Integrating system biology and intratumor gene therapy by trans-complementing the appropriate co-stimulatory molecule as payload in oncolytic herpes virus.

Systems biology has been applied at the multi-scale level within the cancer field, improving cancer prevention, diagnosis and enabling precision medicine approaches. While systems biology can expand the knowledge and skills for oncological treatment, it also represents a challenging expedition due to cancer complexity, heterogeneity and diversity not only between different cancer indications, but also in its evolution process through space and time. Here, by characterizing the transcriptional perturbations of the tumor microenvironment induced by oncolytic, we aimed to rationally design a novel armed oncolytic herpes virus. We found that intratumor oncovirotherapy with HSV-1 induces T-cell activation signatures and transcriptionally activates several costimulatory molecules. We identified differentially expressed costimulatory receptors and binding partners, where inducible co-stimulators (ICOS) resulted in the potentially most beneficial targeted therapy. Through an ex-vivo transcriptomic analysis, we explored the potential of arming an oncolytic virus as a combination therapy strategy; in particular, we engineered a targeted herpes virus encoding ICOSL (THV_ICOSL), which resulted in a significant improvement in tumor size control compared to unarmed parental virus. Also, combination with a PD-1 inhibitor enhanced antitumor efficacy as predictable by upregulation of PD-1 and ligands pair (PD-L1/PD-L2) upon oncolytic virus injection. Generation of the human version of this virus encoding hICOSL orthologue effectively and specifically activated human T cells by triggering the ICOS pathway. Our data support the data-driven generation of armed oncolytic viruses as combination immunotherapeutic with checkpoint inhibitors.

The multi-biomarker disease activity test for assessing response to treatment strategies using methotrexate with or without prednisone in the CAMERA-II trial.

OBJECTIVES: The CAMERA-II trial compared two tight-control, treat-to-target strategies, initiating methotrexate with prednisone (MTX+pred) or MTX with placebo (MTX+plac), in early RA-patients. The multi-biomarker disease activity (MBDA) blood test objectively measures RA disease activity with a score of 1-100. In CAMERA-II, response profiles of the MBDA score, its individual biomarkers, and DAS28 were assessed. METHODS: We evaluated 92 patients from CAMERA-II of whom clinical data and serum for MBDA testing at baseline and ≥ 1 time-point from months 1, 2, 3, 4, 5, 6, 9, or 12 were available. Changes (∆) from baseline for DAS28 and MBDA score and comparisons of ∆DAS28 and ∆MBDA score over time within the MTX+pred versus the MTX+plac strategy were tested for significance with t tests. Changes in biomarker concentration from baseline to months 1-5 were tested with Wilcoxon signed rank test and tested for difference between treatment arms by Mann-Whitney U test. RESULTS: MBDA and DAS28 showed similar response profiles, with gradual improvement over the first 6 months in the MTX+plac group, and in the MTX+pred group faster improvement during month 1, followed by gradual improvement. The 12 MBDA biomarkers could be grouped into 4 categories of response profiles, with significant responses for 4 biomarkers during the MTX+plac strategy and 9 biomarkers during the MTX+pred strategy. CONCLUSIONS: MBDA tracked treatment response in CAMERA-II similarly to DAS28. More individual MBDA biomarkers tracked treatment response to MTX+pred than to MTX+plac. Four response profiles could be observed. TRIAL REGISTRATION: CAMERA-II International Standard Randomised Controlled Trial Number: ISRCTN 70365169 . Registered on 29 March 2006, retrospectively registered.

A multi-biomarker disease activity score can predict sustained remission in rheumatoid arthritis.

BACKGROUND: Reliable assessment of remission is important for the optimal management of rheumatoid arthritis (RA) patients. In this study, we used the multi-biomarker disease activity (MBDA) test to explore the role of biomarkers in predicting point remission and sustained remission. METHODS: RA patients on > 6 months stable therapy in stable low disease activity (DAS28-ESR ≤ 3.2) were assessed every 3 months for 1 year. Baseline, intermittent (IR) and sustained (SR) remission were defined by DAS28-ESR, DAS28-CRP, simple disease activity index (SDAI), clinical disease activity index (CDAI) and ACR/EULAR Boolean criteria. Patients not fulfilling any remission criteria at baseline were classified as 'low disease activity state' (LDAS). Patients not fulfilling any remission criteria over 1 year were classified as 'persistent disease activity' (PDA). MBDA score was measured at baseline/3/6 months. The baseline MBDA score, the 6-month time-integrated MBDA score and MBDA biomarkers were used for analyses. The area under the receiver operating characteristic curve (AUROC) assessed the ability of the MBDA score to discriminate between remission and non-remission. Biomarkers were analysed at baseline using the Mann-Whitney test and over time using the Jonckheere-Terpstra trend test. RESULTS: Of 148 patients, 27% were in the LDAS, 65% DAS28-ESR remission, 51% DAS28-CRP remission, 40% SDAI remission, 43% CDAI remission and 25% ACR/EULAR Boolean remission at baseline. Over 1 year, 9% of patients were classified as PDA. IR and SR were achieved in 42%/47% by DAS28-ESR, 46%/29% by DAS28-CRP, 45%/20% by SDAI, 44%/21% by CDAI and 35%/9% by ACR/EULAR Boolean criteria, respectively. By all remission criteria, baseline MBDA score discriminated baseline remission (AUROCs 0.68-0.75) and IR/SR (AUROCs 0.65-0.74). The 6-month time-integrated MBDA score discriminated IR/SR (AUROCs 0.65-0.79). Baseline MBDA score and concentrations of IL-6, leptin, SAA and CRP were significantly lower in all baseline remission criteria groups vs LDAS. They and the 6-month time-integrated values were lower among patients who achieved IR/SR vs PDA over 1 year. CONCLUSIONS: This study demonstrated that the MBDA score and its biomarkers IL-6, leptin, SAA and CRP differentiated between small differences in disease activity (i.e. between low disease activity and remission states). They were also predictors of remission over 1 year.

Correlation of serum protein biomarkers with disease activity in psoriatic arthritis.

Objective: To assess the correlation of serum protein biomarkers with disease activity across different domains of psoriatic arthritis (PsA).Material and methods: A cross-sectional cohort of 45 adult patients with PsA fulfilling the classification for psoriatic arthritis (CASPAR) criteria was recruited from University of California San Diego (UCSD) Arthritis Clinics. Clinical data and serum samples were collected and serum was analyzed for protein biomarkers hypothesized to be relevant to disease activity in PsA. Correlations were evaluated for clinical disease activity measures across disease domains.Results: Biomarkers with the highest correlation to the composite indices and disease domains were SAA, IL-6, YKL-40, and ICAM-1. In addition, several biomarkers were moderately correlated with individual composite indices and/or disease domains. Low or no correlation was observed with some biomarkers, e.g. MMP-3, MMP-1, EGF, VEGF, and IL-6R. In contrast, the correlation of all biomarkers with certain disease domains was low; specifically, pain, percent body surface area of psoriasis, and patient global assessment. The multi-biomarker disease activity score (MBDA) developed for rheumatoid arthritis (RA) showed high correlations with most composite indices and some disease domains in PsA.Conclusions: These data suggest biomarker analysis can reflect disease activity across disease domains in PsA. Certain domains would likely benefit from the evaluation of additional biomarkers.

Predictive value of a multi-biomarker disease activity score for clinical remission and radiographic progression in patients with early rheumatoid arthritis: a post-hoc study of the OPERA trial.

OBJECTIVES: Measurement of serum biomarkers at disease onset may improve prediction of disease course in patients with early rheumatoid arthritis (RA). We evaluated the multi-biomarker disease activity (MBDA) score and early changes in MBDA score for prediction of 28-joint Disease Activity Score based on C-reactive protein (DAS28-CRP) remission and radiographic progression in the double-blinded OPERA trial. METHOD: Treatment-naïve RA patients (N = 180) with moderate or high DAS28 were randomized to methotrexate (MTX) + adalimumab (n = 89) or MTX + placebo (n = 91) in combination with glucocorticoid injection into swollen joints. X-rays of hands and feet were evaluated at months 0 and 12 (n = 164) by the total Sharp van der Heijde score (TSS). The smallest detectable change (1.8 TSS units) defined radiographic progression (∆TSS ≥ 2). Clinical remission (DAS28-CRP < 2.6) was assessed at baseline and 6 months. MBDA score was determined at 0 and 3 months and tested in a multivariable logistic regression model for predicting DAS28 remission at 6 months and radiographic progression at 1 year. RESULTS: Baseline MBDA score was independently associated with radiographic progression at 1 year [odds ratio (OR) = 1.03/unit, 95% confidence interval (CI) = 1.01-1.06], and changes in MBDA score from baseline to 3 months with clinical remission at 6 months [OR = 0.98/unit, 95% CI 0.96-1.00). In anti-cyclic citrullinated peptide antibody (anti-CCP)-positive patients, 35 of 89 with high MBDA score (> 44) showed radiographic progression (PPV = 39%), compared with 0 of 15 patients (NPV = 100%) with low/moderate MBDA score (≤ 44) (p = 0.003). CONCLUSION: Early changes in MBDA score were associated with clinical remission based on DAS28-CRP at 6 months. In anti-CCP-positive patients, a non-high baseline MBDA score (≤ 44) had a clinical value by predicting very low risk of radiographic progression at 12 months.

Investigation of a multi-biomarker disease activity score in rheumatoid arthritis by comparison with magnetic resonance imaging, computed tomography, ultrasonography, and radiography parameters of inflammation and damage.

OBJECTIVES: To investigate the multi-biomarker disease activity (MBDA) score by comparison with imaging findings in an investigator-initiated rheumatoid arthritis (RA) trial (HURRAH trial, NCT00696059). METHOD: Fifty-two patients with established RA initiated adalimumab treatment and had magnetic resonance imaging (MRI), ultrasonography (US), computed tomography (CT), and radiography performed at weeks 0, 26, and 52. Serum samples were analysed using MBDA score assays and associations between clinical measures, MBDA score, and imaging findings were investigated. RESULTS: The MBDA score correlated significantly with MRI synovitis (rho = 0.65, p < 0.001), MRI bone marrow oedema (rho = 0.36, p = 0.044), and US power Doppler (PD) score at week 26 (rho = 0.35, p = 0.039) but not at week 0 or week 52. In the 15 patients who had achieved a Disease Activity Score based on C-reactive protein (DAS28-CRP) < 2.6 at week 26, MRI and/or US detected subclinical inflammation and 13 (87%) had a moderate/high MBDA score. For the cohort with available data, none of the four patients in MBDA remission (score ≤ 25) at week 26 had progression of imaging damage from baseline to week 52 whereas progression was observed in three out of nine (33%) and seven out of 21 (33%) patients with moderate (30-44) and high (> 44) MBDA scores, respectively. CONCLUSIONS: In this cohort, the MBDA score correlated poorly with MRI/US inflammation. However, the MBDA score and MRI/US were generally concordant in showing signs of inflammation in most patients in clinical remission during anti-tumour necrosis factor (anti-TNF) therapy. MBDA scores were elevated in all patients with structural damage progression.

Vectra DA for the objective measurement of disease activity in patients with rheumatoid arthritis.

Quantitative and regular assessment of disease activity in rheumatoid arthritis (RA) is required to achieve treatment targets such as remission and to optimize clinical outcomes. To assess inflammation accurately, predict joint damage and monitor treatment response, a measure of disease activity in RA should reflect the pathological processes resulting in irreversible joint damage and functional disability. The Vectra DA blood test is an objective measure of disease activity for patients with RA. Vectra DA provides an accurate, reproducible score on a scale of 1 to 100 based on the concentrations of 12 biomarkers that reflect the pathophysiologic diversity of RA. The analytical validity, clinical validity, and clinical utility of Vectra DA have been evaluated for patients with RA in registries and prospective and retrospective clinical studies. As a biomarker-based instrument for assessing disease activity in RA, the Vectra DA test can help monitor therapeutic response to methotrexate and biologic agents and assess clinically challenging situations, such as when clinical measures are confounded by non-inflammatory pain from fibromyalgia. Vectra DA scores correlate with imaging of joint inflammation and are predictive for radiographic progression, with high Vectra DA scores being associated with more frequent and severe progression and low scores being predictive for non-progression. In summary, the Vectra DA score is an objective measure of RA disease activity that quantifies inflammatory status. By predicting risk for joint damage more effectively than conventional clinical and laboratory measures, it has the potential to complement these measures and optimise clinical decision making.

Pattern of care and effectiveness of treatment for glioblastoma patients in the real world: Results from a prospective population-based registry. Could survival differ in a high-volume center?

BACKGROUND: As yet, no population-based prospective studies have been conducted to investigate the incidence and clinical outcome of glioblastoma (GBM) or the diffusion and impact of the current standard therapeutic approach in newly diagnosed patients younger than aged 70 years. METHODS: Data on all new cases of primary brain tumors observed from January 1, 2009, to December 31, 2010, in adults residing within the Emilia-Romagna region were recorded in a prospective registry in the Project of Emilia Romagna on Neuro-Oncology (PERNO). Based on the data from this registry, a prospective evaluation was made of the treatment efficacy and outcome in GBM patients. RESULTS: Two hundred sixty-seven GBM patients (median age, 64 y; range, 29-84 y) were enrolled. The median overall survival (OS) was 10.7 months (95% CI, 9.2-12.4). The 139 patients ≤aged 70 years who were given standard temozolomide treatment concomitant with and adjuvant to radiotherapy had a median OS of 16.4 months (95% CI, 14.0-18.5). With multivariate analysis, OS correlated significantly with KPS (HR = 0.458; 95% CI, 0.248-0.847; P = .0127), MGMT methylation status (HR = 0.612; 95% CI, 0.388-0.966; P = .0350), and treatment received in a high versus low-volume center (HR = 0.56; 95% CI, 0.328-0.986; P = .0446). CONCLUSIONS: The median OS following standard temozolomide treatment concurrent with and adjuvant to radiotherapy given to (72.8% of) patients aged ≤70 years is consistent with findings reported from randomized phase III trials. The volume and expertise of the treatment center should be further investigated as a prognostic factor.

Long-term outcomes of interruption and retreatment vs. continuous therapy with adalimumab for psoriasis: subanalysis of REVEAL and the open-label extension study.

BACKGROUND: REVEAL was a 52-week study of adalimumab for moderate to severe psoriasis. At Week 33, adalimumab-treated patients with sustained responses (PASI ≥75 at Weeks 16 and 33) were re-randomized to receive adalimumab or placebo. Subsequently, they could receive adalimumab in an open-label extension (OLE) study. OBJECTIVE: To compare long-term efficacy and safety of adalimumab 40 mg every other week (eow), given as continuous treatment or with one period of interruption followed by retreatment. METHODS: Patients who were re-randomized to adalimumab or placebo at REVEAL Week 33 and received ≥ 1 dose of OLE adalimumab were analysed as the continuous and retreatment groups, respectively, for >2 years of OLE treatment with adalimumab 40 mg eow. LOCF was used for missing efficacy data. RESULTS: At OLE Weeks 0, 12 and 24, PASI 75 response rates were 84%, 84%, 86% with continuous treatment (N = 233) vs. 45%, 71%, 79% with retreatment (N = 227). Thereafter, efficacies were slightly greater for continuous treatment but similar between groups, with PASI 75 response rates at OLE Week 108 of 75% vs. 73% respectively. Retreatment was most effective for patients with ≥ PASI 50 responses when retreatment was initiated. Adverse event rates for retreatment were equal to or lower than those for continuous treatment. CONCLUSIONS: In psoriasis patients with sustained PASI 75 responses to adalimumab, long-term efficacy of retreatment after a ≤ 19-week interruption was similar to efficacy achieved with > 3 years continuous treatment. Adalimumab retreatment provided the best results when initiated before responses had declined below PASI 50.

Relationship between methotrexate dosing and clinical response in patients with moderate to severe psoriasis: subanalysis of the CHAMPION study.

BACKGROUND: CHAMPION was a phase III trial that compared adalimumab with methotrexate and placebo for chronic plaque psoriasis. OBJECTIVES: To determine the relationship between methotrexate dosing and improvement in psoriasis for patients in CHAMPION. METHODS: Methotrexate-treated patients in CHAMPION received step-up dosing to 15 mg per week during the first 8 weeks. At week 8, PASI 50 responders (early responders, ER) were to continue with 15 mg weekly for the next 8 weeks; PASI 50 nonresponders were to receive 20 mg weekly for the next 4 weeks, followed by 20 mg weekly if they achieved ≥ PASI 50 at week 12 (late responders, LR), or 25 mg weekly if not (late nonresponders, LN). Outcomes were assessed post hoc for patients in these groups who completed CHAMPION. RESULTS: One hundred and three methotrexate-treated patients were analysed: 40 ER, 22 LR and 41 LN. Week 16 mean percentage improvement from baseline in Psoriasis Area and Severity Index was greatest in the ER group, nearly as good in LR, and poor in LN; PASI 75/90/100 response rates were 70%/32%/18%, 41%/9%/5% and 5%/0%/0%, respectively. Among patients with a PASI 75 response at week 16, 72% were in the ER group (comprising 27% of patients overall) and 23% were in the LR group. CONCLUSIONS: Nearly all week 16 PASI 75 responders in CHAMPION achieved a PASI 50 response at week 8 or 12, with maximum methotrexate dosages of 15 or 20 mg per week, respectively. Week 12 may be an appropriate time to discontinue methotrexate treatment in patients who are not achieving good responses.

Adalimumab improves joint-related and skin-related functional impairment in patients with psoriatic arthritis: patient-reported outcomes of the Adalimumab Effectiveness in Psoriatic Arthritis Trial.

OBJECTIVE: To evaluate the effects of adalimumab on patient-reported outcomes of joint-related and skin-related functional impairment, health-related quality of life, fatigue and pain in patients with psoriatic arthritis (PsA). METHODS: Patients with moderately- to severely- active PsA were treated with adalimumab, 40 mg, every other week, or placebo, in this 24-week, randomised, controlled trial. Patient-reported outcomes included the Health Assessment Questionnaire Disability Index (HAQ DI), Short-Form 36 Health Survey (SF-36), the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) Scale and the Dermatology Life Quality Index (DLQI). RESULTS: Adalimumab (n = 151) and placebo (n = 162) groups were comparable with respect to baseline demographics and disease severity. Significant changes from baseline in HAQ DI were reported for adalimumab v placebo (-0.4 v -0.1, p<0.001) at both 12 and 24 weeks. At week 24, significant improvements in the SF-36 domains of physical functioning, role-physical, bodily pain, general health, vitality and social functioning, as well as the physical component summary score, were observed for adalimumab versus placebo (p<0.01). These reported changes in HAQ DI and SF-36 were also clinically important. Significantly more patients treated with adalimumab had complete resolution of functional loss (HAQ DI = 0) and dermatological-related functional limitations (DLQI = 0) compared with placebo at weeks 12 and 24 (p< or =0.001). Adalimumab led to significantly greater improvements in FACIT-Fatigue scores, pain scores, and disease activity measures versus placebo at 12 and 24 weeks (p<0.001 for all). CONCLUSIONS: Adalimumab improved physical-related and dermatological-related functional limitations, HRQOL, fatigue and pain in patients with PsA treated for 24 weeks.

Small fibers peroneal mononeuropathy in a patient with Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune epithelitis characterized by lymphocytic infiltration of exocrine glands and epithelia in multiple sites. One third of the patients present with peripheral nervous system involvement. We describe the case of a woman aged 62 affected by a peroneal nerve mononeuropathy with painful disturbances secondary to a prevalent involvement of small fibers as demonstrated by electrophysiological investigations and skin biopsy. Asymmetric peripheral nerve involvement is not uncommon in SS, though, to our knowledge, it has never been reported of a mononeuropathy involving primarily small fibers.

Immunoglobulin binding properties of the Prosorba immunadsorption column in treatment of rheumatoid arthritis.

Studies of the humoral effects of the Prosorba column were conducted in conjunction with the Phase 3 trial of Prosorba versus sham therapy for rheumatoid arthritis (RA). When perfused with normal human plasma in vitro, Prosorba bound predominantly IgG with a maximal capacity of approximately 462 g of Ig per Prosorba column, equal to about 1.5% of circulating IgG. Prosorba treatment did not alter the concentrations of albumin, IgG, IgM, and IgA in 3 RA patients, except for a small dilutional effect. Kinetic studies demonstrated that Prosorba removed IgG > IgM, IgA, and IgM rheumatoid factor (RF) during the initial moments of apheresis and almost exclusively IgM RF after 15 min. No net protein removal occurred at > or = 60 min. Mean values of circulating immune complexes (CICs) were not significantly decreased by 12 weekly treatments. Complement was activated by the apheresis system upstream of the Prosorba column without changing C3 or C4 levels. We conclude that the Prosorba mechanism of action in RA is not bulk removal of Ig, but might involve modification of the CIC repertoire and could include, but not be limited to, effects related to complement activation.

Effect of 51p1-related gene copy number (V1-69 locus) on production of hepatitis C-associated cryoglobulins.

Monoclonal IgM in type II mixed cryoglobulins (MC) preferentially use 51p1-related immunoglobulin VH genes. In normal preimmune B lymphocytes, 51p1-related gene expression is proportional to the germ-line gene dosage, which can be 0-4. To determine whether 51p1-related gene dosage influences the occurrence of type II MC or the VH gene bias in cryoglobulin IgM, we studied 47 patients chronically infected with hepatitis C virus (HCV), 24 MC+, 23 MC-. By Western analysis, 11 cryoprecipitate IgM (46%) were detected by G6 (a marker for 51p1-related gene products), eight (33%) by Staphylococcal Protein A (a VH3 family marker), and five (21%) by neither, indicating a 23-fold bias favouring 51p1-related genes. All 11 MC+, G6+ patients possessed > or = 1 copy of a 51p1-related gene; nine of the 36 others had none. The mean copy number of 51p1-related genes was greater in MC+ than MC- patients, and in MC+, G6+ patients versus the 36 others (P < 0.04), but significant differences were not seen in analyses restricted to patients with > or = 1 copy of a 51p1-related gene. We conclude that when a 51p1-related gene is present, a strong bias favours G6+ IgM in HCV-associated type II MC, but this bias is not greatly increased by a high dosage of 51p1-related genes. Furthermore, patients lacking 51p1-related genes also produce MC, but with G6- IgM.

Bilateral and simultaneous ischemic optic neuropathy following hip replacement surgery.

Ischemic optic neuropathy, is an exceptional complication of surgery. Moreover, bilateral and simultaneous visual deficit in ischemic optic neuropathy is very rare. We describe two patients who suffered bilateral and simultaneous ischemic optic neuropathy after elective total hip replacement. Anemia and hypotension are the most likely risk factors.

Neuropatía óptica isquémica bilateral y simultánea después de la cirugía para prótesis de cadera / Bilateral and simultaneous ischemic optic neuropathy following hip replacement surgery

La neuropatía óptica isquémica es una complicación perioperatoria infrecuente. Aún más inusual es la neuropatía óptica isquémica con compromiso ocular bilateral y simultáneo. Presentamos los casos de 2 pacientes que sufrieron neuropatía óptica isquémica bilateral y simultánea posterior a reemplazo total de cadera, en los cuales tanto la anemia como la hipotensión fueron identificados como los factores de riesgo más probables (AU)

The rheumatoid factor response in the etiology of mixed cryoglobulins associated with hepatitis C virus infection.

Studies of mixed cryoglobulins (MC) from patients infected with hepatitis C virus (HCV) show that the principal constituents in cryoprecipitate are IgM rheumatoid factors (RF), polyclonal IgG anti-HCV antibodies, and HCV RNA. The HCV-induced RF response is biased to produce IgM RF encoded by a restricted set of Ig V genes, predominantly the VH/VL gene pair 51p1/kv325. The propensity of such IgM RF to cryoprecipitate is likely a coincidental property of their V region sequences, but the clinical effect of this bias is increased by the persistence of circulating HCV-IgG immune complexes. These complexes might induce production of cryoprecipitable IgM RF and furnish multi-molecular structures that favor binding by cryoprecipitable IgM RF. The V gene sequences of HCV-induced IgM RF have features seen in other RF responses, suggesting a common immunological mechanism that is independent of HCV. B cell proliferation is probably enhanced by HCV-specific properties, however, including the ability of HCV proteins to bind to CD81 on the B cell surface, and to influence intracellular regulatory functions following viral entry into B cells. The V gene bias in HCV-induced RF is most apparent among the B cells in monoclonal expansions responsible for type II cryoglobulins, but it might originate early the polyclonal RF response, before MC are detectable. Monoclonal B cell expansions and lymphomatoid bone marrow infiltrates in HCV+ patients predominantly involve CD5-negative IgM RF B cells. Non-RF B cells can also be expanded, including producers of IgG1 and IgG3 that are likely anti-HCV antibodies. The initial site of B cell clonal expansion may be in the liver, where lymphoid aggregates are abundant and RF are produced. Sorting out how MC formation is influenced by properties that are inherent to the RF response, or specific to HCV infection, will be a challenge to future HCV research.