Detection of Antibodies against the Acetylcholine Receptor in Patients with Myasthenia Gravis: A Comparison of Two Enzyme Immunoassays and a Fixed Cell-Based Assay.

The detection of serum anti-acetylcholine receptor (AChR) antibodies is currently an important tool for diagnosing myasthenia gravis (MG) since they are present in about 85% of MG patients. Many serological tests are now available. Nevertheless, results from these tests can be different in some patients. The aim of this study is to compare the sensitivity of a commercially available fixed cell-based assay (F-CBA) to that of enzyme-linked immunosorbent assay (ELISA) kits for anti-AChR detection in patients with a diagnosis of MG. Overall, 143 patients with a confirmed MG diagnosis were included in the study. The detection and measurement of serum anti-AChR antibodies were performed by three analytical methods, namely, a competitive ELISA (cELISA), an indirect ELISA (iELISA), and an F-CBA, according to the manufacturers' instructions. Anti-AChR antibody titers were positive in 94/143 (66%) using the cELISA, in 75/143 (52%) using the iELISA and in 61/143 (43%) using the F-CBA (adult and/or fetal). Method agreement, evaluated by concordant pairs and Cohen's kappa, was as follows: cELISA-iELISA: 110/143 (77%), k = 0.53 (95%CI 0.40-0.66); cELISA-F-CBA: 108/143 (76%), k = 0.53 (95%CI 0.41-0.66); iELISA-F-CBA: 121/143 (85%), k = 0.70 (95%CI 0.57-0.80). Our findings show that the cELISA has better analytical performance than the iELISA and F-CBA. However, the iELISA and F-CBA show the highest concordance.

Progressive right ventricular dysfunction and exercise impairment in patients with heart failure and diabetes mellitus: insights from the T.O.S.CA. Registry.

BACKGROUND: Findings from the T.O.S.CA. Registry recently reported that patients with concomitant chronic heart failure (CHF) and impairment of insulin axis (either insulin resistance-IR or diabetes mellitus-T2D) display increased morbidity and mortality. However, little information is available on the relative impact of IR and T2D on cardiac structure and function, cardiopulmonary performance, and their longitudinal changes in CHF. METHODS: Patients enrolled in the T.O.S.CA. Registry performed echocardiography and cardiopulmonary exercise test at baseline and at a patient-average follow-up of 36 months. Patients were divided into three groups based on the degree of insulin impairment: euglycemic without IR (EU), euglycemic with IR (IR), and T2D. RESULTS: Compared with EU and IR, T2D was associated with increased filling pressures (E/e'ratio: 15.9 ± 8.9, 12.0 ± 6.5, and 14.5 ± 8.1 respectively, p < 0.01) and worse right ventricular(RV)-arterial uncoupling (RVAUC) (TAPSE/PASP ratio 0.52 ± 0.2, 0.6 ± 0.3, and 0.6 ± 0.3 in T2D, EU and IR, respectively, p < 0.05). Likewise, impairment in peak oxygen consumption (peak VO2) in TD2 vs EU and IR patients was recorded (respectively, 15.8 ± 3.8 ml/Kg/min, 18.4 ± 4.3 ml/Kg/min and 16.5 ± 4.3 ml/Kg/min, p < 0.003). Longitudinal data demonstrated higher deterioration of RVAUC, RV dimension, and peak VO2 in the T2D group (+ 13% increase in RV dimension, - 21% decline in TAPSE/PAPS ratio and - 20% decrease in peak VO2). CONCLUSION: The higher risk of death and CV hospitalizations exhibited by HF-T2D patients in the T.O.S.CA. Registry is associated with progressive RV ventricular dysfunction and exercise impairment when compared to euglycemic CHF patients, supporting the pivotal importance of hyperglycaemia and right chambers in HF prognosis. Trial registration ClinicalTrials.gov identifier: NCT023358017.

Clinical Utility of Midregional Proadrenomedullin in Patients with COVID-19.

OBJECTIVE: The aim of the study was to assess the role of midregional proadrenomedullin (MR-proADM) in patients with COVID-19. METHODS: We included 110 patients hospitalized for COVID-19. Biochemical biomarkers, including MR-proADM, were measured at admission. The association of plasma MR-proADM levels with COVID-19 severity, defined as a requirement for mechanical ventilation or in-hospital mortality, was evaluated. RESULTS: Patients showed increased levels of MR-proADM. In addition, MR-proADM was higher in patients who died during hospitalization than in patients who survived (median, 2.59 nmol/L; interquartile range, 2.3-2.95 vs median, 0.82 nmol/L; interquartile range, 0.57-1.03; P <.0001). Receiver operating characteristic curve analysis showed good accuracy of MR-proADM for predicting mortality. A MR-proADM value of 1.73 nmol/L was established as the best cutoff value, with 90% sensitivity and 95% specificity (P <.0001). CONCLUSION: We found that MR-proADM could represent a prognostic biomarker of COVID-19.

Neurogranin as a Novel Biomarker in Alzheimer's Disease.

BACKGROUND: In this study, we investigated the possible role of 2 novel biomarkers of synaptic damage, namely, neurogranin and α-synuclein, in Alzheimer disease (AD). METHODS: The study was performed in a cohort consisting of patients with AD and those without AD, including individuals with other neurological diseases. Cerebrospinal fluid (CSF) neurogranin and α-synuclein levels were measured by sensitive enzyme-linked immunosorbent assays (ELISAs). RESULTS: We found significantly increased levels of CSF neurogranin and α-synuclein in patients with AD than those without AD. Neurogranin was correlated with total tau (tTau) and phosphorylated tau (pTau), as well as with cognitive decline, in patients with AD. Receiver operating characteristic (ROC) curve analysis showed good diagnostic accuracy of neurogranin for AD at a cutoff point of 306 pg per mL with an area under the curve (AUC) of 0.872 and sensitivity and specificity of 84.2% and 78%, respectively. CONCLUSIONS: Our findings support the use of CSF neurogranin as a biomarker of synapsis damage in patients with AD.

Correction to: Human heart shifts from IGF-1 production to utilization with chronic heart failure.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CYP27A1, CYP24A1, and RXR-α Polymorphisms, Vitamin D, and Multiple Sclerosis: a Pilot Study.

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease resulting from a complex interaction of genetic and environmental factors. Hypovitaminosis D seems to contribute to MS susceptibility as both an environmental and a genetic risk factor. The aim of our study was to investigate the association of SNPs in CYP27A1, CYP24A1, and RXR- α genes, vitamin D status, and MS risk. We performed a nested case-control study on patients with multiple sclerosis and healthy controls. Serum 25(OH)D3 levels and genotyping of CYP27A1, CYP24A1, and RXR-α -SNPs were investigated both in MS patients and in healthy controls. Serum 25(OH)D3 levels were measured by a high-performance liquid chromatography (HPLC). Molecular analysis was performed by real-time PCR. The distribution of genotypic and allelic frequencies was not significantly different between patients and controls, except for rs2248137 CYP24A1. In particular, CC genotype (C minor allele) showed a higher frequency in MS patients in comparison to healthy controls. Moreover, we observed significantly lower serum 25(OH)D3 levels in MS patients with CC genotype in comparison to MS patients with GG and GC genotype. The findings of our study suggest a role of rs2248137 CYP24A1 in multiple sclerosis risk.

The complex alteration in the network of IL-17-type cytokines in patients with hereditary angioedema.

Hereditary angioedema (HAE) is a rare autosomic-dominant disorder characterized by a deficiency of C1 esterase inhibitor which causes episodic swellings of subcutaneous tissues, bowel walls and upper airways that are disabling and potentially life-threatening. We evaluated n = 17 patients with confirmed HAE diagnosis during attack and remission state and n = 19 healthy subjects. The samples were tested for a panel of IL (Interleukin)-17-type cytokines (IL-1ß, IL-6, IL-10, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-17, IL-21, IL-22, IL-23) and transforming growth factor-beta (TGF-ß) subtypes. Data indicate that there are variations of cytokine levels in HAE subjects comparing the condition during the crisis respect to the value in the remission phase, in particular type 17 signature cytokines are increased, whereas IL-23 is unmodified and TGF-ß3 is significantly reduced. When comparing healthy and HAE subjects in the remission state, we found a significant difference for IL-17, GM-CSF, IL-21, TGF-ß1 and TGF-ß2 cytokines. These results confirm and extend our previous findings indicating that in HAE there is operating an inflammatory activation process, which involves also T helper 17 (Th17) cytokines and TGF-ß isoforms, associated with localized angioedema attacks and characterized by elevated bradykinin levels.

Association between hypovitaminosis D and systemic sclerosis: True or fake?

BACKGROUND: Vitamin D insufficiency/deficiency is considered a major factor triggering and enhancing several autoimmune disorders; hypovitaminosis D has been reported to be common in Systemic Sclerosis (SSc). Previous studies assessing vitamin D insufficiency/deficiency in SSc have been reviewed, and the relation with pathogenesis and clinical features has been examined. CONTENT: Eligibility criteria were: reporting measurement of Vitamin D serum levels in all participants and evaluating adult onset-SSc individuals as patients group. RESULTS: The association between clinical features and low hormone levels is controversial. Manifold data have shown vitamin D insufficiency/deficiency to have a potential role in the pathogenesis of disease, providing inconclusive findings. SUMMARY: Promoting the onset of SSc depends on the interaction between genetics, environment and infections. It remains a sound question whether Vitamin D insufficiency/deficiency is an environment-linked immunological heckler, making infectious agents taking root.

Fetuin-A is Associated to Serum Calcium and AHSG T256S Genotype but Not to Coronary Artery Calcification.

Vascular calcification has been recently associated to an increased cardiovascular risk and mortality. In few studies, Fetuin-A showed an association to coronary artery calcification (CAC), although the physiopathological mechanism underlying this association has not been fully established yet. Seventy-four patients with one or more cardiovascular risk factor and asymptomatic for coronary vasculopathy were included in the study. CAC was evaluated by Agatston score. Serum Fetuin-A levels were determined by ELISA. Molecular analysis of AHSG T256S gene variant (rs4918) was performed by PCR-RFLP. Serum Fetuin-A was correlated to serum calcium (r = 0,321; P = 0,018), but not to serum phosphorous. Multivariate linear regression analysis confirmed this association and showed that calcium and AHSG genotype were independent predictors of Fetuin-A (P = 0.037, P = 0.014, respectively). In particular, subjects carrying the SS genotype had lower levels of Fetuin-A and calcium (P = 0.037 and P = 0.038, respectively). When we compare subjects with CAC 0-10 with subjects with CAC > 10, we found that only age and male gender (P < 0.001, P = 0.035, respectively), but not Fetuin-A, were associated to CAC. Fetuin-A is not associated to CAC in subjects with low cardiovascular risk profile and asymptomatic for coronary vasculopathy, suggesting that in this setting Fetuin-A, although correlated to serum levels of calcium, could be not involved in mineral deposition on coronary vessels.

Visceral adiposity index (VAI) is predictive of an altered adipokine profile in patients with type 2 diabetes.

AIMS: Although there is still no clear definition of "adipose tissue dysfunction" or ATD, the identification of a clinical marker of altered fat distribution and function may provide the needed tools for early identification of a condition of cardiometabolic risk. Our aim was to evaluate the correlations among various anthropometric indices [BMI, Waist Circumference (WC), Hip Circumference (HC), Waist/Hip ratio (WHR), Body Adiposity Index (BAI) and Visceral adiposity Index (VAI)] and several adipocytokines [Visfatin, Resistin, Leptin, Soluble leptin receptors (sOB-R), Adiponectin, Ghrelin, Adipsin, PAI-1, vascular endothelial growth factor (VEGF), Hepatocyte growth factor (HGF) TNF-α, hs-CRP, IL-6, IL-18] in patients with type 2 diabetes (DM2). MATERIALS AND METHODS: Ninety-one DM2 patients (age: 65.25 ± 6.38 years; 42 men and 49 women) in stable treatment for the last six months with metformin in monotherapy (1.5-2 g/day) were cross-sectionally studied. Clinical, anthropometric, and metabolic parameters were evaluated. Serum adipocytokine levels were assayed with Luminex based kits. RESULTS: At the Pearson's correlation, among all the indices investigated, VAI showed a significant correlation with almost all adipocytokines analyzed [Visfatin, Resistin and hsCRP (all p<0.001); Adiponectin, sOb-R, IL-6, IL-18, HGF (all p<0.010); Ghrelin and VEGF (both p<0.05)]. Through a two-step cluster analysis, 55 patients were identified with the most altered adipocytokine profile (patients with ATD). At a ROC analysis, VAI showed the highest C-statistic [0.767 (95% CI 0.66-0.84)] of all the indices. CONCLUSIONS: Our study suggests that the VAI, among the most common indexes of adiposity assessment, shows the best correlation with the best known adipocytokines and cardiometabolic risk serum markers. Although to date we are still far from clearly identifying an ATD, the VAI would be an easy tool for clearly mirroring a condition of cardiometabolic risk, in the absence of an overt metabolic syndrome.

Cytokine serum profile in a group of Sicilian nonagenarians.

The aim of our study was to evaluate the possibility of using multiplex analysis of the cytokine profile as a marker for successful aging by comparing cytokine plasmatic levels of a group of Sicilian nonagenarians with those of young controls. We analyzed a panel of 17 cytokines, comprehensive of haematopoietic factors T helper 1 (Th1), Th2, inflammation regulatory cytokines, and chemokines. The assay was carried out using the Luminex system. Interleukin (IL)-6 levels (p = 0.01) were increased in nonagenarians, whereas no modifications of other proinflammatory cytokines and chemokines were observed. Interferon-gamma (IFN-γ) and IL-2 levels are unmodified, suggesting a substantial maintenance of relevant T cell functions. In addition, a significant increase of IL-12 serum levels in nonagenarians versus young controls that might be related to the increase of natural killer (NK) cell functions characterizing aging processes was observed. The analysis of Th2 cytokines show an increase of IL-13 and a reduction of IL-4 levels mirroring the maintenance of some effector's mechanisms of the immunoresponse in advanced ages. Our results suggest that the multiplex analysis of cytokine levels might be useful in defining a successful aging profile.

Relevance of gamma interferon, tumor necrosis factor alpha, and interleukin-10 gene polymorphisms to susceptibility to Mediterranean spotted fever.

The acute phase of Mediterranean spotted fever (MSF) is characterized by dramatic changes in cytokine production patterns, clearly indicating their role in the immunomodulation of the response against the microorganism, and the differences in cytokine production seem to influence the extent and severity of the disease. In this study, the single nucleotide polymorphisms (SNPs) of tumor necrosis factor alpha (TNF-alpha) -308G/A (rs1800629) and interleukin-10 (IL-10) -1087G/A (rs1800896), -824C/T (rs1800871), and -597C/A (rs1800872) and the gamma interferon (IFN-gamma) T/A SNP at position +874 (rs2430561) were typed in 80 Sicilian patients affected by MSF and in 288 control subjects matched for age, gender, and geographic origin. No significant differences in TNF-alpha -308G/A genotype frequencies were observed. The +874TT genotype, associated with an increased production of IFN-gamma, was found to be significantly less frequent in MSF patients than in the control group (odds ratio [OR], 0.18; 95% confidence interval [95% CI], 0.06 to 0.51; P corrected for the number of genotypes [Pc], 0.0021). In addition, when evaluating the IFN-gamma and IL-10 genotype interaction, a significant increase of +874AA/-597CA (OR, 5.31; 95% CI, 2.37 to 11.88; P(c), 0.0027) combined genotypes was observed. In conclusion, our data strongly suggest that finely genetically tuned cytokine production may play a crucial role in the regulation of the immune response against rickettsial infection, therefore influencing the disease outcomes, ranging from nonapparent or subclinical condition to overt or fatal disease.

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis.

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)-induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4(+) phenotype. In particular, the T helper 1-type (Th1) subset, as evaluated by chemokine receptor-5 (CCR5) expression, is involved in this process. Cell death in Th1-type uses a CD95-mediated mechanism. Furthermore, Th1-type CCR5(+) cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1-type cells by apoptosis was confirmed by the decrease in interferon-gamma secretion. In conclusion, apoptosis of monocytes, CD4(+) and CD4(+) CCR5(+) T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune-depression in VL.

Estrogen regulates cytokine production and apoptosis in PMA-differentiated, macrophage-like U937 cells.

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) in phorbol-myristate-acetate (PMA)-differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage-like PMA-differentiated U937 cells was also inspected. E2 increased TNF-alpha synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI-182,789 completely abolished the induction of TNF-alpha, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF-alpha production by U937 cells. Exposure of cells to E2 resulted in a dose-dependent decrease of IL-10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage-like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI-182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro-inflammatory action through the modulation of TNF-alpha and IL-10, and regulate the immune effector cells by the induction of programmed cell death.

Anti-inflammatory effects of annexin-1: stimulation of IL-10 release and inhibition of nitric oxide synthesis.

Annexin-1 (ANX-1) is an anti-inflammatory protein induced by glucocorticoids. Like glucocorticoids, ANX-1 and derived peptides inhibit eicosanoid synthesis, block leukocyte migration and induce apoptosis of inflammatory cells. Cytokines may possess either pro-inflammatory, i.e. interleukin(IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12 or anti-inflammatory properties, i.e. IL-4, IL-10. The experiments described in the present study have been performed to answer the question whether the anti-inflammatory action of ANX-1 may be mediated, at least in part, by the release of IL-10. In macrophage (J774) cell line cultures primed with lipolysaccharide (LPS), recombinant ANX-1 stimulated IL-10 release in a dose- and time-dependent manner. In the same cells, the protein and its derived N-terminal peptide (amino acids 2-26) dose-dependently inhibited the release of nitric oxide (NO). Furthermore, both the whole protein and the peptide down-regulated the mRNA expression of the inducible nitric oxide sythase (iNOS). The peptide was also able to inhibit the expression of IL-12 mRNA. These results suggest that some of the anti-inflammatory effects of ANX-1 may be mediated by the release of IL-10, which, in turn, inhibits iNOS mRNA expression and, hence, NO release. In addition, ANX-1-stimulated IL-10 release may also be responsible for the inhibition of IL-12 mRNA expression and, consequently, IL-12 synthesis.

Chemically modified tetracyclines induce cytotoxic effects against J774 tumour cell line by activating the apoptotic pathway.

Here, we have studied the effects of chemically modified tetracyclines (CMTs) on apoptosis both at the level of the cytoplasmic proteolytic caspase cascade, and on Bcl-2 and c-myc mRNA expression in the J774 macrophage cell line. The results indicate that CMTs induce morphological changes consistent with apoptotic events, as clearly demonstrated both by the acridine orange and ethidium bromide staining, and by TUNEL and fragmentation ELISA assays. Furthermore, the analysis of the cell cycle by flow cytometry shows an evident apoptotic sub-G0G1 peak, without important modifications in the cell cycle distribution. CMTs induce programmed cell death (PCD) in a dose-dependent manner and CMT-8 is the strongest among them. CMT-1 and CMT-8 activate mainly caspase-8 as attested by the inhibitory effects of Z-VAD-fmk and Z-IEDT-fmk on CMT-induced apoptosis. Part of CMT-induced PCD is due to the activation of caspase-9, since it is reduced by the specific caspase-9 inhibitor, Z-LEHD-fmk. Besides, CMTs increase Bcl-2 and c-myc mRNA expression. Collectively, these data indicate that CMTs are potentially anti-tumour agents, since they strongly trigger apoptosis both activating the proteolytic system of the caspase family and modulating genes involved in PCD regulation.

Identification of epitopes of Mycobacterium tuberculosis 16-kDa protein recognized by human leukocyte antigen-A*0201 CD8(+) T lymphocytes.

CD8(+) T cells could make an important contribution to protection against tuberculosis (TB), but the antigenic determinants recognized in the context of major histocompatibility complex class I molecules remain ill defined. Our aim was to identify nonamer peptides derived from the acr/16-kDa antigen. Two immunogenic peptides (p21-29 and p120-128) were identified by their ability to elicit cytotoxic CD8(+) T cells from juvenile patients recovering from TB. Epitope-specific recognition was demonstrated by the lysis of both Mycobacterium tuberculosis-infected and peptide-pulsed macrophages, the release of cytotoxic granules, and interferon-gamma and tumor necrosis factor-alpha production. CD8(+) T cell responses to p21-29 and p120-128 were detected ex vivo in freshly isolated peripheral blood mononuclear cells from patients with TB but not in those from healthy control subjects. Our data suggest that these antigenic peptides can play a critical role in effective immunity against mycobacterial infection and TB.