Improved (4 Plus 2) Rituximab Protocol for Severe Cases of Mixed Cryoglobulinemia: A 6-Year Observational Study.

BACKGROUND: In a prospective, single-center open study, we evaluated the very long-term effects of rituximab (RTX) administered to patients with severe mixed cryoglobulinemia (MC). METHODS: RTX was administered to 31 patients with MC (type II in 29 cases and type III in 2) with diffuse membranoproliferative glomerulonephritis (16 cases), peripheral neuropathy (26) and large skin ulcers (7). All but 4 patients had serum anti-hepatitis C virus antibodies. RTX was administered at a dose of 375 mg/m2, according to a '4 + 2' protocol (days 1, 8, 15 and 22 plus 1 dose 1 and 2 months later). No other immunosuppressive drugs were added. Response was evaluated over a very long-term follow-up (mean 72.47 months, range 30-148). RESULTS: Complete remission of pretreatment active manifestations was observed in all cases of purpuric lesions and non-healing vasculitic ulcers, and in 80% of the peripheral neuropathies. Cryoglobulinemic nephropathy significantly improved during follow-up, starting from the 2nd month after RTX (serum creatinine from 2.1 ± 1.7 to 1.5 ± 1.6 mg/dl, p ≤ 0.05; 24-hour proteinuria from 2.3 ± 2.1 to 0.9 ± 1.9 g/24 h, p ≤ 0.05). Improvement of cryoglobulinemic serological hallmarks, such as cryocrit and low complement C4, were observed. No clinically relevant side effects were recorded. Re-induction with RTX was carried out in 9 relapsed patients after a mean of 31.1 months (12-54), again with beneficial effects. The survival rate was 75% at 6 years and the probability of remaining symptom-free for 10 years without any therapy was of about 60% after a single '4 + 2' infusion cycle, while the probability of living symptom-free 5 years after relapsing was 80% if given the same treatment. CONCLUSION: In this open, prospective study, RTX appeared to be very effective and safe in the treatment of the most severe cases of MC.

8-isoprostane, prostaglandin E2, C-reactive protein and serum amyloid A as markers of inflammation and oxidative stress in antiphospholipid syndrome: a pilot study.

OBJECTIVE: To test the inflammation and oxidative stress hypothesis in antiphospholipid syndrome (APS) patients and to identify possible associations with clinical and laboratory features of the disease. METHODS: Serum amyloid A (SAA), C-reactive protein (CRP), 8-isoprostane and prostaglandin E2 (PGE) were assayed in the sera of 45 APS patients and then compared to control groups made up of 15 antiphospholipid antibody (aPL) negative patients with systemic lupus erythematosus, 15 aPL negative subjects with pregnancy-related morbidity, 15 aPL negative patients with thrombosis, 15 subjects with persistently positive aPL with no signs or symptoms of APS, and 15 healthy volunteers from among the hospital staff. RESULTS: APS patients showed significantly higher CRP (p = 0.01), SAA (p < 0.01), 8-isoprostane (p = 0.05) and PGE2 (p = 0.001) plasma levels as compared to controls. Among APS subjects, significantly higher 8-isoprostane and PGE2 levels were observed in patients with triple positivity for aPL (lupus anticoagulant, anticardiolipin and anti-beta2-glycoprotein I antibodies) compared to APS patients with single or double aPL positivity. CONCLUSION: Both inflammation and oxidative stress, as measured by SAA, CRP, 8-isoprostane and PGE2, occur in APS and seem to be related to triple positivity for aPL.

A 4-year observation in lupus nephritis patients treated with an intensified B-lymphocyte depletion without immunosuppressive maintenance treatment-Clinical response compared to literature and immunological re-assessment.

BACKGROUND: B cells (BC) play a critical role in systemic lupus erythematosus (SLE). BC depletion therapy still remains an attractive option, despite the disappointing results of randomized controlled trials (RTCs). METHODS: Twelve patients with SLE [3 males, mean age 43.8 yrs (25-55)] with severe multiorgan involvement all including kidney (3 patients with Class IV, 4 with Class III/V and 5 with Class V, according to the International Society of Nephrology/Renal Pathology Society glomerulonephritis classification), skin lesions [10], severe polyarthralgias with arthritis [10], polyserositis [2], and lymphadenopathy [5] have been prospectively treated with an intensified B cell depletion therapy (IBCDT) protocol due to their resistance or intolerance to previous therapy (six cases) or as a front line immunosuppressive treatment in 6 women with unsatisfactory therapeutic compliance or as a specific request of a short-time immunosuppression for gestational perspectives. PROTOCOL: Rituximab (RTX) 375 mg/sm on days 1, 8, 15, 22, and 2 more doses after 1 and 2 months, associated with 2 IV administrations of 10mg/kg of cyclophosphamide and 3 methylprednisolone pulses (15mg/kg) followed by oral prednisone (0.8 mg/kg/day, rapidly tapered to 5mg/day by the end of the 3rd month after RTX). No further immunosuppressive maintenance therapy has been given. RESULTS: Patients had been followed-up for a mean of 44.5 (24-93)months. Significant decreases (p<0.05) were found in the levels of ESR (baseline mean value: 55.0mm; 3 months: 36; end of follow-up: 13), anti-dsDNA antibodies (baseline: 185 U; 3 months: 107; end of follow-up: 15), and proteinuria (baseline: 4.9 g/24h; 3 months: 0.97; end of follow-up: 0.22). C4 values (baseline 11 mg/dl) significantly increased (p<0.05) after 3 months (22 mg/dl) and at the end of the follow-up (20mg/dl). Of the 12 patients, 9 (75%) have remained well after one cycle of IBCDT, with no flare (mean 51.6 months [25-93]). Three patients relapsed after 36, 41, and 72 months, respectively. Following re-treatment, they again showed complete remission over 18-48 months of observation. CONCLUSIONS: A promising role of RTX in an intensified protocol of induction therapy can be envisaged in patients for whom avoiding immunosuppressive maintenance therapy and sparing steroids are particularly appealing. Moreover, our data confirm in one of the longest follow-up available, the opportunity to reconsider the regimens of BL depletion in the treatment of the most severe or refractory forms of SLE despite the disappointing results of RCTs.

Association between transforming growth factor beta1 gene polymorphisms and IgA nephropathy.

BACKGROUND: Transforming growth factor beta1 (TGF-beta1) plays an important role in the modulation of cellular growth and differentiation in a wide variety of cell types and in the production/degradation of the extracellular matrix (ECM). We investigated whether G-800A, C-509T and Leu10-->Pro polymorphisms in the TGF-beta1 gene could be involved in the development and progression of immunoglobulin A nephropathy (IgAN). METHODS: DNA samples were obtained from 101 patients with biopsy proven IgA mesangial nephropathy and 118 healthy controls. The genotypes of G-800A, C-509T and Leu10-->Pro polymorphisms in the TGF-beta1 gene were determined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) with MaeIII, Eco 81I and Pst I, respectively. RESULTS: No significant differences were observed in the genotype distribution of the three TGF-beta1 polymorphisms between patients and controls. The TAC haplotype (T=Leu10, A-800 and C-509 alleles, respectively) was significantly associated with IgAN (p=0.043; odds ratio (OR) =2.334, 95 % confidence interval (95%CI) 1.01-5.41). CONCLUSION: Our study suggests that the haplotype reconstruction of TGF-beta1 gene polymorphisms could be more informative than the investigation of single nucleotide polymorphisms for defining the associated risk of developing IgAN. Further research is needed on larger cohorts to confirm TGF-beta1 involvement and test other TGF-beta1 variants with possible additive or synergistic effects.

Rituximab as a therapeutic tool in severe mixed cryoglobulinemia.

Type II mixed cryoglobulinemia (MC) is a systemic vasculitis, associated in most cases with hepatitis C virus (HCV) infection, sustained by proliferation of oligoclonal cells. Systemic B cell depletion and clinical remission can be achieved in non-Hodgkin lymphoma by human/mouse chimeric monoclonal antibody that specifically reacts with the CD20 antigen (rituximab). Similar effects could be expected in type II MC. Twelve patients, mean age 61.9 years (range 37-76), 11 with HCV infection genotype 2a2c (4 cases) or 1b (6 cases) and 3 (1 case) and symptomatic type II MC with systemic manifestations, including renal involvement, marrow clonal restriction, large necrotizing ulcers, and polyneuropathy, were considered eligible for rituximab therapy because of resistance or intolerance to conventional therapy or important bone marrow infiltration. Rituximab was administered intravenously at a dose of 375 mg/m2 on days 1, 8, 15, and 22. Two more doses were administered 1 and 2 months later. No other immunosuppressive drugs were added. Response was evaluated by assessing the changes in clinical signs, symptoms, and laboratory parameters. Levels of proteinuria, hematuria, erythrocyte sedimentation rate, cryocrit, rheumatoid factor, and IgM decreased while C4 values increased and HCV viral load remained stable during short- and medium-term observation. Bone marrow abnormalities were found to reverse to normal. Constitutional symptoms disappeared or ameliorated. No acute or delayed side effects were seen. Based on this experience and a number of reports published in the last 5 years, Rituximab appears to be a safe and effective therapeutic option in symptomatic patients with HCV-associated MC with signs of systemic vasculitis.

Absence of NF-kappaB activation by a new polystyrene-type adsorbent designed for hemoperfusion.

AIM: The aim of the study was to evaluate biocompatibility of anew polystyrene-type adsorbent (BetaSorb) designed for hemoperfusion, using second-level biomolecular analyses. The device has recently been developed to enhance beta2-microglobulin removal during hemodialysis. Molecular structure and chemical modifications of the surface beads of this cartridge should prevent exposure of dense hydrophobic surface sites to proteins, and avoid the major drawbacks of previous polystyrene-type adsorbent materials. METHODS: Whole blood of healthy donors was incubated in sterile minicolumns packed with BetaSorb Cuprophan, Hemophan, polysulfone and cellulose acetate. In parallel experiments, whole blood was recirculated for 180 min in a sham dialysis circuit equipped with the study sorbent or Hemophan or polysulfone. Biocompatibility was assessed by means of new biomolecular approaches focused on nuclear factor kappaB (NF-kappaB) activation (assessed by electrophoretic mobility shift assay), TNF-alpha and IL-1beta gene expression (evaluated by real-time PCR), TNF-alpha and IL-1beta production (measured by Western blot assay and ELISA), nitric oxide (NO) generation (detected by electron paramagnetic resonance), free oxygen radical production (by chemiluminescence in a biological assay) and the generation of the complement breakdown product C3d. RESULTS: In coincubation experiments, 5-min contact with any dialysis device, but BetaSorb, was enough to induce activation of NF-kappaB. The amount of TNF-alpha precursor form was found to increase after 5 min of exposure to each tested polymer, but no traces of mature forms of TNF-alpha or IL-1beta were detected in in vitro experimental conditions using healthy blood. NO and free oxygen radical generation were significantly lower in blood samples exposed to BetaSorb than in control dialysis devices. C3d levels were found to be increased with Hemophan, unaffected by polysulfone, and remarkably decreased with the BetaSorb device. In the sham hemodialysis experiments, NF-kappaB activation and C3d and NO profiles were similar to direct incubation experiments. Compared to basal levels, quantitation of TNF-alpha and IL-1beta mRNA revealed a 15- and 9-fold increase, respectively, in samples exposed to Hemophan for 180 min. CONCLUSIONS: The new BetaSorb device not only appears to be highly biocompatible, but shares properties that make it probably able to interfere with the activation of the inflammatory state.