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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.

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-alpha) and anti-inflammatory cytokines (TGF-alpha and -beta), pro-angiogenic molecules (including the Angiopoietins, HGF, PECAM-1, HIF-1 alpha, 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.

Systemic sclerosis (SSc) patients have an increased frequency of CD21low B cells and of serum interleukin-4 (IL-4) and IL-21, each possible markers of joint involvement in inflammatory arthritis. The aim of this study was to investigate the possible influence of CD21low B cells, IL-4, and IL-21 on joint involvement in a cohort of 52 SSc patients. The DAS28-ESR was correlated with CD21low B cells (r = 0.452, p < 0.001), IL-4 (r = 0.478, p < 0.001), and IL-21 (r = 0.415, p < 0.001). SSc patients with a DAS28-ESR > 3.2 had more CD21low B cells (12.65% (IQR: 7.11–13.79) vs. 5.08% (IQR: 3.76–7.45), p < 0.01), higher IL-4 levels (132.98 pg/mL (IQR: 99.12–164.12) vs. 100.80 pg/mL (IQR: 62.78–121.13), p < 0.05), and higher IL-21 levels (200.77 pg/mL (IQR: 130.13–302.41) vs. 98.83 pg/mL (IQR: 35.70–231.55), p < 0.01) than patients with a DAS28-ESR ≤ 3.2. The logistic regression analysis models showed that the DAI (OR: 2.158 (95% CI: 1.120; 4.156), p < 0.05) and CD21low B cells (OR: 1.301 (95% CI: 1.099; 1.540), p < 0.01), the DAI (OR: 2.060 (95% CI: 1.082; 3.919), p < 0.05) and IL-4 level (OR: 1.026 (95% CI: 1.006; 1.045), p < 0.01), and the DAI (OR: 1.743 (95% CI: 1.022; 2.975), p < 0.05) and IL-21 level (OR: 1.006 (95% CI: 1.000; 1.011), p < 0.05) were independently associated with a DAS28-ESR > 3.2. An elevated CD21low B cell percentage, IL-4 level, and IL-21 level was associated with higher articular disease activity in patients, suggesting a possible role in the pathogenesis of SSc joint involvement.

Objective: Chronic Hepatitis C virus (HCV) infection can cause severe extrahepatic manifestations, such as mixed cryoglobulins (MC), up to the development of B cell nonHodgkin's lymphoma (B-NHL). Mechanisms transforming of HCV infection into lymphoproliferative and/or autoimmune disorders are still poorly understood. In course of HCV infection, the sustained virus-driven antigenic stimulation may probably induce a B-cell clonal expansion. Measurements of serum free light chains (FLCs) levels, considered as a direct marker of B cell activity, are analyzed with increasing interest in clinical practice, for diagnosis, monitoring and follow-up of plasma cell dyscrasia. Syndecan-1 (CD138) is a transmembrane heparan sulfate proteoglycan expressed and actively shed by most myeloma cells. Membrane CD138 represents the major receptor protein for HCV attachment to the hepatocyte surface and high levels of circulating sCD138 levels are detected in patients at early stage of B-cell chronic lymphocytic leukemia. This study is aimed to evaluate sCD138 and FLC levels as diagnostic biomarkers of HCV-related MC with B-NHL. Patients and Methods: We enrolled 35 HCV-MC-NHL patients, characterized for the specific type of cryoglobulins, and 25 healthy blood donors (HBD) as negative control. Serum sCD138 levels were determined using ELISA kits specific for human sCD138. Serum FLCs were assessed by means of the turbidimetric assay. Results: We found that serum levels of sCD138, as well as FLCs, were significantly higher in patients than in HBD (p<0.001). Conclusions: In a greement with the definition of HCV-driven lymphoproliferative disorders as the consequence of a multifactorial and multistep pathogenetic process, we suggest that sCD138 and FLCs could be considered putative independent markers of worsening progression of the disease.

Background: Extracellular Vesicles (EVs) are sub-micrometer lipid-bound particles released by most cell types. They are considered a promising source of cancer biomarkers for liquid biopsy and personalized medicine due to their specific molecular cargo, which provides biochemical information on the state of parent cells. Despite this potential, EVs translation process in the diagnostic practice is still at its birth, and the development of novel medical devices for their detection and characterization is highly required. Results: In this study, we demonstrate mid-infrared plasmonic nanoantenna arrays designed to detect, in the liquid and dry phase, the specific vibrational absorption signal of EVs simultaneously with the unspecific refractive index sensing signal. For this purpose, EVs are immobilized on the gold nanoantenna surface by immunocapture, allowing us to select specific EV sub-populations and get rid of contaminants. A wet sample-handling technique relying on hydrophobicity contrast enables effortless reflectance measurements with a Fourier-transform infrared (FTIR) spectro-microscope in the wavelength range between 10 and 3 mu m. In a proof-of-principle experiment carried out on EVs released from human colorectal adenocarcinoma (CRC) cells, the protein absorption bands (amide-I and amide-II between 5.9 and 6.4 mu m) increase sharply within minutes when the EV solution is introduced in the fluidic chamber, indicating sensitivity to the EV proteins. A refractive index sensing curve is simultaneously provided by our sensor in the form of the redshift of a sharp spectral edge at wavelengths around 5 mu m, where no vibrational absorption of organic molecules takes place: this permits to extract of the dynamics of EV capture by antibodies from the overall molecular layer deposition dynamics, which is typically measured by commercial surface plasmon resonance sensors. Additionally, the described metasurface is exploited to compare the spectral response of EVs d

Red blood cells (RBCs) are characterized by a remarkable elasticity, which allows them to undergo very large deformation when passing through small vessels and capillaries. This extreme deformability is altered in various clinical conditions, suggesting that the analysis of red blood cell (RBC) mechanics has potential applications in the search for non-invasive and cost-effective blood biomarkers. Here, we provide a comparative study of the mechanical response of RBCs in patients with Alzheimer's disease (AD) and healthy subjects. For this purpose, RBC viscoelastic response was investigated using atomic force microscopy (AFM) in the force spectroscopy mode. Two types of analyses were performed: (i) a conventional analysis of AFM force-distance (FD) curves, which allowed us to retrieve the apparent Young's modulus, E; and (ii) a more in-depth analysis of time-dependent relaxation curves in the framework of the standard linear solid (SLS) model, which allowed us to estimate cell viscosity and elasticity, independently. Our data demonstrate that, while conventional analysis of AFM FD curves fails in distinguishing the two groups, the mechanical parameters obtained with the SLS model show a very good classification ability. The diagnostic performance of mechanical parameters was assessed using receiving operator characteristic (ROC) curves, showing very large areas under the curves (AUC) for selected biomarkers (AUC > 0.9). Taken all together, the data presented here demonstrate that RBC mechanics are significantly altered in AD, also highlighting the key role played by viscous forces. These RBC abnormalities in AD, which include both a modified elasticity and viscosity, could be considered a potential source of plasmatic biomarkers in the field of liquid biopsy to be used in combination with more established indicators of the pathology.

OBJECTIVE: Bence Jones proteinuria (BJP) refers to monoclonal free immunoglobulin light chains detected in urine, deriving from the clonal expansion of plasma cells in the bone marrow in patients with plasma cell dyscrasias, associated with monoclonal gammopathies of uncertain origin. This review summarizes routinely diagnostic procedures to assess BJP highlighting critical steps of preanalytical, analytical, and post-analytical phases.QUALITATIVE AND QUANTITATIVE METH-ODS: The best option for BJP detection is the first morning void urine sample and immunofixation electrophoresis detection technique (IFE) the recommended method, with the employment of specific polyvalent antisera. Other qualitative tests for a quick evaluation of BJP are currently available. Densitometric analysis per-formed on the 24-hour urine is the recommended method to quantify BJP. To overcome the 24-hour collection, it is possible to use morning urine sample and correlate the assessed value of BJP to creatininuria. In addition to the traditional ones, we here reviewed screening methods currently used to avoid false negatives and reduce the time around test (TAT), together with immunochemical quantification methods for in-creased sensitivity, after checking BJP by IFE. Mass spectrometry emerges as a new challenge in the determination of BJP.CONCLUSIONS: The employment of different based-assays methods may be useful for diagnostic purposes to improve the accuracy of BJP monitoring in monoclonal gammopathies.

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.

– OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identified in China, in December 2019 determines COronaVIrus Disease 19 (COVID-19). Whether or not the virus was present in Italy earlier the first autochthonous COVID-19 case was diagnosed is still uncertain. We aimed to identify anti-SARS-CoV-2 antibodies in sera collected from 4th November 2019 to 9th March 2020, in order to assess the possible spread of the virus in Italy earlier than the first official national diagnosis. PATIENTS AND METHODS: Anti-SARS-CoV-2 antibodies were evaluated in retrospective serum samples from 234 patients with liver diseases (Hep-patients) and from 56 blood donors (BDs). We used two rapid serologic tests which were confirmed by a validated chemoluminescence assay. RESULTS: Via rapid tests, we found 10/234 (4.3%) IgG-positive and 1/234 (0.4%) IgM-positive cases in the Hep-patient group. Two/56 (3.6%) IgG-positive and 2/56 (3.6%) IgM-positive cases were detected in BD group. Chemoluminescence confirmed IgG-positivity in 3 Hep-patients and 1 BD and IgM-positivity in 1 Hep-patient. RNAemia was not detected in any of the subjects, rendering the risk of transfusion transmission negligible. CONCLUSIONS: Our results suggest an early circulation of SARS-CoV-2 in Italy, before the first COVID-19 cases were described in China. Rapid tests have multiple benefits; however, a confirmation assay is required to avoid false positive results.

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.

Metabolic diseases are associated with a higher risk of a severer coronavirus disease 2019 (COVID-19) course, since fatty liver is commonly associated with metabolic disorders, fatty liver itself is considered as a major contributor to low-grade inflammation in obesity and diabetes. Recently a comprehensive term, metabolic (dysfunction) associated fatty liver disease (MAFLD), has been proposed. The hepatic inflammatory status observed in MAFLD patients is amplified in presence of severe acute respiratory syndrome coronavirus 2 infection. Intestinal dysbiosis is a powerful activator of inflammatory mediator production of liver macrophages. The intestinal microbiome plays a key role in MAFLD progression, which results in non-alcoholic steatohepatitis and liver fibrosis. Therefore, patients with metabolic disorders and COVID-19 can have a worse outcome of COVID-19. This literature review attempts to disentangle the mechanistic link of MAFLD from COVID-19 complexity and to improve knowledge on its pathophysiology.

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.

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.

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.

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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.

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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.