Inhibition of Eph/ephrin interaction with the small molecule UniPR500 improves glucose tolerance in healthy and insulin-resistant mice.

Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between ß-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-ßH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-ßH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy ß-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.

Osteogenic response and osteoprotective effects in vivo of a nanostructured titanium surface with antibacterial properties.

In implantology, as an alternative approach to the use of antibiotics, direct surface modifications of the implant addressed to inhibit bacterial adhesion and to limit bacterial proliferation are a promising tactic. The present study evaluates in an in vivo normal model the osteogenic response and the osteointegration of an anodic spark deposition nanostructured titanium surface doped with gallium (ASD + Ga) in comparison with two other surface treatments of titanium: an anodic spark deposition treatment without gallium (ASD) and an acid etching treatment (CTR). Moreover the study assesses the osteoprotective potential and the antibacterial effect of the previously mentioned surface treatments in an experimentally-induced peri-implantitis model. The obtained data points out a more rapid primary fixation in ASD and ASD + Ga implants, compared with CTR surface. Regarding the antibacterial properties, the ASD + Ga surface shows osteoprotective action on bone peri-implant tissue in vivo as well as an antibacterial effect within the first considered time point.

Orexin B inhibits viability and differentiation of stromal cells from swine adipose tissue.

Adipose tissue is recognized as a fundamental endocrine organ. Nowadays, we are also aware that it contains the highest number of stromal cells (ASCs) per unit of volume. These cells can differentiate between different phenotypes among which the adipocytes. The aim of this work was to verify whether orexin B, crucial mediator of the energy balance, modifies the differentiation of cultured ASCs. We used the pig as a model. Our data demonstrate that swine ASCs express prepro-orexin. Orexin B treatment inhibits ASCs proliferation (P < 0.05) and adipogenic differentiation (P < 0.05). Data collected could be interesting both in animal production field because consumers require lean meat, and in human medicine study about obesity because pig can be considered a valuable animal model for translational studies.

Expression and function of the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in the swine ovarian follicle.

The most characterized stromal cell-derived factor-1 (SDF-1) variants are the isoform α, which is the predominant one but undergoes rapid proteolysis, and the ß isoform, which is more resistant. Through the interaction with a specific chemokine receptor called CXCR4, SDF-1 is able to regulate different physiological processes. The aim of this study was to verify the expression and potential functional role of SDF-1 and CXCR4 in the porcine ovary. Firstly, the expression of SDF-1 and its receptor in different ovarian districts was verified for the first time. Thereafter, the effect of SDF-1 ß isoform (51-72) fragment on functional parameters, such as proliferation, metabolic activity, redox status, nitric oxide production, and steroidogenic activity, was assessed on granulosa cells collected from follicles. In addition, the potential effect of this protein in vascular events was verified through investigations on porcine aortic (AOC) endothelial cells, such as the production of nitric oxide and viability tests. The proliferation and metabolic activity were not affected by treatment with the cytokine. As regard to steroidogenesis, the peptide stimulated both estrogen (P = 0.049) and progesterone production (P = 0.039). Redox status was affected by the examined substance since superoxide anion was inhibited (P = 0.001), while antioxidant power (P = 0.034), as well as nitric oxide generation, were stimulated (P = 0.034). Tests performed on AOCs showed significant stimulation of nitric oxide production (P = 0.004) by the examined peptide, while cell viability was unaffected. Therefore, the potential role of cytokine in the mechanisms involved in the regulation of follicular function can be hypothesized.

Orexin system in swine ovarian follicles.

Successful reproduction is strictly linked to metabolic cues. The orexins are a family of hypothalamic neurohormones, well known for their key role in the control of food intake and the involvement in several aspects of the reproductive process. The biological actions of both orexins are carried out through binding to the related Orexin 1 (OX1R) and Orexin 2 (OX2R) G-protein-coupled receptors. The purpose of this study was to investigate the presence of orexin system components in the porcine ovaries, to contribute to expand the knowledge about their pleiotropic role. First, we investigated the localization of orexin A (OXA) and its receptors by immunochemistry in different ovarian districts. Thereafter, we evaluated the expression of the prepro-orexin (PPO) gene and OXA effects on granulosa cell functions. Immunohistochemical study revealed the presence of orexinergic system components in porcine ovarian follicles. Moreover, our data show the expression of PPO messenger RNA in swine ovarian follicles >5 mm. In addition, OXA influences proliferation (P < 0.05), steroidogenic activity (P < 0.05), and redox status of granulosa cells (P < 0.05). Therefore, we hypothesize that OXA could exert a local physiological role in swine ovarian follicles even if further studies are required to deeply define the function of this pleiotropic system.

Orexin A in swine corpus luteum.

Orexin A (OXA) is a hypothalamic neuropeptide which acts on 2 known G-protein-coupled receptors. It has been demonstrated that OXA is a central molecular link between food intake and reproduction. More recently, its peripheral role has been investigated, and we demonstrated its involvement in regulating ovarian follicle function. The present study was undertaken to explore a potential physiological role of orexin system in swine corpus luteum, a transient ovarian endocrine organ. Our aim was, first, to analyze the localization and eventual colocalization of OXA and its 2 receptors within the different cell types composing the corpus luteum structure. Second, we wanted to explore the effects of OXA on isolated luteal cells, and finally to verify a potential involvement of OXA in angiogenesis, a crucial event in corpus luteum development. Our data demonstrate the local expression of OXA and its receptors in swine corpus luteum. Luteal cell functions were affected by treatment with OXA. In particular, progesterone production was inhibited (P < 0.05) and nonenzymatic scavenging activity was increased (P < 0.05). Moreover, OXA inhibited (P < 0.05) new vessel growth. Our results suggest that OXA could act locally to play a role in corpus luteum demise.

PTPN11 mutations are not responsible for the Cardiofaciocutaneous (CFC) syndrome.

Cardiofaciocutaneous (CFC) syndrome is a multiple congenital anomalies/mental retardation syndrome characterized by congenital heart defects, characteristic facial appearance, short stature, ectodermal abnormalities and mental retardation. It was described in 1986, and to date is of unknown genetic etiology. All reported cases are sporadic, born to non-consanguineous parents and have apparently normal chromosomes. Noonan and Costello syndromes remain its main differential diagnosis. The recent finding of PTPN11 missense mutations in 45-50% of the Noonan patients studied with penetrance of almost 100% and the fact that in animals mutations of this gene cause defects of semilunar valvulogenesis, made PTPN11 mutation screening in CFC patients a matter of interest. We sequenced the entire coding region of the PTPN11 gene in ten well-characterised CFC patients and found no base changes. We also studied PTPN11 cDNA in our patients and demonstrated that there are no interstitial deletions either. The genetic cause of CFC syndrome remains unknown, and PTPN11 can be reasonably excluded as a candidate gene for the CFC syndrome, which we regard as molecular evidence that CFC and Noonan syndromes are distinct genetic entities.

Recombinant immunoblot assay for hepatitis C virus antibody in chronic hepatitis.

Testing for hepatitis C virus by ELISA requires confirmation by recombinant immunoblot assay (RIBA). The first-generation RIBA uses the same antigen as used in the ELISA and one further antigen. A second-generation RIBA in which two further antigens are present, detects positivity that is not found by either the ELISA or the original RIBA. Consequently, although it is adequate to test ELISA positive sera with the first-generation RIBA, the second-generation assay is recommended for confirming negativity.

One course versus two courses of recombinant alpha interferon in chronic C hepatitis.

Fifty-five patients with antibodies to HCV and chronic liver disease have been enrolled in the study. Thirty-four patients were treated with recombinant alpha interferon (IFN, 3 MU daily for 10 days followed by 3 MU twice/week for 3 months), and were compared to 21 untreated controls. Alanine aminotransferase (ALT) normalization was observed in a significant proportion of treated patients (52.9%), but 66.6% of them experienced a relapse after discontinuation of the therapy. The evaluation of the early ALT behavior after the 10 days priming with daily IFN administration was useful in predicting the response. The administration of a second IFN course with the same schedule and duration as the first course did not increase the efficacy of the treatment. Increased dosage and/or prolonged administration are probably required.

Isolate antibody to hepatitis C virus core antigen (C22) by RIBA-2: correlation with HCV-RNA and anti-NS5.

The presence of circulating hepatitis C virus genome (HCV-RNA), elevated ALT levels and antibodies to an NS5-derived synthetic peptide have been examined in 13 subjects with isolate positivity for antibodies to the HCV core antigen (C22) on RIBA-2 testing. All subjects were followed up for 8-18 months (mean 12.4 months). In seven subjects (54%), intermittent or persistent viremia was associated with abnormal ALT levels (6 subjects) and with positivity for antibodies to NS5-peptide (6 subjects). On the other hand, in 6 out of 13 subjects (46%) no viral replication, no liver cytonecrosis and no antibodies to NS5 were found. It is concluded that isolate reactivity to C22 by RIBA-2 is a heterogeneous condition that corresponds to two distinct categories of subjects: those with active HCV infection and those without evidence of virus replication. Although HCV-RNA determination is the most reliable means of identifying HCV carriers, antibodies to NS5 can be a useful marker of virus activity. In fact, antibodies to NS5 were detected in 6 out of 7 viremic patients, compared to 0 out of 6 non-viremic patients (P = 0.004). It remains to be elucidated whether the isolate reactivity to core antigen found in non-viremic subjects represents a specific, HCV-induced antibody response, or is an unrelated crossreactivity.

Analysis of ceftazidime and related compounds by micellar electrokinetic chromatography.

A micellar electrokinetic chromatographic method for the separation and quantification of ceftazidime, its delta2-isomer and pyridine (two ceftazidime related impurities) was developed and validated. Optimised conditions were obtained using an electrolyte system consisting of 25 mM sodium tetraborate, at pH 9.2, and 75 mM sodium dodecylsulphate. A limit of detection of 0.2 microg ml(-1) and a limit of quantitation of 0.6 microg ml(-1) were estimated for pyridine and delta2-isomer: this means that levels of < 0.1% of pyridine and delta2-isomer in ceftazidime can be determined. Calibration curves for all analytes were linear over the studied ranges with correlation coefficients >0.999. Good reproducibility for migration times and corrected peak areas were achieved (RSD % 0.3 and 1.0, respectively). The results demonstrate that the method is reproducible, accurate and appropriate for ceftazidime assay in pharmaceutical samples.

Canine bladderworm (Capillaria plica) infection associated with glomerular amyloidosis.

Capillaria plica (Trichuroidea: Capillariidae), commonly known as bladderworm, is a nematode rarely associated with clinical disease that resides in the lower urinary tract of wild and domestic canids. In the present paper a case of canine urinary capillariosis associated with glomerular amyloidosis is described. The dog, an 8-year-old, male, hunting Jagd terrier had a history of weight loss and diarrhoea and was referred to the University of Parma Teaching Veterinary Hospital (UPTVH). Clinical and laboratory tests shown here suggest that C. plica may be a contributing factor to glomerular amyloidosis.

Pathogenesis and subsequent cross-protection of influenza virus infection in pigs sustained by an H1N2 strain.

The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co-circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross-protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre-determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross-protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post-challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross-protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post-infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross-protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub-type induced less protection against re-infection by homologous and heterologous virus than H1N1 sub-type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross-protection against heterologous infection.

A novel microdeletion syndrome with loss of the MSH2 locus and hereditary non-polyposis colorectal cancer.

Constitutional chromosome deletions can predispose to the development of cancer with the phenotypic characteristics of inherited cancer syndromes, when the deleted region encompasses a tumour suppressor gene. Examples of such conditions are represented by the cytogenetic deletions associated with retinoblastoma, Wilms tumour and familial adenomatous polyposis. So far, no constitutional deletions involving the genes implicated in hereditary non-polyposis colorectal cancer (HNPCC) have been identified. This may be at least partially because of the lack of distinctive phenotypic manifestations in HNPCC. We describe the first case of a constitutional microdeletion associated with HNPCC. Suspicion of a microdeletion was prompted by the association of mental retardation, postnatal growth deficiency, minor congenital anomalies and early onset (37 years) sporadic colon cancer. The patient was found to harbour a microdeletion within chromosome 2p16-p21, including the MSH2 gene. Since there are very few reports of deletions of the 2p16-p21 region, our observation sets the grounds for the definition of a novel multiple congenital anomaly/mental retardation/cancer microdeletion syndrome.

Assisted reproductive technology and congenital overgrowth: some speculations on a case of Pallister-Killian syndrome.

We report on a boy with Pallister-Killian syndrome (PKS) who was conceived by assisted reproductive technology (ART), specifically in vitro fertilization (IVF) with parents' gametes. A prenatal diagnosis performed elsewhere by CVS failed to detect the presence of the isochromosome 12p that was demonstrated postnatally in approximately 50% of cultured skin fibroblasts. Given that the patient did not show the congenital overgrowth typical of PKS, we speculate that ART might have restricted overgrowth in this particular case. More broadly, we hypothesize that overgrowth might protect from early demise fetuses conceived by ART, a technology known to cause low and very low birth weight.

Prevalence of hepatitis E in a central Italian town at high endemicity for hepatitis C virus.

In the spring of 1994, the occurrence of Hepatitis E virus antibodies was evaluated in 653 subjects representing all age-groups in the general population of a Central Italian town, where a high hepatitis C virus prevalence had been reported. The overall anti-HEV prevalence was 2.6% ranging from 1.4% in the 30-49 age-group to 5.7% (p < 0.01) in the 60-70 age-group; none of the subjects under 30 years of age were positive. Sociodemographic variables, such as family size and years of schooling were not associated with HEV exposure. Anti-HEV positivity was found in 1.8% (1/56) of the subjects who were positive for anti-HCV and in 2.7% (16/597) of those who were anti-HCV negative (O.R 1.5; C.I.: 95% = 0.2-11.7). Thus no association was found between HEV and HCV infections. These data suggest a past spread of HEV in this area and underline the occurrence of long-lasting antibodies in infected subjects.

High prevalence of hepatitis C virus infection in a small central Italian town: lack of evidence of parenteral exposure.

In the spring of 1994, anti-HCV prevalence and associated risk factors were evaluated in 681 subjects representing all age-groups in the general population of a small central Italian town. The overall anti-HCV prevalence was 8.4%, ranging from 3.7% in the 30-39 age-group to 18.2% (p < 0.01) in the 60-70 age-group; no subject below 30 years of age was positive. Multiple logistic regression analysis showed that the only variables independently associated with anti-HCV positivity were awareness of unspecified liver disease (O.R. 3.58), age > 45 years (O.R. 2.72), and lowest number of years of schooling (O.R. 11.0) while no association was found with any parenteral exposure such as blood transfusion, intravenous drug use, major or minor surgical intervention, use of glass syringes or dental therapy. The HBsAg prevalence in this population was 1.3%, which corresponds to the rate reported in central Italy. These findings show a high level of HCV endemicity, with no evidence of parenteral exposure.