Reliability of video taped interviews with children suspected of being sexually abused.

OBJECTIVE--To determine the reliability of judgments about the likelihood of child sexual abuse based only on video recorded interviews. DESIGN--Blinded rating of likelihood of abuse by seven professional groups and comparison with consensus rating. SETTING--Child and adolescent psychiatry centre. SUBJECTS--Four people from each of seven professional disciplines: specialist psychiatrists, general psychiatrists, experimental psychologists, trainee social workers, trainee clinical psychologists, lawyers, and police. MAIN OUTCOME MEASURE--Rating of 12 recorded interviews. RESULTS--Agreement between the consensus panel and professional groups was 83% (151/183) for high likelihood cases (seven cases) and 89% (118/132) for low likelihood cases (five). Specialist psychiatrists and the police were better able to identify high likelihood cases than were other groups with less experience of interviewing sexually abused children (91% (48/53) v 79% (102/129); p = 0.05). CONCLUSIONS--Raters could accurately distinguish children with low likelihood of abuse on interview evidence alone, but those with more experience of dealing with sexual abuse were better at identifying high likelihood cases.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

Background

The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.

Methodology/Principal Findings

In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC₅₀) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC₅₀ of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant.

Conclusions

Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.     

Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression

Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.     

Dynamic Storage of Dopamine in Rat Brain Synaptic Vesicles In Vitro

Abstract: The dynamics of catecholamine storage were studied in highly purified, small synaptic vesicles from rat brain both during active uptake or after inhibiting uptake with reserpine, tetrabenazine, or removal of external dopamine. To assess turnover during active uptake, synaptic vesicles were allowed to accumulate [³H]dopamine ([³H]DA) for ～10 min and then diluted 20-fold into a solution containing unlabeled DA under conditions such that active uptake could continue. After dilution, [³H]DA was lost with single exponential kinetics at a half-time of ～4 min at 30°C in 8 mM Cl^? medium, in which both voltage and H⁺ gradients are present in the vesicles. In 90 mM Cl^? medium, in which high H⁺ and Cl^? gradients but no voltage gradient are present, [³H]DA escaped at a half-time of ～7 min. In both high and low Cl^? media, ～40% of [³H]DA efflux was blocked by reserpine or tetrabenazine. The residual efflux also followed first-order kinetics. These results indicate that two efflux pathways were present, one dependent on DA uptake (and thus on the presence of external DA) and the other independent of uptake, and that both pathways function regardless of the type of electrochemical H⁺ gradient in the vesicles. The presence of both uptake-dependent and -independent efflux was observed in experiments using DA-free medium, instead of uptake inhibitors, to prevent uptake. Uptake-independent efflux showed molecular selectivity for catecholamines; [¹⁴C]DA was lost about three times faster than [³H]norepinephrine after adding tetrabenazine directly (without dilution) to vesicles that had taken up comparable amounts of each amine. In addition, the first-order rate constant for uptake-independent efflux showed little change over a 60-fold range of internal DA concentrations, which suggests that this pathway had a high transport capacity. All efflux was blocked at 0°C, suggesting that efflux did not occur through a large pore. There was little or no change in the proton gradient in synaptic vesicles, monitored by [¹⁴C]methylamine equilibration, during the experimental manipulations used here. Thus, the driving force for catecholamine uptake remained approximately constant. The physiological role of uptake-independent efflux could be to allow the monoamine content of synaptic vesicles to be regulated over a time range of minutes and, thereby, control the amount released by exocytosis. These results imply that catecholamines turn over with a half-time of minutes during active uptake by brain synaptic vesicles in vitro.