Synthesis, molecular docking and antibacterial activity of an oxadiazole-based lipoteichoic acid inhibitor and its metabolites.

Amongst drug resistant Gram-positive bacteria, Staphylococcus aureus is a pathogen of great concern as it is the leading cause of life-threatening nosocomial and community acquired infections which are often associated with implanted medical devices. The biosynthesis of lipotheicoic acid (LTA) by S. aureus has been recognized as a promising antibacterial target, owing its critical role in the growth and survival of Gram-positive bacteria. Here we report for the first time the chemical synthesis and characterisation of an oxadiazole based compound (1771), previously described as an inhibitor of LTA biosynthesis by targeting Lta synthase enzyme (LtaS). To investigate its controversial mode of action, we also performed molecular docking studies, which indicated that 1771 behaves as a competitive inhibitor against LtaS. We also synthesised and evaluated the antimicrobial activity of 1771 metabolites which we have identified from its decomposition in mouse serum, proving that the biological activity was caused by intact 1771.

Linear, Non-Conjugated Cyclic and Conjugated Cyclic Paraphenylene under Pressure.

The n-paraphenylene family comprises chains of phenylene units linked together by C-C bonds that are between single- and double-bonded, and where n corresponds to the number of phenylene units. In this work, we compare the response of the optical properties of different phenylene arrangements. We study linear chains (LPP), cyclic systems (CPPs), and non-conjugated cyclic systems with two hydrogenated phenylenes (H4[n]CPP). Particularly, the systems of interest in this work are [6]LPP, [12]- and [6]CPP and H4[6]CPP. This work combines Raman and infrared spectroscopies with absorption and fluorescence (one- and two-photon excitations) measured as a function of pressure up to maximum of about 25 GPa. Unprecedented crystallographic pressure-dependent results are shown on H4[n]CPP, revealing intramolecular π-π interactions upon compression. These intramolecular interactions justify the H4[n]CPP singular optical properties with increasing fluorescence lifetime as a function of pressure.

Use of a miniature diamond-anvil cell in a joint X-ray and neutron high-pressure study on copper sulfate pentahydrate.

Single-crystal X-ray and neutron diffraction data are usually collected using separate samples. This is a disadvantage when the sample is studied at high pressure because it is very difficult to achieve exactly the same pressure in two separate experiments, especially if the neutron data are collected using Laue methods where precise absolute values of the unit-cell dimensions cannot be measured to check how close the pressures are. In this study, diffraction data have been collected under the same conditions on the same sample of copper(II) sulfate pentahydrate, using a conventional laboratory diffractometer and source for the X-ray measurements and the Koala single-crystal Laue diffractometer at the ANSTO facility for the neutron measurements. The sample, of dimensions 0.40 × 0.22 × 0.20 mm3 and held at a pressure of 0.71 GPa, was contained in a miniature Merrill-Bassett diamond-anvil cell. The highly penetrating diffracted neutron beams passing through the metal body of the miniature cell as well as through the diamonds yielded data suitable for structure refinement, and compensated for the low completeness of the X-ray measurements, which was only 24% on account of the triclinic symmetry of the sample and the shading of reciprocal space by the cell. The two data-sets were combined in a single 'XN' structure refinement in which all atoms, including H atoms, were refined with anisotropic displacement parameters. The precision of the structural parameters was improved by a factor of up to 50% in the XN refinement compared with refinements using the X-ray or neutron data separately.

CD8+ T-cell responses identify beta-cell autoimmunity in human type 1 diabetes.

Despite the understanding that type 1 diabetes pathogenesis is mediated by T-cells, detection of these rare lymphocytes remains largely elusive. Suitable T-cell assays are highly needed, since they could offer preclinical diagnoses and immune surrogate end points for clinical trials. Although CD4+ T-cell assays have met with limited success, CD8+ T-cells are increasingly recognized as key actors in the diabetes of the NOD mouse. CD8+ T-cells are likely to play a role also in humans and may provide new markers of beta-cell autoimmunity. Taking advantage of a panel of HLA-A2-restricted beta-cell epitopes derived from preproinsulin, GAD, and islet glucose-6-phosphatase catalytic subunit-related protein (IGRP), we have implemented an islet-specific CD8+ T-cell interferon-gamma enzyme-linked immunospot (ISL8Spot) assay. The ISL8Spot assay is capable of detecting and quantifying beta-cell-reactive CD8+ T-cells directly ex vivo, without any preliminary expansion, using either fresh or frozen samples. Positive ISL8Spot responses separate new-onset diabetic and healthy samples with high accuracy (86% sensitivity, 91% specificity), using as few as five immunodominant epitopes. Moreover, sensitivity reaches 100% when the ISL8Spot assay is complemented by antibody determinations. Combination of CD8+ T-cell measurements with immune intervention strategies may open new avenues toward type 1 diabetes prediction and prevention.

Changes over time in the prevalence and quality of care of type 2 diabetes in Italy: the Casale Monferrato surveys, 1988 and 2000.

BACKGROUND AND AIMS: In this study we assessed the prevalence of diagnosed type 2 diabetes and the quality of care during the period 1988-2000 in an Italian population. METHODS AND RESULTS: Two population-based surveys, using similar methods and centralized measurements, were conducted in 1988 and 2000 in a representative Italian area to identify people with known diabetes. The adjusted prevalence (reference, 2001 Italian population) was computed. The age- and sex-adjusted prevalence rates of diabetes in the population of Casale Monferrato were 2.13% (2.05-2.22) in 1988 and 3.07% (2.97-3.17) in 2000. In comparison with diabetic persons recruited in 1988 and independently of age and sex, persons recruited in 2000 had a lower likelihood of having HbA1c > or = 7.0% (OR=0.48; 0.42-0.56), diastolic blood pressure > or = 80 mmHg (OR=0.61; 0.49-0.75), LDL cholesterol > or = 2.59 mmol/l (OR=0.77; 0.63-0.93) and AER > or = 20 microg/min (OR=0.53; 0.45-0.61; they had a higher likelihood of having BMI > or = 25 kg/m(2) (OR=1.49; 1.2-1.74). However, 45.4% of patients still had HbA1c > or = 7.0%, 80% blood pressure > or = 130/80 mmHg and 79% LDL-cholesterol values > or =2.59 mmol/l. CONCLUSION: More than two-thirds of Italians with diabetes are now aged 65 years and more. The quality of control of glycemia, lipids and blood pressure improved and the prevalence of diabetic nephropathy decreased over time, although complete adherence to international guidelines has not yet been achieved.

Incidence of type 1 and type 2 diabetes in adults aged 30-49 years: the population-based registry in the province of Turin, Italy.

OBJECTIVE: Incidence of type 1 diabetes is considered to be low in adults, but no study has been performed in Mediterranean countries. RESEARCH DESIGN AND METHODS: We extended the study base of the registry of the province of Turin, Italy, to subjects aged 30-49 years in the period 1999-2001 to estimate the incidences of type 1 and type 2 diabetes. Diagnosis of type 1 diabetes was based on permanent insulin treatment or a fasting C-peptide level < or =0.20 nmol/l or islet cell (ICA) or GAD (GADA) antibody positivities. RESULTS: We identified 1,135 case subjects with high completeness of ascertainment (99%), giving an incidence rate of 58.0 per 100,000 person-years (95% CI 54.7-61.5). The incidence of type 1 diabetes was 7.3 per 100,000 person-years (6.2-8.6), comparable with the rates in subjects aged 0-14 and 15-29 years (10.3 [9.5-11.2] and 6.8 [6.3-7.4]). Male subjects had a higher risk than female subjects for both type 1 (rate ratio [RR] 1.70 [95% CI 1.21-2.38]) and type 2 (2.10 [1.84-2.40]) diabetes. ICA and/or GADA positivities were found in 16% of the cohort. In logistic regression, variables independently associated with autoimmune diabetes were age 30-39 years (odds ratio [OR] 2.39 [95% CI 1.40-4.07]), fasting C-peptide <0.60 nmol/l (3.09 [1.74-5.5]), and BMI <26 kg/m2 (2.17 [1.22-3.85]). CONCLUSIONS: Risk of type 1 diabetes between age 30 and 49 years is similar to that found in the same area between age 15 and 29 years. Further studies are required to allow geographical comparisons of risks of both childhood and adulthood autoimmune diabetes, the latter being probably higher than previously believed.

HLA-B7-restricted islet epitopes are differentially recognized in type 1 diabetic children and adults and form weak peptide-HLA complexes.

The cartography of ß-cell epitopes targeted by CD8(+) T cells in type 1 diabetic (T1D) patients remains largely confined to the common HLA-A2 restriction. We aimed to identify ß-cell epitopes restricted by the HLA-B7 (B*07:02) molecule, which is associated with mild T1D protection. Using DNA immunization on HLA-B7-transgenic mice and prediction algorithms, we identified GAD and preproinsulin candidate epitopes. Interferon-γ (IFN-γ) enzyme-linked immunospot assays on peripheral blood mononuclear cells showed that most candidates were recognized by new-onset T1D patients, but not by type 2 diabetic and healthy subjects. Some epitopes were highly immunodominant and specific to either T1D children (GAD(530-538); 44% T cell-positive patients) or adults (GAD(311-320); 38%). All epitopes displayed weak binding affinity and stability for HLA-B7 compared with HLA-A2-restricted ones, a general feature of HLA-B7. Single-cell PCR analysis on ß-cell-specific (HLA-B7 tetramer-positive) T cells revealed uniform IFN-γ and transforming growth factor-ß (TGF-ß) mRNA expression, different from HLA-A2-restricted T cells. We conclude that HLA-B7-restricted islet epitopes display weak HLA-binding profiles, are different in T1D children and adults, and are recognized by IFN-γ(+)TGF-ß(+)CD8(+) T cells. These features may explain the T1D-protective effect of HLA-B7. The novel epitopes identified should find valuable applications for immune staging of HLA-B7(+) individuals.

Age-period-cohort analysis of 1990-2003 incidence time trends of childhood diabetes in Italy: the RIDI study.

OBJECTIVE: To investigate age-period-cohort effects on the temporal trend of type 1 diabetes in children age 0-14 years in Italian registries. RESEARCH DESIGN AND METHODS: This report is based on 5,180 incident cases in the period 1990-2003 from the Registry for Type 1 Diabetes Mellitus in Italy (RIDI). Multilevel (random intercept) Poisson regression models were used to model the effects of sex, age, calendar time, and birth cohorts on temporal trends, taking into account the registry-level variance component. RESULTS: The incidence rate was 12.26 per 100,000 person-years and significantly higher in boys (13.13 [95% CI 12.66-13.62]) than in girls (11.35 [10.90-11.82]). Large geographical variations in incidence within Italy were evident; incidence was highest in Sardinia, intermediate in Central-Southern Italy, and high in Northern Italy, particularly in the Trento Province, where the incidence rate was 18.67 per 100,000 person-years. An increasing temporal trend was evident (2.94% per year [95% CI 2.22-3.67]). With respect to the calendar period 1990-1992, the incidence rates increased linearly by 15, 27, 35, and 40% in the following time periods (P for trend < 0.001). With respect to the 1987-1993 birth cohort, the incidence rate ratio increased approximately linearly from 0.63 (95% CI 0.54-0.73) in the 1975-1981 cohort to 1.38 (1.06-1.80) in the 1999-2003 cohort. The best model, however, included sex, age, and a linear time trend (drift). CONCLUSIONS: Large geographical variations and an increasing temporal trend in diabetes incidence are evident among type 1 diabetic children in Italy. Age-period-cohort analysis shows that the variation over time has a linear component that cannot be ascribed to either the calendar period or the birth cohort.

Fasting plasma C-peptide and micro- and macrovascular complications in a large clinic-based cohort of type 1 diabetic patients.

OBJECTIVE: A protective effect of residual beta-cell function on microvascular complications of type 1 diabetes has been suggested. Our aim was to retrospectively evaluate the association of fasting plasma C-peptide values with micro- and macrovascular complications. RESEARCH DESIGN AND METHODS: We recruited a clinic-based cohort of 471 type 1 diabetic patients born after 1945 and cared for in the period 1994-2004. Centralized measurements and standardized procedures of ascertainment of micro- and macrovascular complications were employed. Individual cumulative averages of A1C up to 2007 were calculated. RESULTS: Residual beta-cell secretion was detected even many years after diabetes diagnosis. In multivariate linear regression analysis, fasting plasma C-peptide values were positively associated with age at diagnosis (beta = 0.02; P < 0.0001) and triglycerides (beta = 0.20; P = 0.05) and inversely associated with diabetes duration (beta = -0.03; P < 0.0001) and HDL cholesterol (beta = -0.006; P = 0.03). The final model explained 21% of fasting C-peptide variability. With respect to fasting C-peptide values in the lowest tertile (<0.06 nmol/l), higher values were associated with lower prevalence of microvascular complications (odds ratio [OR] 0.59 [95% CI 0.37-0.94]) independently of age, sex, diabetes duration, individual cumulative A1C average during the study period, hypertension, and cardiovascular diseases. No association was evident with macrovascular complications (0.77 [0.38-1.58]). CONCLUSIONS: Our study shows an independent protective effect of residual beta-cell function on the development of microvascular complications in type 1 diabetes, suggesting the potential beneficial effect of treatment that allows the preservation of even modest beta-cell function over time.

What is the clinical usefulness of the metabolic syndrome? The Casale Monferrato study.

OBJECTIVE: Data on the clinical usefulness of the metabolic syndrome with respect to cardiovascular risk are not conclusive. We have assessed this issue in a large population-based cohort of diabetic and nondiabetic people in Southern Europe. METHODS: An Italian population-based cohort of 3729 individuals (2211 without diabetes and 1518 with diabetes) was examined, with centralized measurements, including the Homeostasis Model Assessment (HOMA) index in nondiabetic people. The usefulness of the metabolic syndrome (ATP III criteria) as an indicator of cardiovascular disease (CVD), independently of classical and novel risk factor [C-reactive protein (CRP) and albumin excretion rate (AER)] was assessed by using unconditional logistic regression. RESULTS: One thousand, seven hundred and fifty-three individuals (47.0%) had neither diabetes nor the metabolic syndrome, 458 (12.3%) had the metabolic syndrome only, 442 (11.8%) had type 2 diabetes only and 1076 (28.9%) had both diabetes and the metabolic syndrome. The highest likelihood of having CVD was conferred by both diabetes and the metabolic syndrome [odds ratio (OR) = 4.37, 95% confidence interval (CI) 3.25-5.87], independently of age, sex, low-density lipoprotein-cholesterol, smoke, AER, and CRP values. After further adjustment for its individual components, the association between CVD and the metabolic syndrome was no more evident. Among people with CRP 3 mg/l or less, ORs were similar in nondiabetic people with the metabolic syndrome and in diabetic people without it, whereas among those with CRP greater than 3 mg/l OR was two-fold higher in the latter. Values in upper quartiles of the HOMA-IR conferred a significant two-fold increased OR of CVD, even after adjustment for individual components of the metabolic syndrome, CRP and AER. CONCLUSIONS: The additional information provided by the metabolic syndrome is limited, in both diabetic and nondiabetic people, whereas the HOMA index is a useful indicator of CVD, independently of individual components of the metabolic syndrome, classical and novel risk factors.

C-reactive protein and 5-year survival in type 2 diabetes: the Casale Monferrato Study.

OBJECTIVE: To determine to what extent plasma C-reactive protein (CRP) values influence 5-year all-cause and cardiovascular mortality in type 2 diabetic individuals, independently of albumin excretion rate (AER) and other cardiovascular risk factors, and its incremental usefulness for predicting individual risk of mortality. RESEARCH DESIGN AND METHODS: Measurements of CRP were performed in 2,381 of 3,249 (73.3%) subjects as part of the population-based Casale Monferrato Study. Its association with 5-year all-cause and cardiovascular mortality was assessed with multivariate Cox proportional hazards modeling. The C statistic and measures of calibration and global fit were also assessed. RESULTS: Results are based on 496 deaths in 11.717 person-years of observations (median follow-up 5.4 years). With respect to subjects with CRP < or =3 mg/l, those with higher values had an adjusted hazard ratio (HR) of 1.51 (95% CI 1.18-1.92) for all-cause mortality and 1.44 (0.99-2.08) for cardiovascular mortality. In normoalbuminuric subjects, respective HRs of CRP were 1.56 (1.13-2.15) and 1.65 (1.00-2.74), AER being neither a modifier nor a confounder of CRP association. In analysis limited to diabetic subjects without cardiovascular disease (CVD), adjusted HRs were 1.67 (1.24-2.24) for all-cause mortality and 1.36 (0.83-2.24) for cardiovascular mortality. The improvement in individual risk assessment was marginal when measured with various statistical measures of model discrimination, calibration, and global fit. CONCLUSIONS: CRP measurement is independently associated with short-term mortality risk in type 2 diabetic individuals, even in normoalbuminuric subjects and in those without a previous diagnosis of CVD. Its clinical usefulness in individual assessment of 5-year risk of mortality, however, is limited.

The frequency and immunodominance of islet-specific CD8+ T-cell responses change after type 1 diabetes diagnosis and treatment.

OBJECTIVE: Islet-reactive CD8(+) T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. RESEARCH DESIGN AND METHODS: We took advantage of a recently validated islet-specific CD8(+) T-cell gamma-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2(+) adult type 1 diabetic patients close to diagnosis and at a second time point 7-16 months later. RESULTS: CD8(+) T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60-67 to 20% (P < 0.02). The previously subdominant IA-2(206-214) and IGRP(265-273) peptides were newly targeted, thus becoming the immunodominant epitopes. CONCLUSIONS: Shifts both in frequency and in immunodominance of CD8(+) T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements.

Immunization of HLA class I transgenic mice identifies autoantigenic epitopes eliciting dominant responses in type 1 diabetes patients.

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells. CD8(+) T cells have recently been assigned a major role in beta cell injury. Consequently, the identification of autoreactive CD8(+) T cells in humans remains essential for development of therapeutic strategies and of assays to identify aggressive cells. However, this identification is laborious and limited by quantities of human blood samples available. We propose a rapid and reliable method to identify autoantigen-derived epitopes recognized by human CD8(+) T lymphocytes in T1D patients. Human histocompatibility leukocyte Ags-A*0201 (HLA-A*0201) transgenic mice were immunized with plasmids encoding the T1D-associated autoantigens: 65 kDa glutamic acid decarboxylase (GAD) or insulinoma-associated protein 2 (IA-2). Candidate epitopes for T1D were selected from peptide libraries by testing the CD8(+) reactivity of vaccinated mice. All of the nine-candidate epitopes (five for GAD and four for IA-2) identified by our experimental approach were specifically recognized by CD8(+) T cells from newly diagnosed T1D patients (n = 19) but not from CD8(+) T cells of healthy controls (n = 20). Among these, GAD(114-123), GAD(536-545) and IA-2(805-813) were recognized by 53%, 25%, and 42% of T1D patients, respectively.