Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

Cryoglobulins: putative effectors of adaptive immune response.

Cryoglobulinaemia consists of circulating monoclonal and/or polyclonal immunoglobulins with rheumatoid factor (RF) activity that precipitate at temperatures <37°C. Cryoglobulinaemic syndrome, characterised by clinical signs of systemic vasculitis, is associated with chronic infection of hepatitis C virus (HCV) and might evolve in B-cell malignancies. In about one third of all HCV infection cases, serum autoantibodies are commonly found. This is probably due directly to the transformation of infected B cells but, also, indirectly, to the viral chronic stimulation of a pool of autoreactive B cells. The pattern of IgG subclasses seems to contribute to the worsening progression of HCV infection into lymphoproliferative and/or autoimmune diseases. Many evidences showed that B cells circulating in patients with HCV-associated mixed cryoglobulinaemia (MC) are profoundly abnormal; moreover, in most of cases, normal B cells are replaced by expanded clonal B cells characterized by the low expression of CD21. After viral eradication, these cells persist in circulation and their occurrence does not correlate with serum cryoglobulins nor with vasculitis response or relapse. It is probably due to the persistence of monoclonal B cells producing RF, that in course of MC can be reactivated by circulating immune complexes, highly produced during infections or tumours. Here, we aimed to review current literature focusing the pathogenesis of MC referring to specificity and immunochemical characteristics of the immunoglobulins involved in cryoprecipitation.

Biomarkers of minimal residual disease in rituximab-treated patients with mixed cryoglobulinemia.

Hepatitis C virus (HCV) represents the major risk factor for mixed cryoglobulinemia (MC), a small-vessel vasculitis that may evolve into an overt B-cell non-Hodgkin's lymphoma. Here, we aimed to identify a biomarker signature for the early diagnosis of minimal residual disease (MRD). We assessed free light chains (FLCs), IgM k,and IgM λ heavy/light chain (HLC) pairs, and vascular endothelial growth factor (VEGF) in sera from 34 patients with MC vasculitis (32 HCV- and 2 HBV-related), treated with low-dose rituximab (RTX). FLCs and IgM HLCs were measured by turbidimetric assay; VEGF by an enzyme-linked immunosorbent assay. After RTX, the positive (complete + partial) clinical and laboratory responses were of 85.29% and 50%, respectively; in contrast, the mean levels of FLCs, IgM HLCs, and VEGF were substantially unaffected in most patients and still above the normal range. In those achieving a reduction of FLCs and IgM k and λ chains values within the range of normality, we found that post-treatment free λ chains and IgM k values correlated with clinical and laboratory response. Our results suggest that high levels of FLCs, IgM HLCs, and VEGF could represent the signature of "dormant" B cell clones' activity that could be very useful to identify MRD indicative of possible relapse or worsening outcome.

Laboratory Investigation of Hybrid IgG4 k/λ in MuSK Positive Myasthenia Gravis.

Myasthenia gravis with antibodies (Abs) against the muscle-specific tyrosine kinase (MuSK) is a rare autoimmune disorder (AD) of the neuromuscular junction (NMJ) and represents a prototype of AD with proven IgG4-mediated pathogenicity. Thanks to the mechanism of Fab-arm exchange (FAE) occurring in vivo, resulting MuSK IgG4 k/λ Abs increase their interference on NMJ and pathogenicity. The characterization of hybrid MuSK IgG4 as a biomarker for MG management is poorly investigated. Here, we evaluated total IgG4, hybrid IgG4 k/λ, and the hybrid/total ratio in 14 MuSK-MG sera in comparison with 24 from MG with Abs against acetylcholine receptor (AChR) that represents the not IgG4-mediated MG form. In both subtypes of MG, we found that the hybrid/total ratio reflects distribution reported in normal individuals; instead, when we correlated the hybrid/total ratio with specific immune-reactivity we found a positive correlation only with anti-MuSK titer, with a progressive increase of hybrid/total mean values with increasing disease severity, indirectly confirming that most part of hybrid IgG4 molecules are engaged in the anti-MuSK pathogenetic immune-reactivity. Further analysis is necessary to strengthen the significance of this less unknown biomarker, but we retain it is full of a diagnostic-prognostic powerful potential for the management of MuSK-MG.

Reversion of anergy signatures in clonal CD21low 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.

Hepatitis B virus causes mixed cryoglobulinaemia by driving clonal expansion of innate B-cells producing a VH1-69-encoded antibody.

OBJECTIVES: To investigate the expression of a VH1-69-encoded idiotype, and the phenotypic and functional features of monoclonal B-cells from patients with type II mixed cryoglobulinaemia (MC) secondary to chronic hepatitis B virus (HBV) infection. METHODS: B-cell immunophenotype and expression of a VH1-69-encoded idiotype were investigated by flow cytometry. B-cell proliferative responses to stimuli were investigated by the CFSE dilution assay. RESULTS: Two out of five patients with chronic HBV studied had massive monoclonal expansion of VH1-69-expressing B-cells. These cells had the peculiar CD21(low) phenotype and low responsiveness to stimuli typical of the VH1-69-expressing B-cells commonly expanded in MC secondary to hepatitis C virus (HCV) infection. In both patients, anti-HBV therapy led to the regression of MC and of VH1-69+ B-cell expansion. CONCLUSIONS: VH1-69-encoded antibodies are known to preferentially recognise a variety of viral proteins including HCV E2, influenza A virus haemagglutinin and HIV gp41/gp120, and may serve as innate first line antiviral defense. Thus, like HCV, HBV may cause MC by protracted antigenic stimulation of VH1-69-expressing B-cells.

Human Leukocyte Antigen Class II associations in late-onset Myasthenia Gravis.

OBJECTIVE: Genetic factors predisposing to late-onset myasthenia gravis (LOMG) have not been clearly defined yet. However, genome-wide association studies identified Human Leukocyte Antigen (HLA) Class II alleles as a hotspot in this disease subtype. The aim of this study was to analyze the correlations of HLA Class II alleles with clinical data and titin antibodies in this patient subgroup. METHODS: This study consecutively enrolled anti-acetylcholine receptor antibody-positive, non-thymoma patients with generalized LOMG. All patients were of Italian ancestry. HLA-DRB1 and -DQB1 genotyping and serum titin antibody testing were performed in this population. RESULTS: A total of 107 patients (females: 28/107, 26.2%; median age of onset: 68 years, range: 50-92) were included. We found a positive association with HLA-DRB1*07 (P = 1.1 × 10-5 ), HLA-DRB1*14 (P = 0.0251) and HLA-DQB1*02 (P = 0.0095). HLA-DRB1*03, HLA-DRB1*11, and HLA-DQB1*03 were protective alleles (P = 7.9 × 10-5 , P = 0.0104, and P = 0.0067, respectively). By conditional haplotype analysis, HLA-DRB1*07-DQB1*02 was found to be the major risk haplotype (OR = 4.10; 95% C.I.: 2.80-5.99; P = 6.01 × 10-11 ). The mean age at onset was 73.4 years in DRB1*07 homozygotes, 69.7 years in heterozygotes, and 66.6 in non-carriers (P = 0.0488). DRB1*07 carriers and non-carriers did not differ in disease severity and response to therapy. Titin antibodies were detected in 61.4% of the cases, having no association with HLA alleles or specific clinical characteristics. INTERPRETATION: In our study, we identified the HLA DRB1*07-DQB1*02 haplotype as a predisposing factor for the development of generalized LOMG in the Italian population.

Long-Lasting Rituximab-Induced Reduction of Specific-But Not Total-IgG4 in MuSK-Positive Myasthenia Gravis.

The use of rituximab (RTX), an anti-CD20 monoclonal antibody (Ab), in refractory myasthenia gravis (MG) is associated with a better response in patients with Abs to the muscle-specific tyrosine kinase (MuSK) than in other MG subgroups. Anti-MuSK Abs are mostly IgG4 with proven pathogenicity and positive correlation with clinical severity. The rapid and sustained response to RTX may be related to MuSK Ab production by short-lived Ab-secreting cells derived from specific CD20+ B cells. Here, we investigated the long-term effects of RTX in nine refractory MuSK-MG patients with a follow-up ranging from 17 months to 13 years. In patients' sera, we titrated MuSK-specific IgG (MuSK-IgG) and MuSK-IgG4, along with total IgG and IgG4 levels. Optimal response to RTX was defined as the achievement and maintenance of the status of minimal manifestations (MM)-or-better together with a ≥ 50% steroid reduction, withdrawal of immunosuppressants, and no need for plasma-exchange or intravenous immunoglobulin. After a course of RTX, eight patients improved, with optimal response in six, while only one patient did not respond. At baseline, MuSK-IgG and MuSK-IgG4 serum titers were positive in all patients, ranging from 2.15 to 49.5 nmol/L and from 0.33 to 46.2 nmol/L, respectively. MuSK Abs mostly consisted of IgG4 (range 63.80-98.86%). RTX administration was followed by a marked reduction of MuSK Abs at 2-7 months and at 12-30 months (p < 0.02 for MuSK-IgG and p < 0.01 for MuSK-IgG4). In patients with a longer follow-up, MuSK Ab titers remained suppressed, paralleling clinical response. In the patient who achieved long-term complete remission, MuSK-IgG4 was no longer detectable within 2 years, while MuSK-IgG remained positive at very low titers up to 10 years after RTX. In the patient who did not respond, MuSK-IgG and MuSK-IgG4 remained unchanged. In this patient series, total IgG and IgG4 transiently decreased (p < 0.05) at 2-7 months after RTX. The different trends of reduction between MuSK-IgG4 and total IgG4 after RTX support the view that short-lived Ab-secreting cells are the main producers of MuSK Abs. The ratio between short-lived Ab-secreting cells and long-lived plasma cells may influence the response to RTX, and B-cell severe depletion may reduce self-maintaining autoimmune reactivity.

Serological profile of asymptomatic HCV positive patients with low level of cryoglobulins.

Clinical spectrum of hepatitis C virus (HCV)-related cryoglobulinemia varies from an asymptomatic presentation to severe vasculitis and lymphoma. A recent study in HCV-negative patients suggests that low cryoglobulins (CGs) levels are responsible for severe renal and neurological complications. The aim of this study was to identify a panel of serological biomarkers associated with low levels of CGs in HCV-positive patients. We studied a population of 79 untreated patients with chronic HCV infection: 13 naïve patients without CGs; 28 patients with asymptomatic mixed cryoglobulinemia (MC) and low levels of CGs (16/28 with polyclonal type III and 12/28 with microheterogeneous type III CGs); 38 patients with symptomatic MC and high levels of type II CGs. Serum samples were collected and examined for rheumatoid factor (RF) IgG and IgM, free light chains (FLCs) and C3 and C4 complement components. We found that RF-IgG and IgM, free k chains and k+λ were increased while C4 component was reduced, both in symptomatic and asymptomatic patients. Our results suggest that, even in absence of MC symptoms, the low levels of CGs may represent a trigger of activation for immune system in course of HCV infection. The identification of a correlated biomarkers panel could improve the clinical management of these patients and pave the way for target treatment strategies. © 2018 BioFactors, 45(3):318-325, 2019.

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.

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 CD21(low) 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 CD21(low) B cells that are spontaneously expanded in a subset of CVID and in some other immunological disorders. The CD21(low) 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 CD21(low) B cells that undergo accelerated apoptosis.

Involvement of the Src-cortactin pathway in migration induced by IGF-1 and EGF in human breast cancer cells.

Cancer cells need to become motile in order to escape the primary tumor and move to distant areas to form metastasis. They move as single cells or as a group, following different stimuli, including growth factors. Among them, insulin-like growth factor­1 (IGF-1) and epidermal growth factor (EGF) and their receptors have been implicated in the development and progression of human breast carcinoma. In this report, we provide evidence that the tyrosine kinase Src is responsible for migration promoted by both IGF-1 and EGF in MDA-MB-231 and MCF7 cells, although with a different effect. Moreover, both IGF-1 and EGF induce reorganization of actin cytoskeleton in lamellipodia and membrane ruffles in a time- and Src-dependent manner. Furthermore, we analyzed the tyrosine phosphorylation status of the actin-binding protein cortactin upon growth factor stimulation, showing that even the activation of cortactin is time- and Src-dependent. In addition, immunofluorescence analysis with anti-paxillin antibody reveals that, after treatment with growth factors, tyrosine phosphorylated cortactin is localized on the plasma membrane in correspondence of focal adhesions. Collectively, our findings suggest a crucial role for Src-mediated activation of cortactin in cell migration, reorganization of actin cytoskeleton and phosphotyrosine cortactin localization to the focal adhesions in human breast cancer cell lines upon both IGF-1 and EGF stimulation.