Therapeutic effects of obeticholic acid (OCA) treatment in a bleomycin-induced pulmonary fibrosis rat model.

PURPOSE: We recently demonstrated a protective effect of the farnesoid X receptor agonist obeticholic acid (OCA) in rat models of bleomycin-induced pulmonary fibrosis (PF). Aim of the present study was to investigate whether the positive effects of OCA treatment are apparent also on ongoing bleomycin-induced PF, i.e., after 2 weeks of bleomycin administration. METHODS: Bleomycin-induced PF rats were treated 2 weeks after bleomycin administration with OCA or pirfenidone for two additional weeks. Pulmonary function test was performed at 2 and 4 weeks in all experimental groups. At the same time points, lung morphological features and mRNA expression profile of genes related to fibrosis, inflammation and epithelial-mesenchymal transition were also assessed. RESULTS: After 2 weeks, bleomycin significantly increased the pressure at the airway opening (PAO), a functional parameter related to fibrosis-induced lung stiffness, and induced diffuse lung interstitium fibrosis, with upregulation of inflammation (IL1ß, MCP1) and tissue remodeling (COL1A1, COL3A1, ET1, MMP7, PDGFa, αSMA, SNAI1) markers. At week four, a further increase of lung fibrosis and PAO was observed, accompanied by upregulation of extracellular matrix-related mRNA expression. OCA administration, even after the establishment of PF, significantly improved pulmonary function, normalizing PAO, and reverted the bleomycin-induced lung alterations, with significant reduction of markers of inflammation (CD206, COX2, HIF1, IL1ß, MCP1), epithelial proliferation (CTGF, PDGFa) and fibrosis (COL1A1, COL3A1, ET1, FN1, MMPs, αSMA, SNAIs, TGFß1, TIMPs). Results with OCA were similar or superior to those obtained with pirfenidone. CONCLUSIONS: In conclusion, our results demonstrate a significant therapeutic effect of OCA in already established PF.

High added value of a population-based participatory surveillance system for community acute gastrointestinal, respiratory and influenza-like illnesses in Sweden, 2013-2014 using the web.

In 2013-2014, the Public Health Agency of Sweden developed a web-based participatory surveillance system, Hӓlsorapport, based on a random sample of individuals reporting symptoms weekly online, to estimate the community incidence of self-reported acute gastrointestinal (AGI), acute respiratory (ARI) and influenza-like (ILI) illnesses and their severity. We evaluated Hӓlsorapport's acceptability, completeness, representativeness and its data correlation with other surveillance data. We calculated response proportions and Spearman correlation coefficients (r) between (i) incidence of illnesses in Hӓlsorapport and (ii) proportions of specific search terms to medical-advice website and reasons for calling a medical advice hotline. Of 34 748 invitees, 3245 (9·3%) joined the cohort. Participants answered 81% (139 013) of the weekly questionnaires and 90% (16 351) of follow-up questionnaires. AGI incidence correlated with searches on winter-vomiting disease [r = 0·81, 95% confidence interval (CI) 0·69-0·89], and ARI incidence correlated with searches on cough (r = 0·77, 95% CI 0·62-0·86). ILI incidence correlated with the web query-based estimated incidence of ILI patients consulting physicians (r = 0·63, 95% CI 0·42-0·77). The high response to different questionnaires and the correlation with other syndromic surveillance systems suggest that Hӓlsorapport offers a reasonable representation of AGI, ARI and ILI patterns in the community and can complement traditional and syndromic surveillance systems to estimate their burden in the community.

Genetic susceptibility to cutaneous melanoma in southern Switzerland: role of CDKN2A, MC1R and MITF.

BACKGROUND: Nearly 10% of all cases of cutaneous melanoma (CM) occur in patients with a personal or family history of the disease. OBJECTIVES: To obtain information about genetic predisposition to CM in Ticino, the southern region of Switzerland, a zone with moderate-to-high CM incidence. METHODS: We identified germline mutations in highly CM-associated genes (CDKN2A and CDK4) and low/medium-penetrance variants (MC1R and MITF) in patients with multiple primary CMs or individuals with one or more CM and a positive family history for CM or pancreatic cancer among first- or second-degree relatives. Healthy blood donors (n = 146) were included as a control group. RESULTS: From July 2010 to July 2012, 57 patients (41 pedigrees) were included. Twenty-six were melanoma-prone families (with at least two cases) and 15 had multiple CMs. Pancreatic cancer was found in six families. The CDKN2A mutation p.V126D was identified in seven patients (four families) with a founder effect, whereas CDKN2A A148T was detected in seven cases (five families) and seven healthy donors (odds ratio 2·76, 95% confidence interval 0·83-9·20). At least one MC1R melanoma-associated polymorphism was detected in 32 patients (78%) and 97 healthy donors (66%), with more than one polymorphism in 12 patients (29%) and 25 healthy donors (17%). The MITF variant p.E318K was identified in four patients from three additional pedigrees (7%) and one healthy control (0·7%). CONCLUSIONS: Inclusion criteria for the Ticino population for genetic assessment should follow the rule of two (two affected individuals in a family or a patient with multiple CMs), as we detected a CDKN2A mutation in almost 10% of our pedigrees (four of 41), MITF p.E318K in 7% (three of 41) and a higher number of MC1R variants than in the control population.

Overexpression of histamine H4 receptors in the kidney of diabetic rat.

OBJECTIVE AND DESIGN: The renal expression of H1 and H2 receptors has previously been demonstrated, while that of the H4 receptor has been poorly investigated, and thus the aim of this research was to investigate the expression of the H4 receptor in the kidney of diabetic rats. MATERIAL OR SUBJECTS: 24 8-week-old male Wistar rats. TREATMENT: Diabetes was induced in 12 rats by a single intravenous injection of streptozotocin, and animals were killed 6 weeks later. METHODS: Kidneys were collected and processed for quantitative PCR or immunohistochemical analyses. To ascertain the renal topology of the H4 receptor, colocalization experiments were performed with a series of markers. RESULTS: H4 receptor is expressed in healthy rats, although at a very low level, and is strongly upregulated in diabetic animals. Immunohistochemical analysis revealed the highest immune-positivity in the medulla. Colocalization experiments revealed a close overlap in expression topology of the H4 receptor and both Tamm-Horsfall glycoprotein and aquaporin 1 was observed. CONCLUSIONS: The results demonstrate, for the first time, that the H4 receptor is expressed in the kidney mainly by resident renal cells of the loop of Henlé and that this receptor is significantly overexpressed in diabetic animals, thus suggesting a possible role in the pathogenesis of diabetes-associated renal disease.

Chemorepulsion of axons in the developing mammalian central nervous system.

During development of the nervous system, distinct populations of nerve cells extend specialized processes, axons and dendrites, over considerable distances to locate their targets. There is strong evidence for two general mechanisms by which these connections are made. The first involves attractive and repulsive interactions, both between cells and between them and their extracellular matrix. The second depends on the release of diffusible chemoattractants by target structures. Evidence is now provided for a mechanism of axon guidance in which diffusible chemorepulsive factors create exclusion zones for developing axons, causing them to turn away from inappropriate territory.

Chemorepulsion of developing motor axons by the floor plate.

In the developing nervous system, motor axons grow away from the ventral midline floor plate, suggesting that the latter might be a source of repulsive axonal guidance cues. In donor to host transplantation experiments, ectopic pieces of floor plate were positioned between chick hindbrain motor neurons and their exit points. Immunohistochemistry and retrograde axonal labeling techniques demonstrated that motor axons diverted from their normal pathways to avoid grafted floor plate, often traversing abnormally long circuitous trajectories to reach exit points. When ventral explants of rat hindbrain and spinal cord were cocultured at a distance from floor plate explants within collagen gel matrices, the outgrowth of motor axons was dramatically reduced from explant borders that faced the floor plate. Thus, the floor plate secretes diffusible repulsive cues in vitro that may exclude motor axons from the midline during development.

Surround repulsion of spinal sensory axons in higher vertebrate embryos.

We have tested whether the orientation of axons sprouting from bipolar dorsal root ganglion neurons is influenced by diffusible cues from surrounding tissues. Surface ectoderm, dermomyotome, and notochord exert strong chemorepulsion on axons growing in collagen gels, operating at separations beyond those found in vivo and active in cocultures of chick and mouse tissues. Basal and alar plates of the neural tube are devoid of activity, as is the posterior-half-sclerotome, which repels in a contact-dependent manner. When ganglia are sandwiched between dermomyotome and notochord placed at a distance, axon growth is channeled in a bipolar trajectory. These results show that gradients of diffusible repulsion molecules flanking axon pathways can generate linear patterns of axon growth. We suggest that such "surround repulsion" may function generally, in concert with contact-dependent guidance mechanisms, to guide axons in the developing nervous system.

POMT1 and POMT2 mutations in CMD patients: a multicentric Italian study.

Mutations in POMT1 and POMT2 genes were originally identified in Walker-Warburg syndrome (WWS) and subsequently reported in patients with milder phenotypes characterised by mental retardation with or without brain abnormalities and without ocular malformations. As part of a multicentric Italian study we screened the POMT1 and POMT2 genes in 61 congenital muscular dystrophy (CMD) patients with alpha-dystroglycan reduction on muscle biopsy and/or clinical and radiological findings suggestive of the known forms of CMD with alpha-dystroglycan deficiency. The aim of the study was to establish how frequently mutations in POMT1 and POMT2 occur in CMD patients in the Italian population and to evaluate the spectrum of associated phenotypes. Thirteen patients showed mutations in POMT1 and five harboured mutations in POMT2, accounting for a total of 20 different mutations, eight of which were novel (two in POMT1 and six in POMT2). Normal brain MRI associated with mental retardation and microcephaly was the most frequent finding in patients with mutations in POMT1 (six out of 13), but was also found in a patient with POMT2 mutations. Predominant cerebellar hypoplasia was also frequent both in patients with POMT1 (three out of 13) and POMT2 (three out of 5) mutations. A MEB phenotype with frontal cortical dysplasia and pons abnormalities was found in two patients with POMT1 and in one with POMT2 mutations, while a WWS phenotype was only found in a case with mutations in POMT1. Mutations causing frameshifts and stop codons were responsible for the more severe phenotypes. Our results provide further evidence that, as previously reported for FKRP, the array of mutations in POMT1 and POMT2 is ample and the spectrum of associated phenotypes is wider than initially thought.

The mTOR inhibitor rapamycin significantly improves facial angiofibroma lesions in a patient with tuberous sclerosis.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with an incidence of approximately one in 6000. It arises from a genetic abnormality involving either the TSC1 gene on chromosome 9 or the TSC2 gene on chromosome 16. The protein product of TSC1 is hamartin and that of TSC2 is tuberin. In cells, hamartin and tuberin form a complex which inhibits the mammalian target of rapamycin (mTOR), a central controller of cell growth and proliferation. Angiofibroma affects 70-80% of patients with TSC, typically on the face. We report a patient with TSC with recurrent life-threatening haemorrhage from both kidneys due to extensive angiomyolipoma formation leading to bilateral nephrectomy and renal transplantation. Immunosuppressive treatment with rapamycin, a specific mTOR inhibitor, initiated because of renal transplantation, reduced facial angiofibroma dramatically.

Laboratory tests for evaluating the level of attenuation of bluetongue virus.

One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK(21c13) or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all animals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained.

Axon guidance. Growth cones say no.

A recently characterized protein stops developing axons from growing; a gene family encoding related molecules has also been described.

Axon guidance: following the Eph plan.

Recently discovered 'Eph' family receptors and their ligands appear likely to provide the 'cytochemical tags' that Sperry speculated enable axons projecting from the retina to find their correct targets in the brain.

The Hammersmith functional score correlates with the SMN2 copy number: a multicentric study.

Previous studies showed that SMN2 copy number correlates inversely with the disease severity. Our aim was to evaluate SMN2 copy numbers and the Hammersmith functional motor scale in 87 patients with SMA II in order to establish whether, within SMAII, the number of copies correlates with the severity of functional impairment. Our results showed a relative variability of functional scores, but a significant correlation between the number of SMN2 genes and the level of function.

An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy.

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10(6.3) TCID(50) of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3-9.3log(2)(ED50%/50 microl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

Clinical, humoral and IFNgamma responses of cattle to infection with Mycoplasma mycoides var. mycoides small colony and attempts to condition the pathogenesis of the infection.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides var. mycoides small colony (MmmSC), is one of the most important diseases of cattle in Africa. The role of innate or acquired cell mediated and humoral immunity in conferring protection against MmmSC infection has not yet been elucidated. On the other hand, the pathological lesions caused by the aetiological agent have been considered indicative of an immunopathological process. In this study ten naïve cattle were exposed to in-contact infection with animals infected by intubation with a strain of MmmSC. Clinical signs, antibody response, IFNgamma release and pathological changes at necropsy were analysed and compared with the events following in-contact infection of an equal number of animals kept under daily treatment with cyclosporine for the entire observation period of 84 days. Cyclosporine is a suppressor of the immune response related to the T-cell system. Under the conditions of the experiment, cyclosporine appeared to condition the pathogenesis of CBPP by delaying the events that follow infection, bringing further support to the possibility that the immune response may have an impact on the disease outcome.

Reliability of the Hammersmith functional motor scale for spinal muscular atrophy in a multicentric study.

The aim of this study was to validate the Hammersmith functional motor scale for children with spinal muscular atrophy in a large cohort of 90 non-ambulant children with spinal muscular atrophy type 2 or 3. All had a baseline assessment (T0) and were reassessed either at 3 months (T1) (n = 66) or at 6 months (T2) (n = 24). Inter-observer reliability, tested on 13 children among 3 examiners, was > 95%. Of the 66 children examined after 3 months 4 had adverse effects in between assessments and were excluded from the analysis. Forty-two (68%) of the remaining 62 reassessed had no variation in scores between T0 and T1 and 13 (21%) were within +/- 1 point. 9 (37.5%) of the 24 children reassessed after 6 months had no variation in scores between T0 and T2 and another 9 (37.5%) had variations within +/- 1 point. Our study confirms previous observations of the reliability of the scale and helps to establish a baseline for assessing changes of functional ability over 3 and 6 month intervals. This information can be valuable in view of therapeutic trials.

Glucagon-like peptide 2 counteracts the mucosal damage and the neuropathy induced by chronic treatment with cisplatin in the mouse gastric fundus.

BACKGROUND: Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone synthesized and secreted by the enteroendocrine 'L' cells able to exert intestine-trophic and anti-inflammatory effects. The antineoplastic drug cisplatin causes gastrointestinal alterations with clinical symptoms (nausea and vomiting) that greatly affect the therapy compliance. Experimentally, it has been reported that chronic cisplatin treatment caused mucosal damage and enteric neuropathy in the rat colon. METHODS: We investigated, through a combined immunohistochemical and functional approach, whether [Gly(2) ]GLP-2, a GLP-2 analog, was able to counteract the detrimental effects of long-term cisplatin administration in the mucosa and myenteric neurons of mouse gastric fundus. KEY RESULTS: Morphological experiments showed a reduction in the epithelium thickness in cisplatin-treated mice, which was prevented by [Gly(2) ]GLP-2 co-treatment. Immunohistochemistry demonstrated that cisplatin caused a significant decrease in myenteric neurons, mainly those expressing neuronal nitric oxide synthase (nNOS), that was prevented by [Gly(2) ]GLP-2 co-treatment. In the functional experiments, [Gly(2) ]GLP-2 co-treatment counteracted the increase in amplitude of the neurally induced contractions observed in strips from cisplatin-treated animals. The NO synthesis inhibitor L-N(G) -nitro arginine caused an increase in amplitude of the contractile responses that was greater in preparations from cisplatin+[Gly(2) ]GLP-2 treated mice compared to the cisplatin-treated ones. CONCLUSIONS & INFERENCES: The results demonstrate that in cisplatin long-term treated mice [Gly(2) ]GLP-2 is able to counteract both the mucosal gastric fundus damage, by preventing the epithelium thickness decrease, and the neuropathy, by protecting the nNOS neurons. Taken together, the present data suggest that [Gly(2) ]GLP-2 could represent an effective strategy to overcome the distressing gastrointestinal symptoms present during the anti-neoplastic therapy.