Milica Mitrevski

Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia.

Hepatitis C virus (HCV)‐associated mixed cryoglobulinemia (MC) vasculitis commonly regresses upon virus eradication, but conventional therapy with pegylated interferon and ribavirin yields approximately 40% sustained virologic responses (SVR). We prospectively evaluated the efficacy and safety of sofosbuvir‐based direct‐acting antiviral therapy, individually tailored according to the latest guidelines, in a cohort of 44 consecutive patients with HCV‐associated MC. In two patients MC had evolved into an indolent lymphoma with monoclonal B‐cell lymphocytosis. All patients had negative HCV viremia at week 12 (SVR12) and at week 24 (SVR24) posttreatment, at which time all had a clinical response of vasculitis. The mean (±standard deviation) Birmingham Vasculitis Activity Score decreased from 5.41 (±3.53) at baseline to 2.35 (±2.25) (P < 0.001) at week 4 on treatment to 1.39 (±1.48) (P < 0.001) at SVR12 and to 1.27 (±1.68) (P < 0.001) at SVR24. The mean cryocrit value fell from 7.2 (±15.4)% at baseline to 2.9 (±7.4)% (P < 0.01) at SVR12 and to 1.8 (±5.1)% (P < 0.001) at SVR24. Intriguingly, in the 2 patients with MC and lymphoma there was a partial clinical response of vasculitis and ∼50% decrease of cryocrit, although none experienced a significant decrease of monoclonal B‐cell lymphocytosis. Adverse events occurred in 59% of patients and were generally mild, with the exception of 1 patient with ribavirin‐related anemia requiring blood transfusion. Conclusion: Interferon‐free, guideline‐tailored therapy with direct‐acting antivirals is highly effective and safe for HCV‐associated MC patients; the overall 100% rate of clinical response of vasculitis, on an intention‐to‐treat basis, opens the perspective for curing the large majority of these so far difficult‐to‐treat patients. (Hepatology 2016;64:1473‐1482)

Efficacy of low-dose rituximab for the treatment of mixed cryoglobulinemia vasculitis: Phase II clinical trial and systematic review

OBJECTIVE To evaluate whether rituximab at a low dose of 250 mg/m(2) × 2 may be as effective as at higher dosages, most commonly 375 mg/m(2)×4, used in previous studies on the treatment of patients with refractory mixed cryoglobulinemia (MC) vasculitis associated with hepatitis C virus (HCV) infection. METHODS We conducted a phase 2, single-arm two-stage trial (EUDRACT n. 2008-000086-38) of low-dose rituximab in 52 patients with HCV-associated MC who were ineligible/intolerant or non-responder to antiviral therapy. The primary outcomes were response of vasculitis evaluated by the Birmingham Vasculitis Activity Score (BVAS) at months 3, 6 and 12, rate of relapses and time to relapse, and rate of adverse events. Our data were compared with those reported in 19 published studies selected among 291 reviewed in a literature search. RESULTS The cumulative response rate (complete and partial) at month 3 was 81% in our patients, and 86% in 208 patients from studies using high-dose rituximab. The relapse rate and median time to relapse were, respectively, 41% and 6 months in our study, and 32% and 7 months in high-dose studies. Treatment-related adverse events were 11.5% in our study and 19.9% in high-dose studies. None of these differences was statistically significant. CONCLUSION Rituximab at a low dosage of 250 mg/m(2) × 2 is as effective as at higher dosages for treating MC vasculitis. This low-dose regimen may improve the cost/benefit profile of rituximab therapy for MC.

Intravenous Immunoglobulin and Immunomodulation of B-Cell – in vitro and in vivo Effects

Intravenous immunoglobulin (IVIG) is used as replacement therapy in patients with antibody deficiencies and at higher dosages in immune-mediated disorders. Although different mechanisms have been described in vitro, the in vivo immunomodulatory effects of IVIG are poorly understood. Different studies have suggested that IVIG modulates B-cell functions as activation, proliferation, and apoptosis. Recently, it was shown that IVIG induces in vitro B-cell unresponsiveness similar to anergy. In accord with this, we recently reported that IVIG therapy in patients affected by common variable immunodeficiency (CVID) interferes in vivo with the B-cell receptor (BCR) signaling by increasing constitutive ERK activation and by reducing the phosphorylated ERK increment induced by BCR cross-linking. Moreover, we observed that IVIG induces in CVID patients an increase of circulating CD21low B-cells, an unusual population of anergic-like B-cells prone to apoptosis. Therefore, IVIG at replacement dose in vivo could prime B-cells to an anergic, apoptotic program. Here, we discuss these recent findings, which may improve our understanding of the immunomodulatory effects of IVIG, individualizing single involved molecules for more specific treatments.

Intravenous immunoglobulin replacement therapy in common variable immunodeficiency induces B cell depletion through differentiation into apoptosis-prone CD21 low B cells

Intravenous immunoglobulin (IVIG), besides its use as replacement therapy in patients with antibody deficiencies, is broadly used as an immunomodulatory agent for the treatment of autoimmune and inflammatory disorders. The mechanisms of action of IVIG include Fc receptor blockade, inhibition of cytokines and growth factors, modulation of macrophages and dendritic cells, enhancement of regulatory T cells, and modulation of B cells through the FcγRIIB receptor and CD22. Recent studies suggest that in vitro exposure of human B cells to IVIG determines functional changes reminiscent of anergy and that IVIG treatment of patients with common variable immunodeficiency (CVID) induces in B cells ERK activation, a feature of anergy. Here, we show that IVIG therapy drives the B cells of patients with CVID to down-regulate CD21 expression and to assume the peculiar phenotype of the anergic-like, apoptosis-prone CD21low B cells that are spontaneously expanded in a subset of CVID and in some other immunological disorders. The CD21low B cells newly generated after IVIG infusion undergo spontaneous apoptosis upon in vitro culture. Furthermore, IVIG infusion is rapidly followed by a significant, although discrete, decrease in the number of circulating B cells, but not of T cells or of natural killer cells. These findings suggest that IVIG therapy may constrain antibody responses by inducing B cell depletion through differentiation into CD21low B cells that undergo accelerated apoptosis.

Reversion of anergy signatures in clonal CD21 low B cells of mixed cryoglobulinemia after clearance of HCV viremia

Hepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of IgM+CD27+ B cells. These cells display both the features of anergy induced by continual engagement of the B-cell receptor (BCR), such as high expression of phosphorylated extracellular signal-regulated kinase (pERK) and reduced lifespan, and of virus-specific exhaustion, such as CD21low phenotype and a defective response to ligation of BCR and Toll-like receptor 9 (TLR9). MC usually regresses after eradication of HCV with interferon, whose immunomodulatory activity might contribute to this effect. We investigated the phenotypic and functional changes in clonal B cells of MC patients with sustained virologic responses to direct-acting antivirals (DAAs), which lack immunomodulatory properties. We found that high pERK expression and accelerated apoptosis revert within 4 weeks after beginning therapy, whereas clonal B cells unresponsive to TLR9 stimulation persist for at least 24 weeks, although they may partially rescue normal CD21 expression. Thus, similar to mouse models, features of anergy in MC B cells rapidly revert after disengagement from HCV, whereas virus-specific exhaustion imparts a durable inhibitory imprint on cell function. Treatment of HCV+ MC with DAAs provides a valuable tool for untangling the molecular mechanisms of anergy and exhaustion in human B cells.

Sterile abscesses complicating monoclonal gammopathy of undetermined significance

A 47-year-old man presented with cough, fever and chest pain. A chest CT scan showed multiple nodules with air cavities in some, and IgGj monoclonal gammopathy of undetermined significance (MGUS) was detected. Blood, sputum and bronchoalveolar lavage cultures were negative for common and acid-fast bacteria or fungi. Antineutrophil cytoplasmic antibodies were negative. Empirical broad-spectrum antibiotic therapy was unsuccessful, while steroid therapy (prednisone 50 mg ⁄d) was followed by rapid clinical and radiological improvement. After steroid withdrawal fever recurred and novel pulmonary nodules developed. An open lung biopsy revealed abscesses with acute and chronic inflammatory areas containing neutrophlis and eosinophils, surrounded by giant epitheloid cells and interstitial fibrosis (Fig. 1A). As steroid therapy was resumed, fever disappeared and nodules regressed. The patient remained well on maintenance steroid therapy for 6 months, but shortly after treatment withdrawal fever relapsed and multiple lung, mesenteric, hepatic and splenic abscesses developed (Fig. 1B). As a predominantly eosinophilic abscess was revealed by liver biopsy, visceral larva migrans was suspected and albendazole was given without success. Based on the absence of microorganisms in the lesions, the failure of antimicrobial therapy, and the efficacy of steroid therapy we diagnosed an ‘aseptic abscesses syndrome,’ a rare condition commonly associated with chronic inflammatory bowel disease or MGUS (1).

Temporary pacemaker in refractory cluster headache treated with verapamil

Sir,We present a 35-year-old malepatient with 10 years’ history ofepisodic cluster headache (3–4attacks per day, previously treatedwith methysergide, lithium and oxy-gen without significant benefit).Therapy with verapamil 240 mg/dayreduced the frequency of attacks (2–3per day). After increasing the dose ofverapamil to 360 mg/day the patientpresented hypotension (75/40 mmHg)and serious bradycardia (HR 32/min),which required the implant of a tem-porary ventricular non-sequentialpacemaker. Once the critical periodhad passed, the blood pressure nor-malised; after the electrophysiologystudy found normal results, it waspossible to remove the pacemaker.Before starting verapamil therapy,cardiac examination and 24-h ECGrecording were normal.In the last 7 years, we have beenobserving four patients treated withverapamil for episodic clusterheadache that have referred hypoten-sion and serious bradycardia. Thepatients have not presented any con-traindication to prophylactic therapywith verapamil. Verapamil is a calci-um channel blocker and its commonadverse effects include constipation,dizziness, nausea, bradycardia andhypotension. It is widely used as afirst-line prophylactic drug for clusterheadache at doses starting at 360 mg[1, 2]. Patients with cluster headachepresent an increase in sympathetictone before the attack of a headache.This “sympathetic storm” could causeshort but hazardous periods ofarrhythmia [3]. On the contrary, aparasympathetic overactivity withconsequent bradycardia is presentduring the attack [4, 5]. Verapamiltherapy prevents episodic clusterheadache but it requires a strict con-trol of blood pressure and heart rate,because an overlap between theeffects of verapamil and the effects ofthe parasympathetic overactivitycharacterising the attack of clusterheadache that could cause serioushypotension and bradycardia can beverified [6].

DEC1/STRA13 is a key negative regulator of activation-induced proliferation of human B cells highly expressed in anergic cells

The transcription factor DEC1/STRA13 (also known as BHLHE40 and SHARP2) is involved in a number of processes including inhibition of cell proliferation and delay of cell cycle, and is a negative regulator of B cell activation and development in mice. We show here that, unlike in mice, DEC1/STRA13 expression is induced in human naïve and memory resting B cells by activation through the B-cell receptor (BCR) or Toll-like receptor 9 (TLR9). siRNA silencing of DEC1/STRA13 increases the capacity of activated B cells to perform a high number of divisions after TLR9 ligation. This identifies DEC1/STRA13 as a critical negative regulator of clonal expansion of activated human B cells. We also show that DEC1/STRA13 is upregulated in human anergic CD21low B cells clonally expanded in patients with HCV-associated mixed cryoglobulinemia, which fail to proliferate in response to BCR or TLR9 ligation. siRNA knockdown of DEC1/STRA13, however, fails to restore responsiveness to stimuli in these cells, although it might improve the proliferative capacity in a subset of anergic cells with less pronounced proliferative defect.

Modulatory Effects of Antibody Replacement Therapy to Innate and Adaptive Immune Cells

Intravenous immunoglobulin administered at replacement dosages modulates innate and adaptive immune cells in primary antibody deficiencies (PAD) in a different manner to what observed when high dosages are used or when their effect is analyzed by in vitro experimental conditions. The effects seem to be beneficial on innate cells in that dendritic cells maturate, pro-inflammatory monocytes decrease, and neutrophil function is preserved. The effects are less clear on adaptive immune cells. IVIg induced a transient increase of Treg and a long-term increase of CD4 cells. More complex and less understood is the interplay of IVIg with defective B cells of PAD patients. The paucity of data underlies the need of more studies on patients with PAD before drawing conclusions on the in vivo mechanisms of action of IVIg based on in vitro investigations.