Search

Chronic liver diseases (CLD) stem from various causes and lead to a gradual progression that ultimately may result in fibrosis and eventually cirrhosis. This process is typically prolonged and asymptomatic, characterized by the complex interplay among various cell types, signaling pathways, extracellular matrix components, and immune responses. With the prevalence of CLD increasing, diagnoses are often delayed, which leads to poor prognoses and in some cases, the need for liver transplants. Consequently, there is an urgent need for the development of novel, non-invasive methods for the diagnosis and monitoring of CLD. In this context, serum biomarkers-safer, repeatable, and more acceptable alternatives to tissue biopsies-are attracting significant research interest, although their clinical implementation is not yet widespread. This review summarizes the latest advancements in serum biomarkers for detecting hepatic fibrogenesis and advocates for concerted efforts to consolidate current knowledge, thereby providing patients with early, effective, and accessible diagnoses that facilitate personalized therapeutic strategies.

Skin is the forestage for a series of many-sided functions of tumor necrosis factor-alpha (TNF-α), a proinflammatory cytokine with staggering versatility and sizable implications for tissue homeostasis, immune responses, angiogenesis, apoptosis, local and systemic inflammation. An aberrant TNF-α-mediated crosstalk has been linked to the pathogenesis of acute and chronic skin inflammatory diseases, and indeed, TNF-α dysregulation can contribute to the development and progression of psoriasis, vitiligo, local damage following exposition to ultraviolet light radiations, cutaneous lupus erythematosus, and acne vulgaris. Therapies that target TNF-α are conspicuously used in the treatment of different skin disorders, aiming to modulate the in vivo immune functions triggered by many cutaneous cells, including keratinocytes, mast cells, or Langerhans cells, and reduce inflammation taking place within the skin. Herein, we focus on the key relationships between TNF-α and distinct skin non-neoplastic inflammatory or physiologic conditions, showing that a natural induction of TNF-α may have a protective significance but that TNF-α overproduction may be harmful or even lethal. Many questions remain unraveled in the therapeutic practice, and caution should be exercised due to eventual backlashes exerted by TNF-α in maintaining skin health or in provoking skin disease.

Introduction: An aberrant immune response involving yet unidentified environmental and genetic factors plays a crucial role in triggering Kawasaki disease (KD). Aims: The aim of this study was to assess general and laboratory data at the onset of KD in a single-center cohort of children managed between 2003 and 2023 and retrospectively evaluate any potential relationship with the development of KD-related cardiovascular abnormalities (CVAs). Patients and methods: We took into account a total of 65 consecutive children with KD (42 males, median age: 22 months, age range: 2-88 months) followed at the Department of Life Sciences and Public Health in our University; demographic data, clinical signs, and laboratory variables at disease onset, before IVIG infusion, including C-reactive protein, hemoglobin, white blood cell (WBC) count, neutrophil count, platelet count, aminotransferases, natremia, albumin, total bilirubin, and 25-hydroxyvitamin D were evaluated. Results: Twenty-one children (32.3% of the whole cohort) were found to have echocardiographic evidence of CVAs. Univariate analysis showed that diagnosis of KD at <1 year or >5 years was associated with CVAs ( p = 0.001 and p = 0.01, respectively); patients with CVAs had a longer fever duration and mostly presented atypical or incomplete presentations. Interestingly, all patients with CVAs had lower levels of vitamin D (less than 30 mg/dL, p = 0.0001) and both higher WBC and higher neutrophil counts than those without CVAs ( p = 0.0001 and p = 0.01, respectively). Moreover, blood levels of albumin were significantly lower in KD patients with CVAs compared to those without (11/21, 52% versus 13/44, 30%, p = 0.02). Multiple logistic regression with correction for sex showed that serum vitamin D < 30 ng/mL, WBC count > 20.000/mm 3 , and age > 60 months at KD onset were the only independent factors statistically associated with CVAs. Conclusions: Hypovitaminosis D, WBC count over 20.000/mm 3 , and age above 5 years at KD onset emerged as independent factors statistically associated with the occurrence of CVAs.

Introduction: Systemic sclerosis (SSc) is characterized by microvascular damage of skin and internal organs with chronic hypoxia and release of cytokines and hormones such as neutrophil gelatinase-associated lipocalin (NGAL), fibroblast growth factor-23 (FGF-23) and Klotho. Aim of the study was to evaluate FGF-23, Klotho and NGAL serum levels in SSc patients and healthy controls (HC) and to evaluate serum levels changes of FGF-23, Klotho and NGAL after Iloprost., Methods: Twenty-one SSc patients and 20 HC were enrolled. In SSc patients, peripheral venous blood samples were collected at the first day before the autumn Iloprost infusion (t0), 60 min (t1) and 14 days after Iloprost infusion (t2)., Results: SSc patients had higher serum level of FGF-23 [18.7 ± 6.4 pg/ml versus 3.6 ± 2.2 pg/ml, p < 0.001], Klotho [5.1 ± 0.8 pg/ml versus 2.3 ± 0.6 pg/ml, p < 0.001] and NGAL [20.9 ± 2.6 pg/ml versus 14.5 ± 1.7 pg/ml, p < 0.001] than HC. Iloprost infusion reduces serum level of FGF-23 (18.7 ± 6.4 pg/ml versus 10.4 ± 5.5 pg/ml, p < 0.001), Klotho (5.1 ± 0.8 pg/ml versus 2.5 ± 0.6 pg/ml, p < 0.001) and NGAL (20.9 ± 2.6 pg/ml versus 15.1 ± 2.3 pg/ml, p < 0.001) between t0 and t1. The Iloprost infusion reduces serum level of FGF-23 (18.7 ± 6.4 pg/ml versus 6.6 ± 5.1 pg/ml), Klotho (5.1 ± 0.8 pg/ml versus 2.3 ± 0.4 pg/ml) and NGAL (20.9 ± 2.6 pg/ml versus 15.5 ± 1.9 pg/ml) between t0 and t2., Conclusions: SSc patients had higher FGF-23, Klotho and NGAL than HC. Iloprost reduces serum levels of FGF-23, Klotho and NGAL., (© 2024. The Author(s).)

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated disorder affecting the peripheral nervous system. Despite the established diagnostic criteria, monitoring disease activity and treatment remains challenging. To address this limitation, we investigated serum neurofilament light chain (sNfL) and serum free light chains (sFLCs) as potential biomarkers. A total of 32 CIDP patients undergoing immunoglobulin therapy and 32 healthy controls enrolled in the present study, and agreed to have their blood plasma sNfL and sFLCs analyzed, while CIDP severity was assessed through the modified Rankin Scale (mRS) and the Overall Neuropathy Limitations Scale (ONLS). In line with the immunoglobulin treatment aimed at limiting neuronal damage administered to the majority of patients, sNfL levels did not exhibit significant differences between the two groups. However, CIDP patients showed significantly elevated sFLC and sFLC ratios, while the marker levels did not correlate with the clinical scores. The study confirms the potential of sFLCs as a sensitive biomarker of inflammatory processes in CIDP. Additionally, the present study results regarding neurofilaments strengthen the role of sNfL in monitoring CIDP treatments, confirming the effectiveness of immunoglobulin therapy. Overall, our results demonstrate how combining these markers can lead to better patient characterization for improved treatment.

Renal involvement is a common occurrence in patients with immuno-rheumatological diseases (IRDs). Several instances of glomerulonephritis (GN) occur in the setting of IRD and complicate the clinical course of an underlying condition. The aim of this study was to observe the spectrum of nephropathies according to age, kidney function, history of IRD at the time of biopsy, and histopathological kidney diagnosis. We evaluated data relating to 699 consecutive kidney native biopsies (female 52.1%) with a median age of 48 years (IQR 34-62) performed in adult patients collected over 15 years. The study population was divided into three groups: patients with kidney histological findings correlated to underlying IRD (Group 1), patients with kidney histological findings not correlated to underlying IRD (Group 2), and patients with kidney histological findings compatible with "de novo" IRD (absent in personal medical history) (Group 3). Kidney involvement related to IRD was found in 25.2% of patients. Group 1 was mostly represented by lupus nephritis (76.6%), with a younger age than Group 3 ( p < 0.001) and by a higher percentage of females than other groups ( p < 0.001). Group 3 was the most represented by microscopic polyangiitis (50.8%) when compared with the other two groups ( p < 0.001). Acute nephritic syndrome ( p < 0.001), acute kidney injury (AKI), and abnormal urinalysis ( p < 0.001) were more represented in Group 3 than the other groups. In conclusion, IRDs are characterized by different clinical presentations and heterogeneous histological findings. Kidney biopsy remains fundamental to achieving the correct diagnosis and starting targeted therapy.

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-1β 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.

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., (© 2023. The Author(s).)

Systemic sclerosis (SSc) patients have an increased frequency of CD21 low 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 CD21 low B cells, IL-4, and IL-21 on joint involvement in a cohort of 52 SSc patients. The DAS28-ESR was correlated with CD21 low 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 CD21 low 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 CD21 low 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 CD21 low 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.

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.

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 (F E NO), 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 FEV 1 /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)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Basile, Santini, Napodano, Macis, Pocino, Gulli, Malerba, Bush, Adcock and Montuschi.)

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 ® .

Objectives: To assess serum immunoglobulin G (IgG) subclasses in a cohort of systemic sclerosis (SSc) patients and to evaluate the influence of IgG subclasses in the main complications of the disease., Methods: The serum level of IgG subclasses was evaluated in 67 SSc patients and 48 healthy controls (HC), matched for sex and age. Serum samples were collected and measured IgG1-4 subclasses by turbidimetry., Results: SSc patients had lower median total IgG [9.88 g/l (IQR 8.18-11.42 g/l) vs. 12.09 g/l (IQR 10.24-13.54 g/l), p < 0.001], IgG1 [5.09 g/l (IQR 4.25-6.38 g/l) vs. 6.03 g/l (IQR 5.39-7.90 g/l), p < 0.001], and IgG3 [0.59 g/l (IQR 0.40-0.77 g/l) vs. 0.80 g/l (IQR 0.46-1 g/l), p < 0.05] serum levels compared to HC. The logistic regression analysis showed IgG3 as the only variable associated with the diffusing capacity of the lung for carbon monoxide (DLco) ≤60% of the predicted [OR 9.734 (CI 95%: 1.312-72.221), p < 0.05] and modified Rodnan skin score (mRSS) [OR 1.124 (CI 95%: 1.019-1.240), p < 0.05], anti-topoisomerase I [OR 0.060 (CI 95%: 0.007-0.535), p < 0.05], and IgG3 [OR 14.062 (CI 95%: 1.352-146.229), p < 0.05] as variables associated with radiological interstitial lung disease (ILD)., Conclusion: SSc patients have reduced levels of total IgG and an altered IgG subclass distribution compared to HC. Moreover, SSc patients show different serum IgG subclasses profiles according to the main involvement of the disease.

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 -β), 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.

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 µm. 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 µm) 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 µm, 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 derived from cancer cells with increasing invasiveness and metastatic potential, suggesting that the average secondary structure content in EVs can be correlated with cell malignancy., Conclusions: Thanks to the high protein sensitivity and the possibility to work with small sample volumes-two key features for ultrasensitive detection of extracellular vesicles- our lab-on-chip can positively impact the development of novel laboratory medicine methods for the molecular characterization of EVs., (© 2022. The Author(s).)

COVID-19 continues to afflict the global population, causing several pathological diseases and exacerbating co-morbidities due to SARS-CoV-2's high mutation. Recent interest has been devoted to some neuronal manifestations and to increased levels of Nerve Growth Factor (NGF) and Brain-derived Neurotrophic Factor (BDNF) in the bloodstream during SARS-CoV-2 infection, neurotrophins that are well-known for their multifactorial actions on neuro-immune-endocrine and visual functions. Nineteen (19) patients were enrolled in this monocentric prospective study and subjected to anamnesis and biosamples collection (saliva and blood) at hospitalization (acute phase) and 6 months later (remission phase). NGF and BDNF were quantified by ELISA, and biochemical data were related to biostrumental measurements. Increased NGF and BDNF levels were quantified in saliva and serum during the acute phase of SARS-CoV-2 infection (hospitalized patients), and reduced levels were observed in the next 6 months (remission phase), never matching the baseline values. Salivary and circulating data would suggest the possibility of considering sera and saliva as useful matrices for quickly screening neurotrophins, in addition to SARS-CoV2 antigens and RNA. Overall, the findings described herein highlight the importance of NGF and BDNF as dynamic biomarkers for monitoring disease and reinforces the possibility of using saliva and sera for quick, non-invasive COVID-19 screening.

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 (β = −0.132, p < 0.01). The reduction in serum adiponectin levels is correlated with skin thickness.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nardini, Ciasca, Lauria, Rossi, Di Giacinto, Romanò, Di Santo, Papi, Palmieri, Perini, Basile, Alcaro, Di Stasio, Bizzarro, Masullo and De Spirito.)

Objective: to assess the influence of SARS-CoV-2 mRNA vaccine on B-cell phenotypes in systemic sclerosis (SSc)., Methods: peripheral blood B-cell subpopulations were evaluated before (t1) and 3 months (t3) after the second dose of vaccine in 28 SSc patients. Peripheral blood B-cell subpopulations were evaluated in 21 healthy controls (HCs) only at t1. Anti-spike IgG levels were evaluated at t3 in both cohorts., Results: SSc patients presented higher naive, double-negative, and CD21 low B cells compared to HCs. IgM-memory and switched-memory B cells were lower in SSc patients than HCs. No differences in anti-spike IgG levels after vaccination were observed between SSc patients and HCs. Anti-spike IgG levels after vaccination were lower in SSc patients with increased CD21 low B cells at baseline compared to SSc patients with normal CD21 low B cells. A positive correlation was found between IgG levels and naive B cells. A negative linear correlation was shown between IgG levels and IgM-memory, switched-memory, double-negative, and CD21 low B cells., Conclusions: SARS-CoV-2 mRNA vaccine response is normal in SSc patients not undergoing immunosuppressive therapy. The normal number of naive B cells is a positive marker of antibody response. The increased percentage of CD21 low B cells represents a negative marker of antibody response.

Extracellular vesicles (EVs) are abundantly released into the systemic circulation, where they show remarkable stability and harbor molecular constituents that provide biochemical information about their cells of origin. Due to this characteristic, EVs are attracting increasing attention as a source of circulating biomarkers for cancer liquid biopsy and personalized medicine. Despite this potential, none of the discovered biomarkers has entered the clinical practice so far, and novel approaches for the label-free characterization of EVs are highly demanded. In this regard, Fourier Transform Infrared Spectroscopy (FTIR) has great potential as it provides a quick, reproducible, and informative biomolecular fingerprint of EVs. In this pilot study, we investigated, for the first time in the literature, the capability of FTIR spectroscopy to distinguish between EVs extracted from sera of cancer patients and controls based on their mid-IR spectral response. For this purpose, EV-enriched suspensions were obtained from the serum of patients diagnosed with Hepatocellular Carcinoma (HCC) of nonviral origin and noncancer subjects. Our data point out the presence of statistically significant differences in the integrated intensities of major mid-IR absorption bands, including the carbohydrate and nucleic acids band, the protein amide I and II bands, and the lipid CH stretching band. Additionally, we used Principal Component Analysis combined with Linear Discriminant Analysis (PCA-LDA) for the automated classification of spectral data according to the shape of specific mid-IR spectral signatures. The diagnostic performances of the proposed spectral biomarkers, alone and combined, were evaluated using multivariate logistic regression followed by a Receiving Operator Curve analysis, obtaining large Areas Under the Curve (AUC = 0.91, 95% CI 0.81-1.0). Very interestingly, our analyses suggest that the discussed spectral biomarkers can outperform the classification ability of two widely used circulating HCC markers measured on the same groups of subjects, namely alpha-fetoprotein (AFP), and protein induced by the absence of vitamin K or antagonist-II (PIVKA-II).

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.

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.

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 (ρ Ang-1 = -0.73, p = 2E-5) and FI (ρ Ang-1 = -0.52, p = 7E-3, ρ Ang-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.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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., (© 2021 British Society for Immunology.)

The ongoing outbreak of coronavirus disease 2019 (COVID-19), which impairs the functionality of several organs, represents a major threat to human health. One of the hardest challenges in the fight against COVID-19 is the development of wide-scale, effective, and rapid laboratory tests to control disease severity, progression, and possible sudden worsening. Monitoring patients in real-time is highly demanded in this pandemic era when physicians need reliable and quantitative tools to prioritize patients' access to intensive care departments. In this regard, salivary biomarkers are extremely promising, as they allow for the fast and non-invasive collection of specimens and can be repeated multiple times., Methods: We compare salivary levels of immunoglobulin A subclasses (IgA1 and IgA2) and free light chains (kFLC and λFLC) in a cohort of 29 SARS-CoV-2 patients and 21 healthy subjects., Results: We found that each biomarker differs significantly between the two groups, with p -values ranging from 10 -8 to 10 -4 . A Receiving Operator Curve analysis shows that λFLC level is the best-suited candidate to discriminate the two groups (AUC = 0.96), with an accuracy of 0.94 (0.87-1.00 95% CI), a precision of 0.91 (0.81-1.00 95% CI), a sensitivity of 1.00 (0.96-1.00 95% CI), and a specificity of 0.86 (0.70-1.00 95% CI)., Conclusion: These results suggest λFLC as an ideal indicator of patient conditions. This hypothesis is strengthened by the consideration that the λFLC half-life (approximately 6 h) is significantly shorter than the IgA one (21 days), thus confirming the potential of λFLC for effectively monitoring patients' fluctuation in real-time.

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/m 2 or < 4 μg/L when BMI was >30 kg/m 2 . 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., (Copyright © 2020. Published by Elsevier Masson SAS.)

The use of rituximab (RTX), an anti-CD20 monoclonal antibody (Ab), in refractory myasthenia gravis (MG) is associated with a better response in patients with Abs to the muscle-specific tyrosine kinase (MuSK) than in other MG subgroups. Anti-MuSK Abs are mostly IgG4 with proven pathogenicity and positive correlation with clinical severity. The rapid and sustained response to RTX may be related to MuSK Ab production by short-lived Ab-secreting cells derived from specific CD20 + B cells. Here, we investigated the long-term effects of RTX in nine refractory MuSK-MG patients with a follow-up ranging from 17 months to 13 years. In patients' sera, we titrated MuSK-specific IgG (MuSK-IgG) and MuSK-IgG4, along with total IgG and IgG4 levels. Optimal response to RTX was defined as the achievement and maintenance of the status of minimal manifestations (MM)-or-better together with a ≥ 50% steroid reduction, withdrawal of immunosuppressants, and no need for plasma-exchange or intravenous immunoglobulin. After a course of RTX, eight patients improved, with optimal response in six, while only one patient did not respond. At baseline, MuSK-IgG and MuSK-IgG4 serum titers were positive in all patients, ranging from 2.15 to 49.5 nmol/L and from 0.33 to 46.2 nmol/L, respectively. MuSK Abs mostly consisted of IgG4 (range 63.80-98.86%). RTX administration was followed by a marked reduction of MuSK Abs at 2-7 months and at 12-30 months ( p < 0.02 for MuSK-IgG and p < 0.01 for MuSK-IgG4). In patients with a longer follow-up, MuSK Ab titers remained suppressed, paralleling clinical response. In the patient who achieved long-term complete remission, MuSK-IgG4 was no longer detectable within 2 years, while MuSK-IgG remained positive at very low titers up to 10 years after RTX. In the patient who did not respond, MuSK-IgG and MuSK-IgG4 remained unchanged. In this patient series, total IgG and IgG4 transiently decreased ( p < 0.05) at 2-7 months after RTX. The different trends of reduction between MuSK-IgG4 and total IgG4 after RTX support the view that short-lived Ab-secreting cells are the main producers of MuSK Abs. The ratio between short-lived Ab-secreting cells and long-lived plasma cells may influence the response to RTX, and B-cell severe depletion may reduce self-maintaining autoimmune reactivity., (Copyright © 2020 Marino, Basile, Spagni, Napodano, Iorio, Gulli, Todi, Provenzano, Bartoccioni and Evoli.)

Several reports have highlighted the abnormal increments of serum immunoglobulin free light chains (FLCs) in the course of systemic autoimmune rheumatic diseases (SARD), but a comparative analysis among different conditions is still lacking. A strong association between elevated FLC and hepatitis C virus (HCV)-related mixed cryoglobulinaemia (HCVMC) has been well established. Here, we aimed to analyse serum FLC levels in patients with four different SARD in comparison with HCVMC. Using a turbidimetric assay, free κ and λ chains were quantified in sera from 198 SARD patients (37 rheumatoid arthritis, RA; 47 systemic lupus erythematosus, SLE; 52 anti-phospholipid syndrome, APS; 62 primary Sjogren's syndrome, pSS), 62 HCVMC and 50 healthy blood donors (HD). All patient groups showed increased κ levels when compared to HD: 33·5 ± 2·6 mg/l in HCVMC, 26·7 ± 2·3 mg/l in RA, 29·7 ± 1·9 mg/l in SLE, 23·8 ± 1·1 mg/l in APS, 24·2 ± 1·1 mg/l in pSS; 10·1 ± 0·6 mg/l in HD. Free λ levels displayed a significant increase only for HCVMC (20·4 ± 1·4 mg/l) and SLE (18·4 ± 1·0 mg/l) compared to HD (13·6 ± 0·9 mg/l). The increase of κ compared to λ takes into account a κ /λ ratio of 1·6 for all groups. Our results substantially analyse and strengthen the association between FLC and SARD focusing the questions regarding their role in the pathogenesis and diagnosis of human diseases. Unfortunately, the biochemical differences distinguishing normal from pathological FLC have not been identified. Production of different isotypes is probably connected to still-unknown pathways., (© 2019 British Society for Immunology.)

Background: Serological levels of free immunoglobulin light chains (FLCs), produced in excess of heavy chains during synthesis of immunoglobulins by plasma cells, can be considered a direct marker of B cell activity in different systemic inflammatory-autoimmune conditions and may represent a useful predictor of rituximab (RTX) therapeutic efficacy, as reported for rheumatoid arthritis and systemic lupus erythematosus. Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction with antibodies (abs) targeting the acetylcholine receptor (AChR) or the muscle-specific tyrosine kinase (MuSK), inducing muscle weakness and excessive fatigability. As MG course may be remarkably variable, we evaluated the possible use of FLCs as biomarkers of disease activity., Subjects and Methods: We assessed FLC levels in 34 sera from 17 AChR-MG and from 13 MuSK-MG patients, in comparison with 20 sera from patients with systemic autoimmune rheumatic diseases and 18 from healthy blood donors, along with titers of specific auto-abs and IgG subclass distribution., Results: We found a statistically significant increase in free κ chains in both AChR- and MuSK-MG patients, while free λ chain levels were increased only in AChR-MG. We also observed a significant reduction of both free κ and λ chains in 1/4 MuSK-MG patients along with specific abs titer, two months after RTX treatment., Conclusions: From our data, FLCs appear to be a sensitive marker of B cell activation in MG. Further investigations are necessary to exploit their potential as reliable biomarkers of disease activity.

Background: Serum free light chains detection assays are consistently meeting greater interest for the diagnosis and monitoring of monoclonal gammopathies and plasma cell dyscrasias. Nowadays, there are neither standardized methods nor reference material for the determination of free light chains; for this reason, it is important to compare two different assays used in clinical laboratory., Methods: We evaluated 300 serum samples from patients with B-cell disorders and compared the analytical performances of both assay. Each test was assayed on both testing platforms (Siemens Dade Behring BN II Nephelometer and SPAPLUS by The Binding Site). κ/λ ratios were determined and compared. Results were analyzed by Passing-Bablok and Bland-Altman plots to evaluate comparability of the two techniques and to determine bias., Results: The reproducibility of both assays is acceptable, reaching minimum and desirable analytical goals derived from biological variability. However, values are not interchangeable between systems. This study shows that the two systems do not allow results to be transferred from one method to the other even if they display good agreement., Conclusion: Our study highlights the importance of elaborating an international standard for free light chains quantification in order to offer homogeneous results as well as guarantee harmonization of values among laboratories. Moreover, the assays should be validated in specific patient groups to determine that they are clinically fit for purpose., (© 2017 Wiley Periodicals, Inc.)

Vitamin D deficiency has been reported to be a negative prognostic factor in elderly patients with aggressive B-cell lymphomas. In vitro data suggest that vitamin D supplementation may enhance rituximab-mediated cytotoxicity. We prospectively assessed 25-hydroxyvitamin D [25(OH)D] levels at diagnosis in a cohort of 155 patients with aggressive B-cell lymphomas of whom 128 had diffuse large B-cell lymphoma (DLBCL) not otherwise specified. 25(OH)D levels were deficient (<20 ng/mL) in 105 (67%), insufficient (20-29 ng/mL) in 32 (21%), and normal (≥30 ng/mL) in 18 (12%) patients with a seasonal variation. Patient characteristics associated with lower 25(OH)D levels were poor performance status, overweight, B-symptoms, elevated LDH, lower albumin and hemoglobin levels. As a result of a change in practice pattern, 116 patients received vitamin D3 (cholecalciferol) supplementation that included a loading phase with daily replacement and subsequent maintenance phase with a weekly dose of 25,000 IU until end of treatment. This resulted in a significant increase in 25(OH)D levels, with normalization in 56% of patients. We analyzed the impact of 25(OH)D levels on event-free survival in patients treated with Rituximab-CHOP. 25(OH)D levels below 20 ng/mL at diagnosis and IPI were independently associated with inferior EFS. Moreover, patients with normalized 25(OH)D levels following supplementation showed better EFS than patients with persistently deficient/insufficient 25(OH)D levels. Our study provides the first evidence that achievement of normal 25(OH)D levels after vitamin D3 supplementation is associated with improved outcome in patients with DLBCL and deficient/insufficient 25(OH)D levels when receiving rituximab-based treatment., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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: 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.

Objectives: A new rapid, automatic, and sensitive screening test useful to detect cryoglobulins in serum samples is proposed., Design and Methods: The increase of turbidity during the cryoglobulin aggregation was monitored spectrophotometrically in sera from 400 patients with clinical evidence of cryoglobulinemia related disorders and 100 controls. Results were correlated to those obtained by the traditional method., Results: Kinetics of the aggregation curves were described by their maximum turbidity increase, lag time, and slope. Despite a partial correspondence between the traditional and the rapid test, patients with symptomatic cryoglobulinemia showed turbidity values significantly higher than the determined cutoff. Moreover, a functional classification of cryoglobulins is proposed., Conclusions: Due to its high reproducibility, operator independence, low cost, and results obtained within 2 hours, the rapid test can be used as a "real time" monitoring of cryoglobulinemia related diseases and for the evaluation of plasmapheresis efficacy.

Pheochromocytoma is a catecholamine-producing tumor of the sympathetic nervous system which in 4-14% of the cases may be malignant. The differentiation between benign and malignant phoechromocytomas is very difficult, the presence of metastatic lesions and gross invasion into surrounding tissues are the lonely accettable parameters to confirm malignancy. Since the patients with malignant pheochromocytomas have a very poor survival rate, care must be taken for an early detections and a careful follow up. Three cases of malignant pheochromocytoma are reported. In one of these cases the tumor was a functioning mass; the other two cases presented by a hemorrhagic complication. A careful review of the pertinent literature allows to outline the correct approach to this rare pathology as for the diagnosis and the treatment, also including the actual criteria to establish its malignancy.

Where practicable, all subjects found with onset-phase-four blood pressure of 90 mmHg or more after referral were allocated, on a random basis, to alternative antihypertensive regimes at a dosage related to response. Of those 110 receiving either guanethidine, methyldopa or debrisoquine, only the group on methyldopa had significantly reduced median blood pressure by two out of four criteria and after 6 months of therapy. It is nevertheless considered that by assessment of the total group more significant conclusions can be drawn for an eventual attempt to control hypertension on a community basis than by studies of highly selected groups treated under unusually favourable conditions.