NLRP3 Inflammasome Involvement in Heart, Liver, and Lung Diseases-A Lesson from Cytokine Storm Syndrome.

Inflammation and inflammasomes have been proposed as important regulators of the host-microorganism interaction, playing a key role in morbidity and mortality due to the coronavirus disease 2019 (COVID-19) in subjects with chronic conditions and compromised immune system. The inflammasome consists of a multiprotein complex that finely regulates the activation of caspase-1 and the production and secretion of potent pro-inflammatory cytokines such as IL-1ß and IL-18. The pyrin containing NOD (nucleotide-binding oligomerization domain) like receptor (NLRP) is a family of intracellular receptors, sensing patterns associated to pathogens or danger signals and NLRP3 inflammasome is the most deeply analyzed for its involvement in the innate and adaptive immune system as well as its contribution to several autoinflammatory and autoimmune diseases. It is highly expressed in leukocytes and up-regulated in sentinel cells upon inflammatory stimuli. NLRP3 expression has also been reported in B and T lymphocytes, in epithelial cells of oral and genital mucosa, in specific parenchymal cells as cardiomyocytes, and keratinocytes, and chondrocytes. It is well known that a dysregulated activation of the inflammasome is involved in the pathogenesis of different disorders that share the common red line of inflammation in their pathogenetic fingerprint. Here, we review the potential roles of the NLRP3 inflammasome in cardiovascular events, liver damage, pulmonary diseases, and in that wide range of systemic inflammatory syndromes named as a cytokine storm.

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).

Type 2 cytokines and scleroderma interstitial lung disease.

Interstitial lung disease (ILD) is a life-threatening complication of systemic sclerosis (SSc). Type 2 (Th2) cytokines play a pivotal role in airway disease. Study aim was to evaluate serum level of Th2 interleukin (IL) and chemokine in SSc-ILD. Serum levels of IL-4, IL-5, IL-11, IL-13, IL-21, IL-31 and CXCL-13 were measured by Bio-Plex Multiplex Immunoassays in 60 SSc patients and 20 healthy controls (HC). Pulmonary function tests with diffusion lung capacity for carbon monoxide (DLco) and high resolution computed tomography (HRCT) were performed in SSc patients. ILD is defined as fibrotic changes (ground glass, reticular and honeycombing), assessed by Computer-Aided Lung Informatics for Pathology Evaluation and Ratings (CALIPER) software, affecting at least 10% of the lungs. Serum levels of Th2 cytokines were higher in SSc patients than HC. A linear correlation was observed between ground glass and IL-13 (r = 0.342, p < 0.01), IL-21 (r = 0.345, p < 0.01), IL-31 (r = 0.473, p < 0.001), IL-4 (r = 0.863, p < 0.001), IL-5 (r = 0.249, p < 0.05) and peripheral blood eosinophils (r = 0.463, p < 0.001). We found a negative correlation between DLco and IL-4 (r = - 0.511, p < 0.001) and peripheral blood eosinophils (r = - 0.446, p < 0.001). In the logistic regression analysis, IL-4 is associated with DLco ≤ 60% of the predicted [OR 1.039 (CI 95%: 1.015-1.064), p < 0.001], whilst mRSS [OR 1.138 (CI 95%: 1.023-1.266), p < 0.05] and IL-4 [OR 1.017 (CI 95%: 1-1.034), p < 0.05] were associated with ILD. Th2 inflammation could play a key role in early phase of SSc-ILD.

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.

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.

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.

SMA-miRs (miR-181a-5p, -324-5p, and -451a) are overexpressed in spinal muscular atrophy skeletal muscle and serum samples.

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2-4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs. Methods: We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score). Results: Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p<10-5). Conclusions: miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients. Funding: Telethon Italia (grant #GGP12116).

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.

COVID-19, adaptative immune response and metabolic-associated liver disease.

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.

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.

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.

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.

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.

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.

Fecal calprotectin and need of multiple microbiota trasplantation infusions in Clostridium difficile infection.

BACKGROUND AND AIM: Fecal microbiota transplantation (FMT) has proven to be very effective in recurrent Clostridium difficile infection (CDI) when compared with standard antibiotic therapy. However, given the lack of validated criteria, decision regarding number and timing of infusions is currently based on the clinician's experience, severity of infection, and clinical response. We performed a longitudinal assessment of fecal calprotectin concentration (FCC) in CDI patients undergoing FMT. FCCs were correlated with the need for multiple infusions and with the clinical status of the patient. METHODS: Fecal calprotectin concentration measurement was performed just before first procedure (T0 ) and 2 (T1 ) and 5 (T2 ) days later. The need for reinfusion was accounted for in the 8 weeks following procedure, and clinical status was evaluated at the end of the given period. Both outcomes were correlated with measured FCCs. RESULTS: A total of 28 CDI patients undergoing FMT were enrolled. Median FCCs at T0 were significantly higher in patients who needed repeat FMT, 540 µg/g versus patients who underwent single FMT, 290 µg/g (P < 0.05). Differences were not significant for FCC at T1 and T2 . Regarding correlation with clinical outcome, median FCC at T0 was found to be lower in responders compared with non-responders with a trend towards statistical significance (P = 0.07). Correlation at T1 and T2 was not significant. CONCLUSIONS: The use of an easily obtainable parameter such as fecal calprotectin could possibly optimize overall management of FMT procedural framework potentially being able to immediately identify patients who may benefit from repeat infusions.

The prognostic impact of monoclonal immune globulin and free light chain secretion in diffuse large B cell lymphoma (DLBCL).

We analyzed the prognostic impact of levels of free light chains (FLC) in 106 patients with DLBCL, selecting 61 patients with a monoclonal (M) protein in serum, and 45 patients without a M protein as an IPI-matched control group. Patients with a M protein had higher levels of FLC, but these were not of prognostic significance in this group. The presence of a M protein nullified associations of κ-FLC with several laboratory parameters indicating immune system activation observed in patients without a M protein. Patients without M protein and κ-FLC >50 mg/L had a significant inferior event-free survival (p = .004). The presence of M protein of an IgM type at diagnosis was a negative outcome predictor (p = .008), while a non-IgM M protein did not significantly impact on prognosis. In conclusion, the prognostic performance of the FLC assay is altered by the presence of a M protein.

The Laboratory Role in anti-TNF Biological Therapy Era.

Along years, the advent of biological therapy widely modified treatment of rheumatic diseases and other disorders. However, many agents may elicit in anti-drug antibodies (ADAbs) upon consecutive infusions, with a loss of response. For the right strategy of a personalized medicine, the therapeutic monitoring of TNF-α inhibitors and ADAbs represents an important effort in diagnostic-therapeutic pathway, to improve overall patient management and favoring an appropriate clinical approach. A raising number of diagnostic tests have been designed to elucidate the efficacy and/or safety of a specific drug or class of drugs for a targeted patient's group. Our paper reviewed the current understanding of the immunogenicity of biological drugs employed in the treatment of inflammatory diseases underlying the laboratory role.

Sentinel biomarkers in HCV positive patients with mixed cryoglobulinemia.

BACKGROUND: Infections, autoimmunity and cancer play a role as determinants of etiology in Hepatitis C virus (HCV) related mixed cryoglobulinemia (MC). Several factors of risk have been suggested as markers of pathogenesis and progression of HCV-related MC into B cell Non-Hodgkin's Lymphoma (B-NHL). Here, we evaluated IgG subclass distribution, free light chains (FLCs) and vascular endothelial growth factor (VEGF) as a new combination of biomarkers. METHODS: We measured IgG1-4 subclasses, FLCs and VEGF levels in sera 53 from HCV-related MC, in comparison with 40 sera from HCV negative patients with rheumatoid arthritis (RA) and 30 from healthy blood donors (HBD). RESULTS: IgG3 levels were significantly higher in HCV-MC patients with a decrement of IgG2 and IgG4; FLC levels significantly increased in both MC and RA patients' groups; serological VEGF was higher in HCV-MC patients than in HBD in correlation with k and λ levels. CONCLUSION: Our results suggest that a specific IgG subclasses pattern together with raised levels of FLCs and VEGF could represent the biomarker "signature" of an inflammation multistage of acquired immune system.

Circulating tumor cell-related transcripts in blood as prognostic biomarkers of early recurrence after liver resection for colorectal metastases.

BACKGROUND: Several prognostic factors were proposed to improve early detection of recurrence after liver resection of metastases of colorectal cancer. Circulating tumor cell-related transcripts were evaluated in colorectal cancer patients with conflicting results. The aim of this study was to investigate usefulness of carcinoembryonic antigen CAM5, epidermal growth factor receptor, and ERCC1 transcripts in the bloodstream as predictive factors of recurrence in patients who underwent liver resection for metastases of colorectal cancer. METHODS: Peripheral blood was collected from 29 patients at the time of the colorectal cancer liver metastasis resection, and from 25 normal controls. Follow-up draws (FUDs) were also performed at 30 days, and 3 and 12 months since surgery. On each sample, carcinoembryonic antigen CAM5, ERCC1, and GAPDH mRNAs were examined by quantitative reverse transcription (qRT). RESULTS: Carcinoembryonic antigen transcript levels were linearly correlated to the number of spiked cells (qRT analytical limit = five cells). Among 29 patients (20 M/9 F; mean age 63 years (range 32-79), highly significant levels of carcinoembryonic antigen, if compared to the baseline, were detected in those relapsing after surgery (P <0.05). The main differences were between the 1st- and 12th-month FUDs. Significantly higher levels of carcinoembryonic antigen were also detected in patients who died from disease progression during the follow-up (as evaluated at 30 days and 90 days FUDs). CONCLUSIONS: Blood carcinoembryonic antigen-mRNA absolute copy number overtime variation can represent a valid early predictor of relapse after liver resection in colorectal liver metastases patients. Prospective studies, in the context of large clinical trials, will provide further data to also qualify ERCC1 as a predictive biomarker for decisions on therapeutic strategies.