, 15 of remaining 25 had undetectable serum HCV RNAA statistically significant and progressively increasing difference between responder and non-responder was observed from the d16th week of treatment onwardUnfortunately,On follow up, among the 15 responders with undetectable HCV RNA (treatment for 24 weeks)Viremia recurred within six months with accompanying clinical manifestation of cryoglobulimenaPercent Changes in the Protein Concentration of Cryoprecipitate in Patients Receiving Interferon Alfa-2a, According to Whether Viremia Persisted or Disappeared by the End of the Treatment Period
31Peg-IFN We don’t know the genotype of responders in NEJM study Even with response, 100% relapsed in six months
32Treatment of HCV-related Cryoglobulinemic Glomerulonephritis Benefit of antiviral treatment is often transient and restricted to patients with mild and/or quiescent renal diseaseINF tx may be associated with worsening GNRibavirin may be contraindicated in the presence on renal failure and anemiaIs there no hope for our patient?
34Pathogenesis of Mixed Cryoglobulinemia MC is a non-neoplastic B cell lymphoproliferative process induced by HCV in an antigen-driven mechanismB cell activation and/or hyperproliferation with selective expansion of CG-producing B cellsthe mechanisms leading to B cell expansion are still unknownThe interaction btwn HCV envelope protein E2 and the CD81 receptor on B cells may lower the activation threshold of these cells, thus facilitating the production of various autoantibodies, including IgM RF.By itself, this process could lead to type III (polyclonal) cryoglobulinemia as monoclonal RF rarely arises in chronic human immune complex diseases.Transformation of the IgM RF fraction from polyclonal (type III) to the oligoclonal (type II-type III variant) and then to the monoclonal (type II) may occur over a decade or more of chronic disease, and is probably the consequence of a genetic mutation.The high prevalence of Bcl-2 rearrangement in patients with HCV-related MC is consistent with this hypothesis.T(14;18) translocation of the Bcl-2 oncogene results in overexpression of the gene, which may prolong the lifespan of B lymphocytes, favoring the emergence of a dominant clone capable of producing monoclonal IgM-κ RF34
35Pathogenesis of cryoglobulinaemic nephritis and rationale for Rituximab treatment B lymphocytes are targets of HCV infection due to cell expression of the CD81 receptors, which also allow hepatocyte infectionCD81, a tetraspanin expressed on hepatocytes and B lymphocytesRecombinant molecules containing extracellular loop of CD81 bound HCV and inhibited virus binding to CD81 in vitroThe IgM-k that has rheumatoid activity towards anti-HCV IgG forms mega-complexes that do not bind to the erythrocyte transport system,remain free to circulate and saturate the phagocyte’s ability to remove ICs from the blood.(impaired reticuloendothelial system removal of IgM-IgG-HCV complexes, via radiolabelled CG)Phagocyte cell blockade is favoured by HCV infection,which makes cells unable to digest cryoglobulins following phagocytosis.Due to the affinity to the mesangial matrix of the monoclonal IgM component, cryoprecipitable ICs deposit in the glomeruli, where cytokine production favours leukocyte diapedesis and endothelial injury.HOW RITUXIMABRituximab is a chimeric mouse/human mAb specific for the CD20 antigen found on the surface of malignant B lymphocytes in CLL and non-Hodgkin’s lymphomaRituximab targets cell-surface CD20 and acts as a bridge connecting the malignant B-cells to naturally occurring immune effector cells (natural killer cells, macrophages or neutrophils).When engaged by rituximab, the immune effector cells can kill the leukemia cells. This mechanism is called antibody-dependent cell-mediated cytotoxicity (ADCC) because, unless bridged to the tumor by rituximab, the immune effector cells are much less able to kill the tumor cellsThe anti-CD20 monoclonal antibody acts at the very first step of this cascade, blocking B-cell proliferation and, thus, IgM production, which is critical for both cryoglobulin production and deposition in the glomeruli
36Mechanism of rituximab Why Rituximab?Chimeric monocloanl abBinds to the B-cell surface Ag CD20Stop it before it starts
37Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinemic glomerulonephritis (CGGN) (Roccatello_Nephrol Dial Transplant_2004)N = 6Two with bone marrow lymphocyte infiltrationFour with either intolerance or resistance to standard immunosuppressive txHCV genotype1b = 22a2c = 2Tx:Rituximab 375 mg/m2days 1, 8, 15, and 22. Two additional doses were given 1 and 2 months later.No other immunosuppressive drugsEndpointsLaboratory parametersProteinuria, ESR, cryocrit, HCV VLClinical sxs and symptomsSkin ulcers, purpura, arthralgia, weakness, praesthesia and feverBMIgM-kappa clonal restricted lymphocyte interstitial infiltrationPresence of foci of CD20 positive lymphocyteFailed immunosuppressionSteroid (1mg/kg and cyclophosphomide 1.5mg/kg)Glucose intolerance, leukopenia
38Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinemic glomerulonephritis (CGGN) (Roccatello_Nephrol Dial Transplant_2004)Compared with the initial values,a significant decrease ofproteinuriacryocrit was observed 2, 6 and 12 months after first administration of Rituximab (Figure 1).A significant decrease forrheumatoid factor and IgM was also observed at 6 months.IgG levels and viral load did not changeOther study: dec in IgM, while IgA unaffected and IgG was reduced only slightlyProbably due to the lack of CD20 expression on mature plasma cellsFUNineteen months following initiation of therapy,patient three complained of relapsing arthralgia and worsening paraesthesia.She had increased rheumatoid factor and dropped C4 levels.Fourteen months after first Rituximab administration,serum creatinine in patient 5 rose to 5.3 mg/dl without other changes in biochemical parameters
39Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinemic glomerulonephritis (CGGN) (Roccatello_Nephrol Dial Transplant_2004)Patients showedreduction in proteinuriaParalleled by a decrease in the ESR, RF, IgM levels and cryocritAs well as increas in C4 levelsWith no substantial changes in liver cytolytic enzymesMoreover, no increase in VL was detected
40No increase in VL detected Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinemic glomerulonephritis (CGGN) (Roccatello_Nephrol Dial Transplant_2004)No increase in VL detected
41N=15, with type II MC unresponsive to conventional treatments Efficacy and safety of rituximab in type II mixed cryoglobulinemia, Zaja, Blood, 2003N=15, with type II MC unresponsive to conventional treatments11/15 were HCV relatedone with Sjogren syn and two were essentialF/U for 6 monthsTx: Rituximab (days 1, 8, 15, 22)Conventional therapy:IFN, Cyclophosphamide, plasmapheresis, IVIG, Azathiprine,
42Efficacy and safety of rituximab in type II mixed cryoglobulinemia, Zaja, Blood, 2003 Median values(with standard error bars)at baseline and during the 6-month follow-up in the studied patientsA significant reduction in the serum levels of RF, cryoglobulins,and IgM was noticed after treatment, in conjunction with a significant increase in the serum levels of C4By contrast, no significant decrease in serum IgG and IgA was noticedFor the course of ….Changes were evaluated with respect to the baseline value in any single patient.The course ofrheumatoid factor,cryoglobulin, and immunoglobulin serum levelsin the studied patients after rituximab therapy
43ConclusionOptimal strategy for HCV-associated MC nephritis is still undefinedFor our patientINF/Ribavirin - prohibitiveINF-alpha with high relapseCorticosteroid in combination with Rituximab