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HCV is a hepatotropic and lymphotropic virus and is the most frequent cause of "benign" mono-oligoclonal B-lymphocyte proliferation, observed in mixed cryoglobulinemia (MC). The study aims to investigate the presence, prevalence and characteristics of the subclasses of cryoglobulins in HCV-patients to look for a relationship with MC. Fifty HCV-infected patients with cryoglobulins were enrolled. IgG subclasses were characterized in cryoprecipitate, and serum IgG and IgM Rheumatoid Factor (RF) were determined. Patients were stratified into two subgroups according to the presence of IgG3 subclass. Differences were observed in supernatant IgM, IgG3-positive and IgG3-negative patients with a higher IgM concentration in the IgG3-negative cohort (p=0.03). Higher IgM-RF was detected in the IgG3-negative group (p=0.01). IgG3-positive group showed higher IgG-RF compared to the IgG3-negative group (p<0.0001). IgG3-negative/monoclonal-IgM patients had higher cryocrit compared to IgG3-negative/polyclonal-IgM patients (p<0.01). C4 levels were higher in the polyclonal-IgM group compared to monoclonal-IgM group (p<0.01). We speculate that cryoglobulins are part of a progressive clonal selection process in which, B-cells are stimulated to produce oligoclonal IgG3 with RF activity. The persistence of the antigenic stimulus elicits the production of polyclonal IgM-RF and subsequently the formation of oligoclonal IgG/polyclonal IgM containing cryoglobulins. In the last stage, a monoclonal IgM-RF clone is formed which may coexist with a monoclonal IgG3-RF clone., (Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: The etiology of interstitial cystitis (IC) is often idiopathic but can be due to Hunner's ulcers. Hyperbaric oxygen (HBO) is used to treat ulcerative disease of the superficial skin. We hypothesized that HBO can treat ulcerative IC (UIC) but would be less efficacious for non-ulcerative IC (NIC)., Methods: Patients with NIC and UIC enrolled in this study. Following informed consent, demographic information was collected. A visual analog pain scale and validated questionnaires were collected; each patient underwent cystoscopy prior to treatment. Each subject met with a hyperbaric specialist and after clearance underwent 30 treatments over 6 weeks. Adverse events were monitored. Patients repeated questionnaires, visual analog pain scale and global response assessment (GRA) immediately, 2 weeks, 3, 6 and 12 months after treatment. Patients also underwent cystoscopy 6 months after treatment. Differences before and after treatment were compared., Results: Nine patients were recruited to this study. One was unable to participate, leaving two subjects with NIC and six with UIC. All patients completed HBO without adverse events. Three patients completed HBO but pursued other therapies 7, 8.5 and 11 months after treatment. On GRA, 83% of patients with UIC were improved. This treatment effect persisted, as 66% of UIC patients remained better at 6 months. In contrast, only one patient in the NIC group improved. Questionnaire scores improved in both groups. Pain scores improved by 2 points in the UIC group but worsened by 1.5 points in the NIC group. Two patients with ulcers resolved at 6-month cystoscopy., Conclusion: HBO appeared beneficial for both UIC and NIC. Data shows slightly better benefit in patients with UIC compared to NIC; both groups showed improvement. Given the small sample size, it is difficult to draw definitive conclusions from these data. Larger studies with randomization would be beneficial to show treatment effect., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest.

The critical relevance of the lysosomal compartment for normal cellular function can be proved by numbering the clinical phenotypes that arise in lysosomal storage disorders (LSDs), a group of around 70 different monogenic autosomal or X-linked syndromes, caused by specific lysosomal enzyme deficiencies: all LSDs are characterized by progressive accumulation of heterogeneous biologic materials in the lysosomes of various parts of the body such as viscera, skeleton, skin, heart, and central nervous system. At least a fraction of LSDs has been associated with mixed abnormalities involving the immune system, while some patients with LSDs may result more prone to autoimmune phenomena. A large production of proinflammatory cytokines has been observed in Gaucher and Fabry diseases, and wide different autoantibody production has been also reported in both. Many immune-mediated reactions are crucial to the pathogenesis of different inflammatory signs in mucopolysaccharidoses, and subverted heparan sulphate catabolism might dysregulate cellular homeostasis in the brain of these patients. Furthermore, an inappropriate activation of microglia is implicated in the neurodegenerative foci of Niemann-Pick disease, in which abnormal signalling pathways are activated by impaired sphingolipid metabolism. In addition, not the simple impaired catabolism of gangliosides per se, but also the production of anti-ganglioside autoantibodies contributes to the neurological disease of gangliosidoses. Even if the exact relationship between the modification of lysosomal activities and modulation of the immune system remains obscure, there is emerging evidence of different impaired immunity responses in a variety of LSDs: in this review we investigate and summarize the immune abnormalities and/or clinical data about immune system irregularities which have been described in a subset of LSDs., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: In order to establish a diagnosis of monoclonal gammopathy, it is necessary to detect and identify monoclonal components. To confirm the immunological nature of the proteins, the next step is to define their composition in heavy and light chains using immunofixation. The purpose of this study was to compare two different instruments, one semiautomated and the other fully automated for serum and urine immunofixation., Methods: We selected 150 sera and 100 urines from patients admitted for routine analysis, which were analyzed by immunofixation to characterize monoclonal components., Results and Conclusion: Comparison study showed a difference in the identification of small monoclonal components and hypogammaglobulinemia, in serum and urine, between the two analyzers. We also observed a difference in the length of the electrophoretic pattern that is of considerable importance as it leads to a better resolution of the gamma region, allowing to identify even the smallest monoclonal component that can be easily hide in an oligoclonal pattern. For this reason, there is need to ameliorate commercial immunofixation assays. It is essential to improve data harmonization and standardize measurement procedures in order to guarantee a correct diagnosis for the right patient care., (© 2016 Wiley Periodicals, Inc.)

Cryoglobulins are immunoglobulins that precipitate in serum at temperatures below 37°C and resolubilize upon warming. The clinical syndrome of cryoglobulinemia usually includes purpura, weakness, and arthralgia, but the underlying disease may also contribute other symptoms. Blood samples for cryoglobulin are collected, transported, clotted and spun at 37°C, before the precipitate is allowed to form when serum is stored at 4°C in a Wintrobe tube for at least seven days. The most critical and confounding factor affecting the cryoglobulin test is when the preanalytical phase is not fully completed at 37°C. The easiest way to quantify cryoglobulins is the cryocrit estimate. However, this approach has low accuracy and sensitivity. Furthermore, the precipitate should be resolubilized by warming to confirm that it is truly formed of cryoglobulins. The characterization of cryoglobulins requires the precipitate is several times washed, before performing immunofixation, a technique by which cryoglobulins can be classified depending on the characteristics of the detected immunoglobulins. These features imply a pathogenic role of these molecules which are consequently associated with a wide range of symptoms and manifestations. According to the Brouet classification, Cryoglobulins are grouped into three types by the immunochemical properties of immunoglobulins in the cryoprecipitate. The aim of this paper is to review the major aspects of cryoglobulinemia and the laboratory techniques used to detect and characterize cryoglobulins, taking into consideration the presence and consequences of cryoglobulinemia in Hepatitis C Virus (HCV) infection.

Background: HCV can lead to both chronic liver disease and B-cell lymphoproliferative disorders. A strong association exists between HCV and mixed cryoglobulinaemia (MC)., Methods: Anti-nuclear antibodies (ANA), rheumatoid factor Ig-G (RF-IgG), free light chain κ and λ (FLC-κ, FLC-λ) levels and κ/λ ratio were evaluated in 50/420 subjects unexpectedly resulted anti-HCV positive after routine screenings for non-hepathological procedures., Results: Three/fifty patients had HCV-RNA undetectable in the serum and were excluded from the analysis. Thirty-nine/fifty patients had laboratory evidence of circulating cryoglobulins without liver disease and MC-related symptoms. Among them, 17 resulted ANA-positive. The mean cryocrit was higher in ANA-positive patients, while no other demographic/clinical differences were observed between the groups. Significantly higher levels of RF-IgG were observed in ANA-positive vs ANA-negative patients. κ and λ FLC were higher in ANA-positive patients. A ROC analysis, based on ANA-positivity vs ANA-negativity, confirmed a high sensitivity and specificity of RF-IgG test., Conclusions: Published data concerning MC come mostly from symptomatic vasculitis. We analyzed HCV-patients without MC symptoms, founding cryoglobulins in the majority of them. The increased levels of FR-IgG and FLC in CGs-ANA-positive patients, suggest these test could be used to identify a state of silent autoimmune and/or lymphoproliferative condition before the transition to a frank disease in naïve HCV-patients without symptoms of extrahepatic manifestations., (Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Mixed cryoglobulinaemia (MC) is an HCV-related lymphoproliferative disorder characterized by the presence of circulating immune complexes called cryoglobulins. Treatment with anti-CD20 monoclonal antibody rituximab is proved to be very useful, especially in patients ineligible to interferon-based antiviral therapy. Recently, free light chain (FLC) κ/λ ratio and FLC patterns were associated with MC. The aim of this study was to evaluate changes in FLC-κ, FCL-λ, FLC ratio following rituximab treatment in patients with HCV-related MC and to correlate FLC-κ, FCL-λ and FLC ratio values with therapy response., Patients and Methods: We retrospectively enrolled 46 patients with HCV infection (26 females, 20 males), including 10 patients without signs/symptoms of MC-related vasculitis, 36 with MC vasculitis. Clinical and biological data were recorded at baseline and 6 months after RTX treatment. Nephelometric measurement of serum FLCs was taken., Results: The mean serum FLC-κ level and FLC ratio were significantly higher in patients with MC, compared to HCV patients without MC and to blood donors. An abnormal FLC ratio at baseline correlated with the presence of cryoglobulins, C4 consumption, higher RF level and higher vasculitis rate. To evaluate the predictive value of FLCs, patients with MC were divided into two groups according to RTX therapy outcome (responders and no/partial responders). Abnormal baseline FLC ratio was significantly associated with no/partial response., Conclusions: RTX treatment in HCV-related MC induces a reduction in FLC-κ and RF levels. Moreover, pretreatment FLC ratio, which can be easily assessed by a routine test, may be useful to predict response to this expensive treatment for patients with HCV-related MC ineligible to IFN-based therapy., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Monoclonal gammopathies encompass a wide range of diseases characterized by the monoclonal expansion of a B-cell clone. Despite emerging therapeutic strategies, chances of survival of patients who are affected are still scarce, which implies that new tools are necessary not only for the diagnosis but also for the follow-up of patients affected by such diseases. In this context, the use of free light chains (FLCs) has been incorporated into many guidelines. Likewise, tumor microenvironment is consistently gaining importance as role player in tumor pathogenesis. Specifically, Syndecan-1 (CD138), a heparan-sulfate proteoglycan is attracting interests as it is highly expressed and shed by myeloma plasma-cells. The aim of our study was to analyze CD138 levels in the serum of patients affected by multiple myeloma or light chain only disease, and to compare the values obtained with free light chain (FLC) kappa, lambda and FLC ratio in both groups of patients., Methods: 84 patients affected by Multiple Myeloma and Light Chain Myeloma were recruited for this study. Serum CD138 was assessed by ELISA (Diaclone Research, France) and FLC values were quantified by nephelometry (Freelite TM Human Kappa and Lambda Free Kits, The Binding Site, UK). Data was analyzed by GraphPad Prism software and Statgraph., Results: We observed higher CD138 mean values in myeloma patients compared to the light chain only myeloma group. A positive linear regression of CD138 and FLC was observed in the light chain only cohort as opposed to myeloma patients which show an inverse trend., Conclusions: The study highlighted an existing relationship between FLCs and CD138 and wishes to seek also a correlation in order to rapidly and efficiently perform diagnosis and different diagnostic schemes.

Background: Anti-nuclear antibodies are immunoglobulins directed against nuclear antigens. They are associated with many autoimmune disorders, but are frequently found in patients infected with hepatitis C virus, possibly indicating an underlying common origin. Likewise, mixed cryoglobulinemia often accompanies autoimmune diseases and hepatitis C infection., Aim: To compare anti-nuclear antibodies and immunoglobulin content of cryoprecipitates from hepatitis C virus-positive patients in order to assess their predictive value in the onset of hepatitis C virus-driven extrahepatic disorders., Methods: Serum from 40 hepatitis C virus-positive patients and 50 controls with rheumatoid arthritis was processed for cryoglobulin detection: all subjects presented with Type III mixed cryoglobulinemia. Immunoglobulin content and immunoglobulin subclasses of cryoprecipitates were assessed by immunofixation and tested by ELISA for rheumatoid factor. Cryoprecipitates were also analysed for anti-nuclear antibodies by indirect immuno-fluorescence to identify specific patterns typical of each condition., Results: Anti-nuclear antibody patterns differed significantly; 26 infected subjects (65%) were IgG3 positive: of these, 25 were also anti-nuclear antibody-positive (96.1%)., Conclusions: IgG3 are autoreactive clones unrelated to viral recognition and possibly involved in autoimmune disorders. Altogether, these results may represent useful diagnostic device for early detection of hepatitis C virus-induced autoimmune diseases., (Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Calciphylaxis is a relatively rare disorder associated with calcification of small- and medium-sized blood vessels, progressive ischemic skin necrosis, and ulcerations. It is usually seen in patients with end-stage renal disease with secondary hyperparathyroidism and is occasionally seen in primary hyperparathyroidism, hypercalcemia of malignancy, and end-stage liver disease. We report an unusual case of calciphylaxis seen in association with metastatic breast carcinoma in the absence of end-stage renal or parathyroid disease. A 73-year-old woman presented with painful leg ulcers. Serum calcium levels and parathormone levels were within normal limits. Skin biopsies from the ulcers revealed small- to medium-sized subcutaneous arteries with calcification of the media. Some of the arteries were narrowed by fibrointimal hyperplasia and fibrin thrombi. Calcification of the subcutaneous fat, fat necrosis, and suppuration were also seen. Calciphylaxis associated with metastatic osteolytic breast carcinoma is rare. Although end stage renal disease with secondary hyperparathyroidism is the most common cause of calciphylaxis, this case demonstrates that less common conditions with normal serum calcium and parathormone levels may also cause it.