Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

Evaluation of a mouse model for the West Nile virus group for the purpose of determining viral pathotypes.

West Nile virus (WNV; family Flaviviridae; genus Flavivirus) group members are an important cause of viral meningoencephalitis in some areas of the world. They exhibit marked variation in pathogenicity, with some viral lineages (such as those from North America) causing high prevalence of severe neurological disease, whilst others (such as Australian Kunjin virus) rarely cause disease. The aim of this study was to characterize WNV disease in a mouse model and to elucidate the pathogenetic features that distinguish disease variation. Tenfold dilutions of five WNV strains (New York 1999, MRM16 and three horse isolates of WNV-Kunjin: Boort and two isolates from the 2011 Australian outbreak) were inoculated into mice by the intraperitoneal route. All isolates induced meningoencephalitis in different proportions of infected mice. WNVNY99 was the most pathogenic, the three horse isolates were of intermediate pathogenicity and WNVKUNV-MRM16 was the least, causing mostly asymptomatic disease with seroconversion. Infectivity, but not pathogenicity, was related to challenge dose. Using cluster analysis of the recorded clinical signs, histopathological lesions and antigen distribution scores, the cases could be classified into groups corresponding to disease severity. Metrics that were important in determining pathotype included neurological signs (paralysis and seizures), meningoencephalitis, brain antigen scores and replication in extra-neural tissues. Whereas all mice infected with WNVNY99 had extra-neural antigen, those infected with the WNV-Kunjin viruses only occasionally had antigen outside the nervous system. We conclude that the mouse model could be a useful tool for the assessment of pathotype for WNVs.

Descriptive epidemiology of spotty liver disease in Australian cage-free brown egg layer chicken flocks with a scratch area.

Spotty Liver Disease (SLD), caused by Campylobacter hepaticus or C. bilis infection in adult female chickens continues to emerge as a major disease problem in cage-free production systems. Free range production has become the predominant system in Australian egg production and SLD is widespread in these farms. Previous studies have identified having a scratch area as a key determinant for SLD occurrence. An Australia-wide survey of egg production flocks with scratch areas was conducted regarding SLD including 48 individual flocks. Descriptive information on the facilities and flock management practices was reported. The incidence of SLD, age of first outbreak, initial mortality rate, duration of elevated mortality, and magnitude and duration of any associated egg production decline are described. Recurrence of SLD in the same flock was also reported and discussed. Therapies applied were recorded and assessed across SLD severity and duration. SLD occurred in 66.7% of layer flocks whose facility included a scratch area. Recurrent SLD outbreaks occurred in 31% of flocks experiencing SLD. Antibiotic medication reduced duration of mortality and egg production decline. Antibiotic therapy was associated with reduced duration of mortality and a less severe and shorter duration of egg production drops compared to untreated flocks. PCR detection of C. hepaticus in cloacal swabs and house dust samples and a serological ELISA test were compared and evaluated as diagnostic aids or as possible predictors of SLD outbreaks. The ELISA showed substantial agreement with detection of C. hepaticus in cloacal swabs by PCR. Examining composite house dust samples by PCR for C. hepaticus DNA appeared to be the most convenient and cost-effective aid to diagnosis and as a putative predictor for SLD outbreaks.

The membrane effects of melittin on gastric and colorectal cancer.

The cytotoxic effects of melittin, a bee-venom peptide, have been widely studied towards cancer cells. Typically, these studies have examined the effect of melittin over extended-time courses (6-24 hours), meaning that immediate cellular interactions have been overlooked. In this work, we demonstrate the rapid effects of melittin on both gastric and colorectal cancer, specifically AGS, COLO205 and HCT-15 cell lines, over a period of 15 minutes. Melittin exhibited a dose dependent effect at 4 hours of treatment, with complete cellular death occurring at the highest dose of 20 µg/mL. Interestingly, when observed at shorter time points, melittin induced cellular changes within seconds; membrane damage was observed as swelling, breakage or blebbing. High-resolution imaging revealed treated cells to be compromised, showing clear change in cellular morphology. After 1 minute of melittin treatment, membrane changes were observed, and intracellular material could be seen expelled from the cells. Overall, these results enhance our understanding of the fast acting anti-cancer effects of melittin.

Management of factitious disorders: a systematic review.

BACKGROUND: The literature regarding the management of factitious disorder (FD) is diverse and generally of case reports or case series. To date there has been no systematic review of the effectiveness of management techniques. METHODS: Systematic review of all evidence reporting the management and subsequent outcome in FD. Data were extracted and outcomes were assessed using an adaptation of the Global Improvement Scale. Results were analysed by parametric statistical tests; a meta-analysis was not possible. RESULTS: Thirty-two case reports and 13 case series were eligible for inclusion. Analysis of the case reports found no significant difference in outcomes between confrontational and non-confrontational approaches [t(29) = 0.72, p = 0.48], between treatment with psychotherapy compared to no psychotherapy [t(30) = 0.69, p = 0.48], and when psychiatric medication had been prescribed compared with not [t(30) = 0.35, p = 0.73]. A trend was observed that a longer length of treatment lead to better outcomes, but this was not significant [F(5, 26) = 1.17, p = 0.35]. The consecutive case series demonstrated that many FD sufferers were not engaged in treatment and were lost to follow-up but did not provide any strong evidence regarding the effectiveness of different management approaches. CONCLUSIONS: There is an absence of sufficient robust research to determine the effectiveness of any management technique for FD. The establishment of a central reporting register to facilitate the development of evidence-based guidelines is recommended.

Osteoclastogenesis-related cytokines and peri-prosthetic osteolysis in revision metal-on-metal total hip replacements.

PURPOSE: Peri-prosthetic osteolysis is a major cause for revision hip arthroplasty; various cytokines including those in the osteoclastogenesis pathway have been identified as potentially key in the osteolysis process. Adverse reactions to metal debris in metal-on-metal total hip replacements have led to an increase in revision procedures. This study examines the levels of osteoclastogenesis-related cytokines in serum and synovial fluid samples obtained from patients at the time of revision metal-on-metal total hip replacement and compares between patients with and without radiographic evidence of peri-prosthetic osteolysis. METHODS: Sandwich ELISA techniques were used to detect IL-6, IL-18, M-CSF, sRANKL and OPG in the samples. Results were analysed with linear regression, Fisher's tests and t-tests; p<0.05 considered significant. Samples from 36 patients (18 with osteolysis, 18 without osteolysis) were analysed. RESULTS: There was wide variation in the detectable levels of cytokines. No significant differences were found between patients with and without osteolysis in mean synovial fluid levels of IL-6 (p = 0.863), IL-18 (p = 0.324), M-CSF (p = 0.508), sRANKL (p = 0.884), OPG (p = 0.776) or mean serum levels of OPG (p = 0.993) or sRANKL (p = 0.565) (insufficient detection of IL-6, IL-18 or M-CSF in serum samples). A correlation was found between synovial fluid levels of IL-6 and OPG in patients without osteolysis (r2 = 0.618, p<0.001) but not with osteolysis (r2 = 0.0004). CONCLUSIONS: These results indicate that the process of peri-prosthetic osteolysis is complex and multifactorial; there may also be an influence of metallosis. Further research is needed to increase understanding of peri-prosthetic osteolysis and influence clinical practice.

Removal of a below knee plaster cast worn for 28 months: a case report.

INTRODUCTION: An unusual situation in which a below knee cast was removed after 28 months is reported. To the best of our knowledge no similar cases have been reported in the literature. CASE PRESENTATION: The cast was removed from the leg of a 45-year-old Caucasian woman. Significant muscle atrophy and dense skin scales were present but the underlying skin surface was relatively healthy with only small pitted 1-2 mm ulcers. No pathogenic organisms were cultured from this environment. CONCLUSION: It seems likely that skin can tolerate cast immobilization for prolonged duration.