Serum 25-hydroxy vitamin D levels in essential hypertension.

Vitamin D may have prognostic value in hypertension patients and, in addition to conventional biomarkers, could be a valuable tool for disease management. The aim of this study was to assess the association of vitamin D status in patients with essential hypertension and to evaluate its prognostic utility. Forty-eight consecutive patients (40 Caucasian and 8 Asian) aged between 30 and 80 years (mean 61.5, range 34-84 years), were enrolled in the study. The main exclusion criteria were age less than 18 years, kidney failure, onco-hematologic disease, hypo-hyperparathyroidism, osteoporosis, treatment with bisphosphonate or 25(OH) vitamin D supplementation. Of the 48 patients included in the study, hyperlipidemia was described in 28, diabetes type 2 in 8, and ischemic heart disease in 14. Serum electrolytes, calcium, sodium, and potassium concentrations were within normal range. Low 25(OH) vitamin D levels inversely correlated with essential hypertension values (p less than 0.001) were considered extremely significant. The determination of 25(OH) vitamin D levels in patients with essential hypertension could improve the research for possible underlying conditions, which should be managed meticulously according to current guidelines.

Performance assessment of a fully automated electro-chemiluminescence immunoassay system for serum S100B protein.

The altered expression levels of S100 proteins can lead to four different categories of diseases: diseases of the heart and of the central nervous system, inflammatory disorders and cancer. Various studies have shown the lack of harmonization of the results obtained with different methods, mainly due to different performances and measurements of S100B. The purpose of this work was to compare quantitatively the fully automated Elecsys® immunoassay with the reference immunoenzimatic method CanAg® EIA for serum S100B protein. In the study serum samples were analyzed of 161 patients: 85 females (aged 22-83 years) and 76 males (aged 16-90 years), affected by oncological and non-oncological pathologies. Passing–Bablok regression was used to analyze the comparison between the assays; it showed a strong interassay correlation: r = 0.9350 (95% CI =0.9122 – 0.9520), with an intercept of 0.02063 (95% CI=-0.02850 – 0.01400) and a slope of 1.1125 (95% CI=1.0200 – 1.2417). Elecsys® S100 assay should be preferred to CanAg® S100 for better standardization, good reliability and precision but also with the aim to reduce costs and obtain results in a shorter time.

Serum 25-hydroxy vitamin D concentration in acute coronary syndrome.

Vitamin D may have prognostic value in cardiovascular disease (CVD) patients and, in addition to conventional biomarkers, could be a valuable tool for disease management. The aim of this study was to assess the association of vitamin D status in patients with acute coronary syndrome (ACS) and to evaluate its prognostic utility. The levels of 25(OH) vitamin D were correlated with troponin T hs. Forty-eight consecutive outpatients (40 Caucasian and 8 Asian) aged between 40 and 70 years (mean 61.5, range 43-77 years) were enrolled in the study. All patients were admitted to the Emergency Department with chest pain and suspected ACS. The main exclusion criteria were age <18 years, kidney failure, onco-haematological disease, hypo-hyperparathyroidism, hypo/hyperthyroidism, osteoporosis, treatment with bisphosphonate or 25(OH) vitamin D supplementation. Of the 48 subjects included in the study, thoracic pain symptoms were described in 12 patients with unstable angina (UA) and in 6 patients with ST elevation myocardial infarction (STEMI) and in 30 patients with non-ST-elevation myocardial infarction (NSTEMI). Low 25(OH) vitamin D levels correlated with the presence of ACS (p< 0.02) and inversely correlated with Troponin T hs (TnT hs) levels (p< 0.03). The determination of 25(OH) vitamin D levels in combination with TnT hs could improve the research for possible underlying conditions, and these should be managed meticulously according to current guidelines.

Assessing the association between 25-OH vitamin D levels and ROMA score in a population of obese women.

The “Risk of Malignancy Algorithm” (ROMA) combines the diagnostic power of the CA125 and HE4 markers with menopausal status to predict the risk for developing epithelial ovarian cancer (EOC). The aim of this study was to evaluate the association between 25-OH vitamin D levels and ROMA score in obese women. One hundred and eighteen patients with a Body Mass Index (BMI) > 30 kg/m2 (Group 1) and 80 women with a BMI less than 25 kg / m² (Group 2) were studied. The 25-OH vitamin D was quantified with LUMIPULSE® G 1200. As a threshold value, identified by ROC curve analysis, 20.2 ng/ mL (sensitivity 73.3%, specificity 84%) was chosen corresponding to the limit between sufficient and insufficient 25-OH vitamin D according to the World Health Organization (WHO). Low 25-OH vitamin D levels were observed in 64% of obese women and in 11% of normal-weight women (p less than 0.001). ROMA score above 13% was detected only in obese women (19%). An association between low levels of 25-OH vitamin D and ROMA score was observed. Indeed, 64% of obese women with ROMA score >13% had concomitant insufficient levels of 25-OH vitamin D, while only 36% of obese women with ROMA score >13% had sufficient 25-OH vitamin D levels (p less than 0.0001). This study suggests that the deficiency of 25- OH vitamin D in obese women has a possible correlation with high ROMA score.

Endometriosis and Glanzmann’s thrombasthenia.

Glanzmann’s thrombasthenia (GT) is a rare bleeding syndrome characterized by deficiency or defect of platelet aggregation complex. The pathogenesis of endometriosis is controversial but the strongest evidence leans towards retrograde menstruation. GT probably predisposes to endometriosis. The management of women affected by this disease can be difficult due to the risk of bleeding complications, especially during surgical treatment. We describe the cases of three sisters affected by endometriosis and GT, referred to our Department, who received different therapeutic management.

CLEIA CA125 evidences: good analytical performance avoiding "Hook effect".

Cancer antigen 125 (CA125) is a coelomic epithelium-related antigen carried by a high molecular weight glycoprotein complex. It is commonly used as a tumor marker for ovarian cancer to monitor disease progression and response to therapy and as an early detection for recurrence after treatment. The aim of this study was to test the reliability of two different assay methods, a radioimmunometric assay (RIA) and an automated chemiluminescent enzyme immunoassay (CLEIA) system, by measuring CA125 serum levels using both methods in 357 patients and comparing the results. Patients were recruited from Oncologic Unit A, Policlinico Umberto I, Roma. Eighty-six were healthy donors, while 271 were oncologic patients representing a variety of diagnoses. Within this group, 76 patients were diagnosed with an ovarian related pathology (28 cancerous and 48 benign). The evaluation of CA125 marker blood levels showed a high agreement in healthy donors group (R (2) = 0.9003). Interesting results emerged when sera collected from oncologic patients were assessed: significant differences between the two assays were found in nine samples. When assayed again with RIA after a dilution, new values agreed with undiluted CLEIA values (R (2) = 0.9847). Our data suggest an overall good comparison between the two methods. However, some artifacts were obtained with RIA and indicate an underlying presence of "hook effect". CLEIA automated assay showed a good reliability and should be preferred to one-step radioimmunoassays in order to minimize errors.

Pathogenic Escherichia coli found in sewage treatment plants and environmental waters.

We previously demonstrated that some Escherichia coli strains with uropathogenic properties survived treatment stages of sewage treatment plants (STPs), suggesting that they may be released into the environment. We investigated the presence of such strains in the surrounding environmental waters of four STPs from which these persistent strains were isolated. In all, 264 E. coli isolates were collected from 129 receiving water sites in a 20-km radius surrounding STPs. We also included 93 E. coli strains collected from 18 animal species for comparison. Isolates were typed using a high-resolution biochemical fingerprinting method (the PhPlate system), and grouped into common (C) types. One hundred forty-seven (56%) environmental isolates were identical to strains found in STPs' final effluents. Of these, 140 (95%) carried virulence genes (VGs) associated with intestinal pathogenic E. coli (IPEC) or uropathogenic E. coli (UPEC) and were found in a variety of sites within areas sampled. Of the remaining 117 environmental strains not identical to STP strains, 105 belonged to 18 C types and 102 of them carried VGs found among IPEC or UPEC strains. These strains belonged mainly to phylogenetic groups A (A0 and A1) and B1 and to a lesser extent B2(2), B2(3), D1, and D2. Eight of 18 environmental C types, comprising 50 isolates, were also identical to bird strains. The presence of a high percentage of environmental E. coli in waters near STPs carrying VGs associated with IPEC and UPEC suggests that they may have derived from STP effluents and other nonpoint sources.

Transient plasma cell dyscrasia in COVID-19 patients linked to IL-6 triggering.

An unusual clonal gammopathy was reported in COVID-19 patient but whether this anomaly is related or not to the disease has not yet been clarified. To this aim, we selected a cohort of 35 COVID-19 patients swab positive and investigated serological levels of IL-6, immune response to major viral antigens and electrophoretic profile. Elevated levels of IL-6 were accompanied by a significative humoral response to viral Spike protein, revealing an altered electrophoretic profile in the gamma region. We can conclude that elevated levels of IL-6 triggers humoral response inducing a transient plasma cell dyscrasia in severe COVID-19 patients.

Prevalence and persistence of Escherichia coli strains with uropathogenic virulence characteristics in sewage treatment plants.

We investigated the prevalence and persistence of Escherichia coli strains in four sewage treatment plants (STPs) in a subtropical region of Queensland, Australia. In all, 264 E. coli strains were typed using a high-resolution biochemical fingerprinting method and grouped into either a single or a common biochemical phenotype (S-BPT and C-BPT, respectively). These strains were also tested for their phylogenetic groups and 12 virulence genes associated with intestinal and extraintestinal E. coli strains. Comparison of BPTs at various treatment stages indicated that certain BPTs were found in two or all treatment stages. These BPTs constituted the highest proportion of E. coli strains in each STP and belonged mainly to phylogenetic group B2 and, to a lesser extent, group D. No virulence genes associated with intestinal E. coli were found among the strains, but 157 (59.5%) strains belonging to 14 C-BPTs carried one or more virulence genes associated with uropathogenic strains. Of these, 120 (76.4%) strains belonged to seven persistent C-BPTs and were found in all four STPs. Our results indicate that certain clonal groups of E. coli with virulence characteristics of uropathogenic strains can survive the treatment processes of STPs. These strains were common to all STPs and constituted the highest proportion of the strains in different treatment tanks of each STP.

Circulating anti-immunoglobulin antibodies in recent-onset type I diabetic patients.

Human sera from 51 recent-onset insulin-dependent (type I) diabetic patients and 47 unrelated control subjects were screened for the possible presence of circulating factors reacting with several anti-pancreatic islet monoclonal antibodies (MoAb.ISL) in solid-phase radioimmunoassay methods (the original goal being the detection of anti-idiotypic islet cell antibodies and/or specific islet cell antigen-bearing immune complexes). MoAbs from the parental myeloma cell line and purified immunoglobulins (Igs) from different animal species were controls. Type I diabetic sera showed significantly increased binding to MoAb.ISL-coated wells compared with normal subjects (P less than .001). However, the same sera also tended to show a higher binding to the control (non-islet-related) MoAb. Sera from type I diabetic patients also reacted with horse, bovine, pig, rabbit, and goat IgG. Displacement of the binding has been obtained by F(ab')2 and/or Fc fragments of IgG. Evidence has been obtained regarding a similar reaction with human IgM. All the sera were negative when tested for rheumatoid factor by nephelometry. The circulating antibodies described have been proven to be different from islet cell autoantibodies. An anti-Ig antibody is thus present in the sera of recent-onset diabetic patients and represents an additional immunological phenomenon with possible physiopathological and clinical significance.

The acquisition of an insulin-secreting phenotype by HGF-treated rat pancreatic ductal cells (ARIP) is associated with the development of susceptibility to cytokine-induced apoptosis.

The elucidation of mechanisms regulating the regeneration and survival of pancreatic beta cells has fundamental implications in the cell therapy of type 1 diabetes. The present study had the following three aims: 1. to investigate whether pancreatic ductal epithelial cells can be induced to differentiate into insulin-producing cells by exposing them to hepatocyte growth factor (HGF); 2. to characterize some of the molecular events leading to their differentiation toward a beta-cell-like phenotype; 3. to evaluate the susceptibility of newly differentiated insulin-secreting cells to cytokine-induced apoptosis, a mechanism of beta-cell destruction occurring in type 1 diabetes. We demonstrated that HGF-treated rat pancreatic ductal cell line (ARIP) cells acquired the capability to transcribe the insulin gene and translate its counterpart protein. HGF-treated cells also exhibited a glucose-dependent capability to secrete insulin into the cultured medium. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent transcription of neurogenin-3 and Neuro-D in response to HGF. Finally, we determined the susceptibility to proinflammatory cytokine (PTh1)-induced apoptosis by incubating HGF-treated and untreated ARIP cells with a cocktail of interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Such treatment induced apoptotic death, as determined by the TUNEL technique, in about 40% of HGF-treated, insulin-secreting ARIP cells, while untreated ARIP cells were resistant to PTh1-induced apoptosis. In conclusion, we showed that HGF promotes the differentiation of ARIP cells into pancreatic beta-cell-like cells, and that the differentiation toward an insulin-secreting phenotype is associated with the appearance of susceptibility to cytokine-induced apoptosis.

Pancreatic islet ganglioside expression in nonobese diabetic mice: comparison with C57BL/10 mice and changes after autoimmune beta-cell destruction.

Recent observations have shown that the presumed target antigen of cytoplasmic islet cell antibodies (ICA) has properties of a monosialo-ganglioside migrating between GM2 and GM1 standards (GM2-1) and that ICA binding is higher in nonobese diabetic (NOD) than in C57BL/10SnJ mouse pancreatic frozen sections. This study aimed to characterize the ganglioside expression in NOD mouse islets in comparison with the control C57BL/10SnJ strain, taking into account possible sex differences, variations with age, and changes after autoimmune beta-cell destruction. Thus, acidic glycolipid composition was analyzed 1) in isolated islets from 11-week-old female and male NOD mice and age-matched female and male C57BL/10SnJ mice, and 2) in whole pancreas of both NOD and control mouse strains at different ages (4, 8, and 18 weeks) and of female NOD mice before and after diabetes onset. The acidic glycolipid GM2-1 is expressed in isolated female NOD islets, male NOD islets, and C57BL/10SnJ mouse islets, but quantitative analysis showed an increased amount of GM2-1 in NOD vs. C57BL/10 islets. GM3 is a ganglioside fraction expressed in female and male NOD mice and not in the C57BL/10 strain, whereas GD3 characterizes the C57BL/10 strain islets. GM2-1 is the sole ganglioside fraction in the whole pancreas to clearly decrease with age in the NOD mouse, and diabetes onset in this strain is associated with a significant decrease in the expression of this component as well as of GM3, whereas other pancreatic ganglioside (GD3, GD1a, and GT1b) levels did not significantly decrease; no age-related ganglioside change was observed in the C57BL/10SnJ mouse. Interestingly, the observed increased ICA binding in NOD islets is paralleled by the increased expression of GM2-1 islet ganglioside, and beta-cell destruction in NOD mice is associated with a significant decrease in the amount of this ganglioside in the pancreas.

Cultured pancreatic ductal cells undergo cell cycle re-distribution and beta-cell-like differentiation in response to glucagon-like peptide-1.

The intestinal hormone glucagon-like peptide-1 (GLP-1) has been shown to promote an increase in pancreatic beta-cell mass via proliferation of islet cells and differentiation of non-insulin-secreting cells. In this study, we have characterized some of the events that lead to the differentiation of pancreatic ductal cells in response to treatment with human GLP-1. Rat pancreatic ductal (ARIP) cells were cultured in the presence of GLP-1 and analyzed for cell counting, cell cycle distribution, expression of cyclin-dependent-kinase (Cdk) inhibitors, transcription of beta-cell-specific genes, loss of ductal-like phenotype and acquisition of beta-cell-like gene expression profile. Exposure of ARIP cells to 10 nM GLP-1 induced a significant reduction in the cell replication rate and a significant decrease in the percentage of cells in S phase of the cell cycle. This was associated with an increase in the number of cells in G0-G1 phase and a reduction of cells in G2-M phase. Western blot analysis for the Cdk inhibitors, kinase inhibitor protein 1 (p27(Kip1)) and Cdk-interacting protein 1 (p21(Cip1)), demonstrated a significant increase in p27(Kip1) and p21(Cip1) levels within the first 24 h from the beginning of GLP-1 treatment. As cells slowed down their proliferation rate, GLP-1 also induced a time-dependent expression of various beta-cell-specific mRNAs. The glucose transporter GLUT-2 was the first of those factors to be expressed (24 h treatment), followed by insulin (44 h) and finally by the enzyme glucokinase (56 h). In addition, immunocytochemistry analysis showed that GLP-1 induced a time-dependent down-regulation of the ductal marker cytokeratin-20 (CK-20) and a time-dependent induction of insulin expression. Finally, GLP-1 promoted a glucose-dependent secretion of insulin, as demonstrated by HPLC and RIA analyses of the cell culture medium. The present study has demonstrated that GLP-1 induces a cell cycle re-distribution with a decrease in cell proliferation rate prior to promoting the differentiation of cells towards an endocrine-like phenotype.

A new solid-phase radioimmunoassay to detect anti-GAD65 autoantibodies.

This paper describes a simple, rapid, routine method to detect anti-GAD65 autoantibodies by a solid-phase radioimmunoassay using human recombinant GAD65 coated microwells and 125I-protein A to reveal antibody binding. Both recombinant and radiolabelled proteins are commercially available. This new method was validated by investigating the presence of GAD65 autoantibodies in two different studies (A and B); the first including subjects originating from our own case histories (group A sera), the second made up of recoded subjects and standards sent to our lab by the Second International GAD Antibody Workshop organizers (group B sera). In study A we tested sera from 52 normal subjects, 25 newly diagnosed type 1 diabetics and 3 stiff man syndrome (SMS) subjects detecting GAD65 autoantibodies in 72% of IDDM and 100% of SMS patients. In study B we tested (in blind fashion) 89 recoded sample sera or standards that were part of the larger group used in the Second International GAD Antibody Workshop, finding GAD65 autoantibodies in 3.3% of healthy control subjects (1/30), 60% of IDDM patients (18/30), 100% of ICA + nondiabetic subjects (3/3) but in none of 4 nondiabetic patients with Graves disease. Comparing our solid-phase RIA results with those published for the same sera from the Second International GAD Antibody Workshop we obtained for our method a sensitivity of 85.7%, a specificity of 93.9% and a consistency of 100%. These result indicate that our assay, which is based on commercially available reagents, should be a useful tool for the detection of GAD65 autoantibodies in large scale studies.

Autoimmune markers and neurological complications in non-insulin-dependent diabetes mellitus.

To verify whether autoimmune markers related to nervous system structures and other autoimmunity indexes present in diabetes mellitus are associated with subclinical neuropathy, we examined 48 non-insulin-dependent diabetic patients with and without neuroelectrophysiological alterations. Nerve conduction velocity at the external sciatic-popliteal nerve, at the sural nerve, at the median and ulnar nerves level has been evaluated. Autoimmunity was investigated by evaluating glutamic acid decarboxylase (GAD-Ab), insulin (IAA), GM3, GD3 and GT1b gangliosides, pancreatic islet cell (IC-A) and anti-nervous-tissue autoantibody presence. Nerve conduction velocities were decreased in 72.9% of diabetic patients. Anti-insulin antibodies were detected in seven non-insulin created diabetic patients and in higher amount in subjects with (17.1%) than in those without (7.7%) asymptomatic neuropathy. Anti-GM3 antibodies were detected in four diabetic patients all of whom presented neurological complication. A significant correlation has been found between neurological damage and presence of anti-insulin antibodies (p<0.05). In the case of GM3 autoantibody, a similar result was obtained, but the data failed to reach statistical significance. Our data demonstrate that autoimmunity might play a role in the development of peripheral neuropathy.

Expression of Reg and cytokeratin 20 during ductal cell differentiation and proliferation in a mouse model of autoimmune diabetes.

OBJECTIVE: To evaluate the existence of beta-cell differentiation and proliferation in the low-dose streptozotocin (ld-STZ) mouse model of autoimmune diabetes. DESIGN: We studied the expression of Reg protein and cytokeratin 20 (CK20), the presence of proliferative phenomena (judged by the incorporation of bromodeoxyuridine (BrdU)), and the co-expression of Reg, CK20 or BrdU with insulin. MATERIALS AND METHODS: Diabetes was induced in male C57Bl6/J mice by administration of ld-STZ. The animals were killed at days 10 and 23 from the beginning of the induction of disease. Five animals were used at each time point and each group was evaluated for blood glucose concentrations, insulitis, expression of Reg and CK20 pancreatic proteins and BrdU incorporation, together with staining for insulin by immunohistochemistry and laser confocal microscopy. RESULTS: All mice treated with ld-STZ were hyperglycemic and histological investigation showed a mild or severe insulitis both at day 10 and at day 23. At day 10, immunochemistry revealed an intense expression of Reg and CK20 in pancreatic ducts in ld-STZ mice, but not in control mice. Reg and CK20 immunoreactive cells were also positive for insulin. In contrast, at day 23, pancreatic sections reacted weakly with anti-Reg and anti-CK20 antibody; co-localization with insulin was observed for both Reg and CK20. The incorporation of BrdU was observed only in insulin-positive cells in pancreatic sections from mice killed at day 10. CONCLUSIONS: These observations show an islet regeneration mechanism in response to an autoimmune attack, and that the ld-STZ mouse is a suitable model in which to evaluate intervention strategies.

Insulin prophylaxis down-regulates islet antigen expression and islet autoimmunity in the low-dose Stz mouse model of diabetes.

The aims of this study were to evaluate in an autoimmune diabetes animal model [low-dose streptozotocin (LD-STZ) mouse] (a) the efficacy of a prophylactic insulin treatment as a diabetes prevention tool, and (b) its possible mechanisms through both the insulitis evaluation and islets antigen expression. Diabetes was induced in male C57Bl6/J mice with STZ (50 mg/kg b/w for five consecutive days); insulin (1 U/day) was injected subcutaneously for ten consecutive days before the induction of diabetes and for a further ten days. Seventy-one male C57Bl6/J mice were grouped as follows: Group 1 (n = 25) made diabetic with i.p. STZ, Group 2 (n = 21) made diabetic with i.p. STZ and injected subcutaneously with insulin, Group 3 (n = 15) injected with insulin, while Group 4 (n = 10) comprised normal animals as controls. The animals of each group were killed at two intervals: half of them at day 12 and the remainder at day 24 from the beginning of the STZ treatment. A significant reduction of glycemia levels and insulitis severity was observed between mice of Group 1 vs. Group 2 at day 12 and day 24. Down-regulation of islet antigen expression (insulin, A2B5, GM2-1, ICA Ag) was achieved even without a complete metabolic suppression of beta-cell activity. In conclusion, prophylactic insulin treatment is effective to reduce glycemia levels and insulitis severity and down-regulates islet antigen expression in the LD-STZ model.