Calprotectin: two sides of the same coin.

Calprotectin (CLP) is a calcium-binding protein produced by neutrophils and monocytes in the course of inflammation. Today, the role of faecal CLP in chronic IBD is well known, but in recent years attention has shifted towards circulating CLP. In fact, this molecule can be measured in different biological fluids: blood, saliva and urine, using different analytic methods that are described in this review. Furthermore, different data confirm the relevant role of serum CLP in autoimmune diseases. In this review we will highlight the correlation between high levels of circulating CLP and specific autoantibodies of major autoimmune pathologies paving the way to the employment of CLP measurement as useful biomarker for monitoring outcome in different pathologies.

Elevated serum polyclonal immunoglobulin free light chains in patients with severe asthma.

Background: Inflammation plays a pivotal role in the pathophysiology of asthma. Free light chains (FLC) can cause inflammation by mast cell antigen-activation. Serum immunoglobulin (Ig) FLC κ, but not λ, were shown elevated in adult males with asthma. We sought to investigate if serum Ig FLC concentrations are affected by asthma severity and their relationships with inflammatory outcomes. Methods: By using immunoassays, we measured serum κ and λ Ig FLCs in 24 severe persistent asthma patients, 15 patients with moderate persistent asthma, 15 steroid-naïve mild persistent asthma patients and 20 healthy control subjects in a cross-sectional observational study. Total and specific serum IgE concentrations, fractional exhaled nitric oxide (FENO), lung function, peripheral blood eosinophils and neutrophils, and C reactive protein (CRP) were also measured. Results: Serum κ FLC concentrations were elevated in severe asthma patients compared mild asthma patients (p < 0.05) and healthy subjects (p < 0.05). Serum λ FLCs were higher in severe asthma patients than in healthy subjects (p < 0.05) and correlated with blood eosinophil counts (percentage, κ: r = 0.51, p = 2.9678-6; λ: r = 0.42, p = 1.7377-4; absolute values, κ: r = 0.45, p = 6.1284-5; λ: r = 0.38, p = 7.8261-4), but not with total or specific serum IgE. In severe asthma patients, serum Ig FLC correlated with serum CRP (κ: r = 0.33; p = 0.003; λ: r = 0.38, p = 8.8305-4) and blood neutrophil cell counts (percentage, κ: r = 0.31; p = 0.008; λ: r = 0.29, p = 0.01; absolute values, κ: r = 0.40; p = 3.9176-4; λ: r = 0.40, p = 4.5479-4), were elevated in subjects with blood eosinophilia (≥300 cells/µL) (n = 13) compared with non-eosinophilic subjects (n = 10) (κ: 19.2 ± 1.2 mg/L versus 12.1 ± 1.3 mg/L, p < 0.001; λ: 27.2 ± 2.6 mg/L versus 16.8 ± 2.5 mg/L, p < 0.01), but were similar in atopic (n = 15) versus nonatopic subjects (n = 9) (κ: p = 0.20; λ: p = 0.80). Serum FLC were negatively correlated with lung function tests, including forced expiratory volume in one second (FEV1) (κ: r = -0.33; p = 0.0034; λ: r = -0.33; p = 0.0035), and FEV1/forced vital capacity ratio (κ: r = -0.33; p = 0.0034; λ: r = -0.33; p = 0.0036). Conclusion: Serum Ig FLCs are elevated in severe asthma adults and might represent new surrogate markers of inflammation. The pathophysiological implications of these findings require further research. This study was approved by the ethics committee of the University Hospital Agostino Gemelli Foundation and Catholic University of the Sacred Heart (approval number P/1034/CE2012).

Serological and Molecular Characterization of Hepatitis C Virus-Related Cryoglobulinemic Vasculitis in Patients without Cryoprecipitate.

Prolonged B cells stimulation due to the Hepatitis C virus (HCV) can result in autoimmunity, stigmatized by rising levels of cryoglobulins (CGs), the rheumatoid factor (RF), and free light chains (FLC) of immunoglobulins (Ig) associated with a range of symptoms, from their absence to severe cryoglobulinemic vasculitis and lymphoma. Here, we aimed to identify an immunological signature for the earliest stages of vasculitis when cryoprecipitate is still not detectable. We firstly analyzed the IgG subclasses, FLC, and RF in 120 HCV-RNA-positive patients divided into four groups according to the type of cryoprecipitate and symptoms: 30 asymptomatic without cryoprecipitate (No Cryo), 30 with vasculitis symptoms but without CGs that we supposed were circulating but still not detectable (Circulating), 30 type II and 30 type III mixed cryoglobulinemia (Cryo II and Cryo III, respectively). Our results revealed that patients with supposed circulating CGs displayed a pattern of serological parameters that closely resembled Cryo II and Cryo III, with a stronger similarity to Cryo II. Accordingly, we analyzed the groups of Circulating and Cryo II for their immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements, finding a similar mixed distribution of monoclonal, oligoclonal, and polyclonal responses compared to a control group of ten HCV-RNA-negative patients recovered from infection, who displayed a 100% polyclonal response. Our results strengthened the hypothesis that circulating CGs are the origin of symptoms in HCV-RNA-positive patients without cryoprecipitate and demonstrated that an analysis of clonal IGH and TCR rearrangements is the best option for the early diagnosis of extrahepatic complications.

Laboratory and Clinical Settings of Heavy/Light Chain (HLC) Assays in the Management of Monoclonal Gammopathies and Multiple Myeloma.

The antibody-related immune response is mediated by immunoglobulins (Igs), soluble circulating glycoproteins produced by activated B cells that, upon the recognition of specific epitopes on pathogen surfaces, activate, proliferate, and differentiate into antibody-secreting plasma cells. Although the antibodies are effectors of the humoral immune adaptive response, their overproduction in response to a dysregulated proliferation of clonal plasma cell production in tumoral conditions (i.e., multiple myeloma), enriches the serum and urinary matrices, assuming the crucial role of biomarkers. Multiple myeloma (MM) is a plasma cell dyscrasia characterized by the expansion and accumulation of clonally activated plasma cells in bone marrow, determining the release of high amounts of monoclonal component (MC) that can be detected as intact immunoglobulin (Ig), immunoglobulin fragments, or free light chains (FLCs). The importance of detecting biomarkers for the diagnosis, monitoring, and prognosis of diseases is highlighted by the international guidelines that recommend specific assays for the analysis of intact Igs and FLC. Moreover, a developed assay called Hevylite® allows for the quantification of immunoglobulins that are both involved (iHLC) and not involved (uHLC) in the tumor process; this is a fundamental aspect of following up the patient's workup and evaluating the progression of disease, together with the treatments response. We here summarize the major points of the complex scenario involving monoclonal gammopathies and MM clinical management in view of advantages derived for the use of Hevylite®.

Serum Adiponectin, a Novel Biomarker Correlates with Skin Thickness in Systemic Sclerosis.

The aim was to evaluate the longitudinal association between basal serum adiponectin and repeated measurements of skin thickness during 12 months of follow-up in systemic sclerosis (SSc) patients. We enrolled SSc patients with disease duration > 2 years in a prospective observational study. Skin thickness was measured at baseline and after 12 months of follow-up with modified Rodnan skin score (mRSS). Baseline serum adiponectin was determined using a commercial ELISA kit. We enrolled 66 female SSc patients (median age 54 years, IQR 42−62 years). The median disease duration was 12 (IQR 8−16) years and median baseline serum adiponectin was 9.8 (IQR 5.6−15.6) mcg/mL. The median mRSS was 10 (IQR 6−18) at baseline and 12 (IQR 7−18) at follow-up. A significant correlation was observed between baseline serum adiponectin and disease duration (r = 0.264, p < 0.05), age (r = 0.515, p < 0.0001), baseline mRSS (r = −0.303, p < 0.05), and mRSS at follow-up (r = −0.322, p < 0.001). In multiple regression analysis, only mRSS at follow-up showed an inverse correlation with baseline serum adiponectin (ß = −0.132, p < 0.01). The reduction in serum adiponectin levels is correlated with skin thickness.

Inflammation and Oxidative Stress in Seminal Plasma: Search for Biomarkers in Diagnostic Approach to Male Infertility.

Oxidative and inflammatory damage underlie several conditions related to male infertility, including varicocele. Free light chains of immunoglobulins (FLCs) are considered markers of low-grade inflammation in numerous diseases. Coenzyme Q10 (CoQ10), a lipidic antioxidant and anti-inflammatory compound, is involved in spermatozoa energy metabolism and motility. We aimed to evaluate FLCs' seminal levels in patients with varicocele in comparison to control subjects and to correlate them with CoQ10 and Total Antioxidant Capacity (TAC) in human semen. Sixty-five patients were enrolled. Semen analysis was performed; patients were divided into three groups: controls, 12 normozoospermic patients, aged 34 (33-41) years; varicocele (VAR), 29 patients, aged 33 (26-37) years; and idiopathic, 24 oligo-, astheno- and oligoasthenozoospermic patients aged 37 (33.5-40.5) years. FLCs (κ and λ) were assayed by turbidimetric method; CoQ10 by HPLC; TAC by spectrophotometric method. λ FLCs showed a trend toward higher levels in VAR vs. controls and the idiopathic group. VAR showed a trend toward lower κ FLCs levels vs. the other two groups. When comparing κ/λ ratio, VAR showed significantly lower levels vs. controls and idiopathic. Moreover, CoQ10 seminal levels showed higher levels in VAR and idiopathic compared to controls. Data reported here confirm lower levels of κ/λ ratio in VAR and suggest a possible application in personalized medicine as clinical biomarkers for male infertility.

Mono/polyclonal free light chains as challenging biomarkers for immunological abnormalities.

Free light chain (FLC) kappa (k) and lambda (λ) consist of low molecular weight proteins produced in excess during immunoglobulin synthesis and secreted into the circulation. In patients with normal renal function, over 99% of FLCs are filtered and reabsorbed. Thus, the presence of FLCs in the serum is directly related to plasma cell activity and the balance between production and renal clearance. FLCs are bioactive molecules that may exist as monoclonal (m) and polyclonal (p) FLCs. These have been detected in several body fluids and may be key indicators of ongoing damage and/or illness. International guidelines now recommend mFLC for screening, diagnosis and monitoring multiple myeloma and other plasma cell dyscrasias. In current clinical practice, FLCs in urine indicate cast nephropathy and other renal injury, whereas their presence in cerebrospinal fluid is important for identifying central nervous system inflammatory diseases such as multiple sclerosis. Increased pFLCs have also been detected in various conditions characterized by B cell activation, i.e., chronic inflammation, autoimmune disease and HCV infection. Monitoring the coronavirus (COVID-19) pandemic by analysis of salivary FLCs presents a significant opportunity in clinical immunology worthy of scientific pursuit.

Increased Complement Activation in Systemic Sclerosis Patients with Skin and Lung Fibrosis.

INTRODUCTION: The involvement of complement system in the phenotypic expression of systemic sclerosis (SSc) is a debated topic. We aimed to assay complement fractions in SSc patients and to correlate their levels with the clinical course of disease. KEY POINTS: 1. CH50 is increased in SSc patients compared to HC; 2. Serum C2 levels are increased in SSc patients compared to HC; 3. CH50 may represent a biomarker of skin and lung fibrosis severity in SSc patients. METHOD: Complement hemolysis 50% (CH50), C2, C3 and C4 levels have been assessed in 85 SSc patients and 47 healthy controls (HC). RESULTS: SSc patients displayed a statistically significant higher value of CH50 [76.3 U/mL (IQR 65.8-89.4 U/mL) vs. 29.6 U/mL (IQR 24.7-34 U/mL); p < 0.0001] and of C2 [26.1 mg/L (IQR 24.1-32.1 mg/L) vs. 22.7 mg/L (IQR 20.6-24.4 mg/L); p < 0.0001] if compared to HC. Patients with diffuse cutaneous SSc (dcSSc) had higher levels of CH50 than patients with limited cutaneous SSc (lcSSc) [83.6 U/mL (IQR 72.3-102.7 U/mL) vs. 71.3 U/mL (IQR 63.7-83.6 U/mL); p = 0.003]. SSc patients with interstitial lung disease (ILD) had higher CH50 levels if compared to SSc patients without ILD [79.6 U/mL (IQR 68.3-97.4 U/mL) vs. 69.7 U/mL (54.6-85.7 U/mL); p = 0.042]. A positive linear correlation existed between CH50 and the modified Rodnan Skin Score (mRSS) (r = 0.285, p = 0.008) and disease severity scale (DSS) (r = 0.285, p = 0.005); a negative linear correlation was demonstrated between CH50 and the diffusing capacity of carbon monoxide (DLco) (r = -0.252, p = 0.012). In multiple linear regression analysis, only DSS was significant (p = 0.01, beta coefficient 2.446). CONCLUSIONS: Our results show an increment of CH50 and serum C2 levels in SSc patients in comparison to HC; we retain that CH50 may represent a biomarker of disease severity and of skin and lung fibrosis in these patients.

Evaluation of free light chains of immunoglobulins in normal and pathological seminal fluids: Preliminary data.

Immunoglobulins free light chains (FLCs) are assayable in several biological fluids. Currently, there are no reports on FLCs in seminal plasma. The aims of our study were to investigate the presence and detectability of FLCs in seminal plasma and to evaluate the usefulness of this assay in the diagnostic approach to infertile patients. We enrolled 61 patients aged 18-50 years. Semen analysis was performed. They were divided into four groups: controls-normozoospermic, 10 patients, mean ± SEM age 35 ± 1.5 years; varicoceles (VAR), 18 patients aged 24.3 ± 0.96 years; inflammatory (INF) seminal fluids, 24 patients, aged 38.8 ± 2.2 years; and varicoceles and inflammatory (VAR/INF) seminal fluids, 9 patients, aged 29.5 ± 0.71 years. A trend towards higher λ FLCs levels was evidenced in the INF and VAR/INF groups. κ FLCs were higher in normozoospermic patients with lower levels in VAR, both isolated and associated with inflammatory parameters. This differential pattern of the two types of FLCs reached statistical significance when comparing κ/λ ratio, with significant lower levels in VAR vs controls. This is the first report of FLCs assay in seminal plasma. We can hypothesize that λ FLCs are increased in inflammatory processes, whether κ FLCs seem to be influenced by other molecular mechanisms related to varicocele.

Cytokines and Hepatocellular Carcinoma: Biomarkers of a Deadly Embrace.

Hepatocellular carcinoma (HCC) represents a worldwide health matter with a major care burden, high prevalence, and poor prognosis. Its pathogenesis mainly varies depending on the underlying etiological factors, although it develops from liver cirrhosis in the majority of cases. This review summarizes the role of the most interesting soluble factors as biomarkers for early diagnosis and as recommended targets for treatment in accordance with the new challenges in precision medicine. In the premalignant environment, inflammatory cells release a wide range of cytokines, chemokines, growth factors, prostaglandins, and proangiogenic factors, making the liver environment more suitable for hepatocyte tumor progression that starts from acquired genetic mutations. A complex interaction of pro-inflammatory (IL-6, TNF-α) and anti-inflammatory cytokines (TGF-α and -ß), pro-angiogenic molecules (including the Angiopoietins, HGF, PECAM-1, HIF-1α, VEGF), different transcription factors (NF-kB, STAT-3), and their signaling pathways are involved in the development of HCC. Since cytokines are expressed and released during the different stages of HCC progression, their measurement, by different available methods, can provide in-depth information on the identification and management of HCC.

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.

Cryoglobulins: Identification, classification, and novel biomarkers of mysterious proteins.

Cryoglobulins consist of serum immunoglobulins that precipitate below 37°C and resolubilize upon warming. The clinical triad of cryoglobulinemia usually includes purpura, weakness, and arthralgia. Cryoglobulinemic syndrome, clinically defined as a systemic vasculitis, is associated with chronic infection with hepatitis C virus (HCV) and autoimmune disorders and can evolve into B-cell malignancies. While the current literature about HCV-associated cryoglobulinemia is not very limited, little is known about the immunologic and serologic profiles of affected patients. Therefore, comprehension of the pathogenetic mechanisms underlying cryoprecipitation could be very helpful. Due to the persistence of viral antigenic stimulation, biomarkers to use after the worsening progression of HCV infection to lymphoproliferative and/or autoimmune diseases are widely needed. Laboratory methods used to detect and characterize low concentrations of cryoprecipitates and immunotyping patterns could improve patient management. The most critical factor affecting cryoglobulin testing is that the pre-analytical phase is not fully completed at 37°C.

Serum and urine free light chains measurements in patients with systemic sclerosis: novel biomarkers for disease activity.

Circulating free light chains (FLCs), considered biomarkers of B cell activity, are frequently elevated in patients affected by systemic inflammatory autoimmune diseases. As the systemic sclerosis (SSc) clinical course can be variable, this study is aimed at evaluating FLCs levels in affected individuals as biomarkers of disease activity. We assessed FLC levels in serum and urine of 72 SSc patients and 30 healthy controls (HC). Results were analyzed in comparison with overall clinical and laboratory findings, disease activity index (DAI) and disease severity scale (DSS). SSc patients displayed increased levels of κ and λ FLC in serum significantly higher than HC (p = 0.0001) alongside the mean values of free κ/λ ratio and κ + λ sum (p = 0.0001). SSc patients showed increased free κ in urine with a κ/λ higher than HC (p = 0.0001). SSc patients with increased κ + λ in serum showed that erythro-sedimentation rate (p = 0.034), C-reactive protein (p = 0.003), DAI (p = 0.024) and DSS (p = 0.015) were higher if compared to SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free κ and DAI (r = 0.29, p = 0.014). In addition, SSc patients with increased free κ in urine had higher DAI (p = 0.048) than SSc patients with normal κ levels. Our results strengthen the role of serum FLC as useful biomarker in clinical practice to early diagnosis and monitor disease activity, showing for the first time that also urine FLC levels correlated with disease activity in SSc patients.

Solving the mystery of HBV-related mixed cryoglobulinemia: potential biomarkers of disease progression.

OBJECTIVES: The biomarkers of an immunological dysregulation due to a chronic HBV infection are indeed understudied. If untreated, this condition may evolve into liver impairment co-occurring with extrahepatic involvements. Here, we aim to identify a new panel of biomarkers [including immunoglobulin G (IgG) subclasses, RF, and Free Light Chains (FLCs)] that may be useful and reliable for clinical evaluation of HBV-related cryoglobulinemia. METHODS: We retrospectively analysed clinical data from 44 HBV-positive patients. The patients were stratified (according to the presence/absence of mixed cryoglobulinemia) into two groups: 22 with cryoglobulins (CGs) and 22 without CGs. Samples from 20 healthy blood donors (HDs) were used as negative controls. Serum samples were tested for IgG subclasses, RF (-IgM, -IgG, and -IgA type), and FLCs. RESULTS: We detected a strikingly different distribution of serum IgG subclasses between HDs and HBV-positive patients, together with different RF isotypes; in addition, FLCs were significantly increased in HBV-positive patients compared with HDs, while no significant difference was shown between HBV-positive patients with/without mixed cryoglobulinemia. CONCLUSION: The immune-inflammatory response triggered by HBV may be monitored by a peculiar profile of biomarkers. Our results open a new perspective in the precision medicine era; in these challenging times, they could also be employed to monitor the clinical course of those COVID-19 patients who are at high risk of HBV reactivation due to liver impairment and/or immunosuppressive therapies.

A Comparative Study of Serum Angiogenic Biomarkers in Cirrhosis and Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a global health problem associated with chronic liver disease. Its pathogenesis varies according to the underlying etiological factors, although in most cases it develops from liver cirrhosis. The disease progression is accompanied by pathological angiogenesis, which is a prerequisite that favors the development of HCC. AIMS: This study aims at contributing to our understanding of the role of angiogenic factors in the progression of liver disease. For this purpose, we evaluate the clinical significance of serum angiogenic markers (VEGF, Ang-1, Ang-2, the angiopoietin receptor Tie1/2, HGF, and PECAM-1) first in cirrhotic and HCC patients separately, and then comparing cirrhotic patients with and without HCC. MATERIALS AND METHODS: We enrolled 62 patients, out of whom 33 were diagnosed with HCC and 29 with liver cirrhosis without signs of neoplasia. Patients underwent venous blood sampling before and after receiving treatments for the diagnosed disease. Serum markers were evaluated using ELISA assays for Tie1 and the Bio-Plex Multiplex system for the remaining ones. Biomarker levels were investigated as a function of clinical scores for disease staging (MELD and Fibrosis Index, FI). RESULTS: In cirrhotic patients, Ang-1 and Ang-2 correlate with MELD (ρAng-1 = -0.73, p = 2E-5) and FI (ρAng-1 = -0.52, p = 7E-3, ρAng-2 = 0.53, p = 3E-3). A reduction of Ang-2 levels (p = 0.047) and of the Ang-2/Ang-1 ratio (p = 0.031) is observed in cirrhotic patients diagnosed with viral hepatitis after antiviral treatments. In HCC patients, Ang-1 negatively correlates with FI (ρ = -0.63, p = 1E-4), and PECAM-1 positively correlates with MELD (ρ = 0.44, p = 0.01). A significant Ang-1 reduction was observed in deceased patients during the study compared to ones who survived (p = 0.01). In HCC patients, VEGF levels were increased after tumor treatment (p = 0.037). Notably, HGF levels in cirrhotic patients with HCC are significantly raised (p = 0.017) compared to that in those without HCC. CONCLUSIONS: Our results suggest that serum angiogenic markers, with emphasis on Ang-1/2, can contribute to the development of quantitative tools for liver disease staging and therapy monitoring. The comparison between cirrhotic patients with and without HCC suggests that HGF levels are potentially useful for monitoring the insurgence of HCC after a cirrhosis diagnosis. High Ang-1 levels in HCC patients appear to have a protective role as well as prognostic significance.

COVID-19 and hepatic involvement: The liver as a main actor of the pandemic novel.

In the natural history of SARS-CoV-2 infection, liver injury is frequent but quite mild and it is defined as any liver damage occurring during disease progression and treatment of infection in patients with or without pre-existing liver diseases. The underlying mechanisms for hepatic injury in patients with COVID-19 are still unclear but the liver damage in SARS-CoV-2 infection seems to be directly caused by virus-induced cytopathic effects. In this review, we will summarize all data of updated literature, regarding the relationship between SARS-CoV-2 infection, acute response and liver involvement. An overview will be given on liver injury, liver transplant and the possible consequences of COVID-19 in patients with pre-existing liver diseases.

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.

Immunological Role of IgG Subclasses.

The loss of tolerance to self-antigens is the unequivocal "red line" of autoimmunity: both development of autoreactive T and B cells and production of polyclonal autoantibodies represent seminal keys to the pathogenesis of protean autoimmune diseases. Most of these autoantibodies are immunoglobulins G (IgG), functionally distinguished in four subclasses named IgG1, IgG2, IgG3, and IgG4, due to structural differences in the hinge and heavy chain constant regions. Different studies analyzed serum levels of IgG subclasses in the course of different disorders, showing that they might have a pathogenic role by regulating interactions among immunoglobulins, Fc-gamma receptors, and complement. To date, the mechanisms promoting different IgG subclasses distribution during the natural history of most autoimmune diseases remain somewhat unclear. Evidence from the medical literature shows that the serum IgG profile is peculiar for many autoimmune diseases, suggesting that different subclasses could be specific for the underlying driving autoantigens. A better knowledge of IgG subsets may probably help to elucidate their pathological task, but also to define their relevance for diagnostic purposes, patients' personalized management, and prognosis assessment.

Immune dyscrasia in adult growth hormone deficiency: Evaluation of hemolytic complement activity (CH50) and IgG subclasses.

CH50 is a screening assay for the activation of the classical complement pathway, the immunoglobulins-mediated one, activated in several inflammatory diseases. Adult growth hormone deficiency (aGHD) is recognized as a chronic inflammatory condition, although poorly evaluated under the profile of inflammatory biomarkers. The aim of this case-control observational study is to analyze CH50 and immunoglobulins G (IgG) subclasses production in aGHD, comparing this condition to healthy controls. 38 subjects were included and divided as follows: aGHD (n = 18, 6 females and 12 males); healthy controls (n = 20, 10 females and 10 males). GHD was diagnosed with dynamic test using Growth Hormone-Releasing Hormone (GHRH 50 µg i.v. + arginine 0,5 g/Kg), with a peak GH response < 9 µg/L when BMI was <30 kg/m2 or < 4 µg/L when BMI was >30 kg/m2. The two groups were evaluated for hormonal and metabolic parameters, CH50 and IgG subtypes. IgG1 and IgG2 were significantly higher in controls than in aGHD, while IgG3 and IgG4 showed a trend to higher levels in controls, although not significant. Furthermore, CH50 levels were significantly higher in aGHD. These data substantiate the hypothesis of a dyscrasia in IgG subclasses production in aGHD. As IgG levels decrease, CH50 levels do not.