Correction: Alopecia areata patients show deficiency of FOXP3+CD39+ T regulatory cells and clonotypic restriction of Treg TCRß-chain, which highlights the immunopathological aspect of the disease.

[This corrects the article DOI: 10.1371/journal.pone.0210308.].

Alopecia areata patients show deficiency of FOXP3+CD39+ T regulatory cells and clonotypic restriction of Treg TCRß-chain, which highlights the immunopathological aspect of the disease.

Alopecia areata (AA) is a hair loss disorder resulting from an autoimmune reaction against hair follicles. T-helper 1 cells are a major contributor to this disorder, but little is known about the role of T-regulatory cells (Tregs) in AA. Here, we analysed the distribution of circulating Treg subsets in twenty AA patients with active hair loss and fifteen healthy subjects by flow cytometry. The Treg suppressor HLA-DR+ subpopulation was significantly reduced in the patients (P<0.001) and there were significantly fewer cells expressing CD39 among the CD4+CD25+Foxp3+ Treg subpopulation in patients (P = 0.001). FOXP3 CD39 Treg cells were also reduced in hair follicles; by 75% in non-lesional skin and 90% in lesional skin, when compared to control healthy skin. To further characterise Treg cells in AA; Tregs (CD4+CD25+FOXP3+) were investigated for their TCRß sequence. PCR products analysed by Next Generation Sequencing techniques, showed that all frequent public clonotypes in AA Tregs were also present in controls at relatively similar frequencies, excepting two public clonotypes: CATSRDEGGLDEKLFF (V15 D1 J1-4) and CASRDGTGPSNYGYTF (V2 D1 J1-2), which were exclusively present in controls. This suggests that these Treg clonotypes may have a protective effect and that they may be an exciting subject for future therapeutic applications.

Outcomes of toric supplementary intraocular lenses for residual astigmatic refractive error in pseudophakic eyes.

PURPOSE: To evaluate rotational stability and visual and refractive outcomes of supplementary toric IOLs (Sulcoflex Toric 653T, Rayner Intraocular Lenses Ltd) for residual astigmatic refractive error in pseudophakic eyes. METHODS: A retrospective interventional case series was conducted in a single surgeon practice. Charts of patients who had Sulcoflex Toric supplementary IOLs inserted between June 2009 and September 2015 were reviewed. Outcomes were compared between eyes with and without prior corneal transplant. Patients with at least 3-months follow-up were included. RESULTS: In 51 eyes, mean UDVA improved from 20/86 to 20/43 (p = 0.002), though UDVA was better in eyes without corneal grafts (20/31) than eyes with (20/62). The proportion of eyes achieving 20/20 UDVA was 43%, 61% and 17% overall, in eyes with prior graft and in eyes with no prior graft, respectively. Sixty-four percentage achieved a spherical equivalent of within 0.5D of target (84% no graft, 34% prior graft). Fifty-three percentage of eyes achieved a cylinder of within 0.5D of target (no graft: 73%, prior graft: 0%). Mean lens rotation was 8.23° on day 1, and mean maximal rotation during follow-up was 17.63°. Sixty-two percentage of IOLs required repositioning. Of those that required repositioning, this was conducted a mean of 2.3 times. The mean final IOL rotation (following repositioning if required) was 6.17°. CONCLUSION: Sulcoflex Toric supplementary IOLs result in good visual and refractive outcomes in eyes with no prior corneal graft. However, outcomes are sub-optimal in eyes with prior corneal transplantation, and the majority of lenses require repositioning.

Epigallocatechin-3 Gallate Inhibits STAT-1/JAK2/IRF-1/HLA-DR/HLA-B and Reduces CD8 MKG2D Lymphocytes of Alopecia Areata Patients.

BACKGROUND: Alopecia areata (AA) is associated with Interferon- γ (IFN-γ) mediated T-lymphocyte dysfunction and increased circulating Interleukine-17 (IL-17) levels. Epigallocatechin-3-gallate (EGCG) specifically inhibits IFN-γ pathways and unlike Janus Kinase 1 and 2 (JAK1/JAK2) inhibitors (tofacitinib, ruxolitinib), EGCG is safer, more cost-effective, and is a topically active agent. Our objective is to test the mode of action of EGCG in vitro and ex vivo using HaCat, Jurkat cell lines, and peripheral blood mononuclear cells (PBMCs) of AA patients and healthy controls (HCs), respectively. METHODS: distribution of T helper cells (Th1, Th17), and cytotoxic cells (CD8) in PBMCs isolated from 30 AA patients and 30 HCs was investigated by flowcytomterty. In vitro treatment of HaCat and Jurkat cells with 40 µm EGCG for 48 h was performed to measure the level of phosphorylation of signal transducer and activator of transcription protein STAT1, and replicated in ex vivo model using PBMCs of AA patients. RESULTS: Interestingly, 40 µm EGCG is capable of completely inhibiting phosphorylation of STAT1 after 48 h in HaCat and Jurkat cells and ex vivo in PBMCs of AA patients. Based on QPCR data, the action of EGCG on p-STAT1 seems to be mediated via downregulation of the expression of JAK2 but not JAK1 leading to the inhibition of human leukocyte antigens (HLA-DR and HLA-B) expression probably via IRF-1. On the other hand, AA patients have significantly increased levels of Th1, Th17, and CD8 cells and the production of IFN-γ and IL-17 by PBMCs in AA patients was significantly higher compared to HC; p = 0.008 and p = 0.006, respectively. Total numbers of CD8+ cells were not significantly different between treated and untreated samples. However, CD8+ cells with positive Natural killer group 2 member D (NKG2D) transmembrane receptor (CD8+ NKG2D+ subset) was significantly reduced when PBMCs were treated with 20 µm EGCG for 48 h. CONCLUSION: These results suggest that EGCG has a synergistic action that inhibits expression of HLA-DR and HLA-B molecules via the IFN-γ pathway to maintain immune privilege in HF; also it reduces CD8+ NKG2D+ subset.

Whole Genome Sequencing in an Acrodermatitis Enteropathica Family from the Middle East.

We report a family from Tabuk, Saudi Arabia, previously screened for Acrodermatitis Enteropathica (AE), in which two siblings presented with typical features of acral dermatitis and a pustular eruption but differing severity. Affected members of our family carry a rare genetic variant, p.Gly512Trp in the SLC39A4 gene which encodes a zinc transporter; disease is thought to result from zinc deficiency. Similar mutations have been reported previously; however, the variable severity within cases carrying the p.Gly512Trp variant and in AE overall led us to hypothesise that additional genetic modifiers may be contributing to the disease phenotype. Therefore whole genome sequencing was carried out in five family members, for whom material was available to search for additional modifiers of AE; this included one individual with clinically diagnosed AE. We confirmed that the p.Gly512Trp change in SLC39A4 was the only candidate homozygous change which was sufficiently rare (ExAC allele frequency 1.178e-05) and predicted deleterious (CADD score 35) to be attributable as a fully penetrant cause of AE. To identify other genes which may carry relevant genetic variation, we reviewed the relevant literature and databases including Gene Ontology Consortium, GeneMANIA, GeneCards, and MalaCards to identify zinc transporter genes and possible interacting partners. The affected individual carried variants in RECQL4 and GPAA1 genes with ExAC allele frequency <0.01 and CADD score >10. p.Gly512Trp is highly likely to be the pathogenic variant in this family. This variant was previously detected in a Tunisian proband with perfect genotype-phenotype segregation suggestive of pathogenicity. Further research is required in this area due to small sample size, but attention should be given to RECQL4 and GPAA1 to understand their role in the skin disease.

Meta-Analysis of Autoimmune Regulator-Regulated Genes in Human and Murine Models: A Novel Human Model Provides Insights on the Role of Autoimmune Regulator in Regulating STAT1 and STAT1-Regulated Genes.

Autoimmune regulator (AIRE) regulates promiscuous expression of tissue-restricted antigens in medullary epithelial cells (mTEC) of the thymus. To understand the diverse effects of AIRE, it is crucial to elucidate the molecular mechanisms underlying the process of AIRE-regulated gene expression. In this study, we generated a recombinant AIRE expression variant of the TEC 1A3 human cell line, TEC 1A3 AIREhi, to determine genes targeted by AIRE, and using microarray analysis, we identified 482 genes showing significant differential expression (P < 0.05; false discovery rate <5%), with 353 upregulated and 129 downregulated by AIRE expression. Microarray data were validated by quantitative PCR, confirming the differential expression of 12 known AIRE-regulated genes. Comparison of AIRE-dependent differential expression in our cell line model with murine datasets identified 447 conserved genes with a number of transcription regulatory interactions, forming several key nodes, including STAT1, which had over 30 interactions with other AIRE-regulated genes. As STAT1 mutations cause dominant chronic mucocutaneous candidiasis and decreased STAT1 levels in monocytes of autoimmune polyglandular syndrome 1 (APS-1) patients, it was important to further characterize AIRE-STAT1 interactions. TEC 1A3AIREhi were treated with the STAT1 phosphorylation inhibitors fludarabine and LLL3 showed that phosphorylated STAT1 (p-STAT1) was not responsible for any of the observed differential expression. Moreover, treatment of TEC 1A3 AIREhi with STAT1 shRNA did not induce any significant variation in the expression of unphosphorylated STAT1 (U-STAT1) downstream genes, suggesting that these genes were directly regulated by AIRE but not via U-STAT1. The novel model system we have developed provides potential opportunities for further analysis of the pathogenesis of (APS-1) and the wider roles of the AIRE gene.

Genome-Wide Analysis of Circulating Cell-Free DNA Copy Number Detects Active Melanoma and Predicts Survival.

BACKGROUND: A substantial number of melanoma patients develop local or metastatic recurrence, and early detection of these is vital to maximise benefit from new therapies such as inhibitors of BRAF and MEK, or immune checkpoints. This study explored the use of novel DNA copy-number profiles in circulating cell-free DNA (cfDNA) as a potential biomarker of active disease and survival. PATIENTS AND METHODS: Melanoma patients were recruited from oncology and dermatology clinics in Sheffield, UK, and cfDNA was isolated from stored blood plasma. Using low-coverage whole-genome sequencing, we created copy-number profiles from cfDNA from 83 melanoma patients, 44 of whom had active disease. We used scoring algorithms to summarize copy-number aberrations and investigated their utility in multivariable logistic and Cox regression analyses. RESULTS: The copy-number aberration score (CNAS) was a good discriminator of active disease (odds ratio, 3.1; 95% CI, 1.5-6.2; P = 0.002), and CNAS above or below the 75th percentile remained a significant discriminator in multivariable analysis for active disease (P = 0.019, with area under ROC curve of 0.90). Additionally, mortality was higher in those with CNASs above the 75th percentile than in those with lower scores (HR, 3.4; 95% CI, 1.5-7.9; P = 0.005), adjusting for stage of disease, disease status (active or resected), BRAF status, and cfDNA concentration. CONCLUSIONS: This study demonstrates the potential of a de novo approach utilizing copy-number profiling of cfDNA as a biomarker of active disease and survival in melanoma. Longitudinal analysis of copy-number profiles as an early marker of relapsed disease is warranted.

Small population bias and sampling effects in stochastic mortality modelling.

We propose the use of parametric bootstrap methods to investigate the finite sample distribution of the maximum likelihood estimator for the parameter vector of a stochastic mortality model. Particular emphasis is placed on the effect that the size of the underlying population has on the distribution of the MLE in finite samples, and on the dependency structure of the resulting estimator: that is, the dependencies between estimators for the age, period and cohort effects in our model. In addition, we study the distribution of a likelihood ratio test statistic where we test a null hypothesis about the true parameters in our model. Finally, we apply the LRT to the cohort effects estimated from observed mortality rates for females in England and Wales and males in Scotland.

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy.

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of "general" immune checkpoint drugs in this subset of patients.

Complexities of abdominoperineal surgery: synchronous resection of an ano-rectal adenocarcinoma and pelvic schwannoma.

Abdominoperineal resection (APR) is indicated for low rectal/ano-rectal cancers. It necessitates fastidious pelvic dissection posing certain operative difficulties. We present the surgical challenges in a unique case of a patient presenting with a low rectal adenocarcinoma and a synchronous pelvic schwannomas, both requiring resection. A 71-year-old gentleman presented for surveillance colonoscopy following previous excision of colonic polyps. This investigation revealed a polypoid mass at the ano-rectal junction which was histologically proven as an adenocarcinoma with high-grade dysplasia. A staging computed tomography scan revealed an incidental 10 × 15 cm homogeneous, pre-sacral mass. After meticulous operative planning, the patient underwent successful open resection of this mass and concurrent APR for his low rectal lesion. This case demonstrates a rare presentation of a low rectal adenocarcinoma and concurrent pelvic schwannoma. We discuss the technical difficulties encountered in the management of such complex pelvic tumours and highlight the successful outcomes of the synchronous resection.

Expression of Cancer/Testis genes in ductal carcinoma in situ and benign lesions of the breast.

Cancer/testis (CT) genes represent a unique class of genes, which are expressed by germ cells, normally silenced in somatic cells, but activated in various cancers. CT proteins can elicit spontaneous immune responses in cancer patients and this feature makes them attractive targets for immunotherapy-based approaches. We have previously reported that CTs are relatively commonly expressed in estrogen receptor (ER) negative, high risk carcinomas. In this study, we examined the expression of selected CT genes in ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS) and benign proliferative lesions of the breast. ER negative DCIS were found to be associated with significant CT gene expression together with HER2 positivity and a marked stromal immune response.

Centrosomal localisation of the cancer/testis (CT) antigens NY-ESO-1 and MAGE-C1 is regulated by proteasome activity in tumour cells.

The Cancer/Testis (CT) antigen family of genes are transcriptionally repressed in most human tissues but are atypically re-expressed in many malignant tumour types. Their restricted expression profile makes CT antigens ideal targets for cancer immunotherapy. As little is known about whether CT antigens may be regulated by post-translational processing, we investigated the mechanisms governing degradation of NY-ESO-1 and MAGE-C1 in selected cancer cell lines. Inhibitors of proteasome-mediated degradation induced the partitioning of NY-ESO-1 and MAGE-C1 into a detergent insoluble fraction. Moreover, this treatment also resulted in increased localisation of NY-ESO-1 and MAGE-C1 at the centrosome. Despite their interaction, relocation of either NY-ESO-1 or MAGE-C1 to the centrosome could occur independently of each other. Using a series of truncated fragments, the regions corresponding to NY-ESO-1(91-150) and MAGE-C1(900-1116) were established as important for controlling both stability and localisation of these CT antigens. Our findings demonstrate that the steady state levels of NY-ESO-1 and MAGE-C1 are regulated by proteasomal degradation and that both behave as aggregation-prone proteins upon accumulation. With proteasome inhibitors being increasingly used as front-line treatment in cancer, these data raise issues about CT antigen processing for antigenic presentation and therefore immunogenicity in cancer patients.

Effects of CT-Xp gene knock down in melanoma cell lines.

Cancer/testis (CT) genes are encoded by genes that are normally expressed only in the human germ line but which are activated in various malignancies. CT proteins are frequently immunogenic in cancer patients and their expression is highly restricted to tumors. They are thus important targets for anticancer immunotherapy. In several different tumor types, the expression of CT-X genes is associated with advanced disease and poor outcome, indicating that their expression might contribute to tumorigenesis. CT-X genes encoding members of the MAGE protein family on Xq28 have been shown to potentially influence the tumorigenic phenotype. We used small interfering RNA (siRNA) to investigate whether CT-X mapping to the short arm of the X-chromosome might also have tumorigenic properties and therefore be potentially targeted by functional inhibitors in a therapeutic setting. siRNAs specific to GAGE, SSX and XAGE1 were used in cell proliferation, migration and cell survival assays using cell lines derived from melanoma, a tumor type known to present high frequencies of expression of CT antigens. We found that of these, those specific to GAGE and XAGE1 most significantly impeded melanoma cell migration and invasion and those specific to SSX4 and XAGE1 decreased the clonogenic survival of melanoma cells. Our results suggest that GAGE, XAGE1 and SSX4 might each have a role in tumor progression and are possible therapeutic targets for the treatment of melanoma and other malignancies.

An assessment of the impact of behavioural cognitions on function in patients partaking in a trial of early home-based progressive resistance training after total hip replacement surgery.

BACKGROUND: Control cognitions have been directly related to positive engagement with rehabilitation regimes. The impact of such cognitions on recovery following surgery is not well understood. PURPOSE: To assess whether perceived control cognitions predict function 9-12 months following total hip replacement (THR). METHODS: Prospective cohort study performed as part of a randomised controlled trial. Behavioural cognitions (BC) (recovery locus of control (RLOC); perceived external behavioural control (PEBC))) and subjective functional outcome measures (Oxford hip score (OHS) and a reduced version of the Western Ontario and McMasters University Osteoarthritis Function scale (rWOMAC PF)) were administered pre-operatively and up to 12 months post-operatively to 50 patients randomised to home-based progressive resistance training (N = 26) or standard rehabilitation (N = 24), post-THR. Regression analysis investigated variance in functional scores. RESULTS: Group randomisation had no effect on BC. RLOC and OHS (6 months) correlated significantly with 12-month OHS, with 6-month OHS predicting 62.3% of the variance in 12-month OHS. 12-month rWOMAC PF was determined by each of its three previous assessments (pre-operative 8.8%, 6 weeks 17.8% and 6 months 67.3%). Variance in functional gain at 12 months (OHS and rWOMAC PF) was explained by pre-operative OHS and rWOMAC PF (63.7% and 63.8%, respectively). CONCLUSIONS: BC had no impact on functional outcome in this population. Subjectively assessed function at 12 months, as well as the levels of functional gain over time, was best explained by the patients' earlier functional status. Implications for Rehabilitation It is important to assess psychological factors such as poor pre-operative mental health and pain catastrophising in patients undergoing joint replacement surgery as these factors have an adverse effect on subjective patient outcomes. Pre-operative behavioural cognitions appear to have no impact on subjective functional outcome at 12 months post-THR. The pre-existing functional status of the patient appears to be most predictive of subjective function at 12 months post-THR, implying that perhaps earlier surgery may be optimal before the onset of a decline in function.

Do high doses of AT(1)-receptor blockers attenuate central sympathetic outflow in humans with chronic heart failure?

In patients with CHF (chronic heart failure) sympathetic activity increases as cardiac performance decreases and filling pressures increase. We hypothesized that in patients with mild-to-moderate CHF, higher than conventional doses of an AT1-receptor [AngII (angiotensin II) type 1 receptor] antagonist would achieve greater central AT1-receptor blockade, resulting in diminished MSNA (muscle sympathetic nerve activity) and augmented MSNA variability, two indices of central effects on sympathetic outflow. In total, 13 patients with ischaemic cardiomyopathy [NYHA (New York Heart Association) class II-III] were weaned off all pharmacological RAS (renin-angiotensin system) modifiers, and then randomized to receive a low (50 mg/day) or high (200 mg/day) dose of losartan. Central haemodynamics, MSNA and its variability, plasma catecholamines, AngI (angiotensin I) and AngII and aldosterone were assessed both before and 3 months after randomization. Neither dose altered BP (blood pressure), PCWP (pulmonary capillary wedge pressure) or CI (cardiac index) significantly. Compared with 50 mg daily, losartan 200 mg/day decreased MSNA significantly (P<0.05), by approximately 15 bursts/min, and increased MSNA variability within the 0.27-0.33 Hz high-frequency range by 0.11 units(2)/Hz (P=0.06). PNE [plasma noradrenaline (norepinephrine)] fell in parallel with changes in MSNA (r=0.62; P<0.05). These findings support the hypothesis that higher than conventional doses of lipophilic ARBs (AT1-receptor blockers) can modulate the intensity and variability of central sympathetic outflow in patients with CHF. The efficacy and safety of this conceptual change in the therapeutic approach to heart failure merits prospective testing in clinical trials.

NY-ESO-1 expression in meningioma suggests a rationale for new immunotherapeutic approaches.

Meningiomas are the most common primary intracranial tumors. Surgical resection remains the treatment of choice for these tumors. However, a significant number of tumors are not surgically accessible, recur, or become malignant, necessitating the repetition of surgery and sometimes radiation. Chemotherapy is rarely used and is generally not recognized as an effective treatment. Cancer/testis (CT) genes represent a unique class of genes, which are expressed by germ cells, normally silenced in somatic cells, but activated in various cancers. CT proteins can elicit spontaneous immune responses in patients with cancer and this feature makes them attractive targets for immunotherapy-based approaches. We analyzed mRNA expression of 37 testis-restricted CT genes in a discovery set of 18 meningiomas by reverse transcription PCR. The overall frequency of expression of CT genes ranged from 5.6% to 27.8%. The most frequently expressed was NY-ESO-1, in 5 patients (27.8%). We subsequently analyzed NY-ESO-1 protein expression in a larger set of meningiomas by immunohistochemistry and found expression in 108 of 110 cases. In some cases, NY-ESO-1 expression was diffused and homogenous, but in most instances it was heterogeneous. Importantly, NY-ESO-1 expression was positively correlated with higher grade and patients presenting with higher levels of NY-ESO-1 staining had significantly worse disease-free and overall survival. We have also shown that NY-ESO-1 expression may lead to humoral immune response in patients with meningioma. Considering the limited treatment options for patients with meningioma, the potential of NY-ESO-1-based immunotherapy should be explored.

Differential evolution of MAGE genes based on expression pattern and selection pressure.

Starting from publicly-accessible datasets, we have utilized comparative and phylogenetic genome analyses to characterize the evolution of the human MAGE gene family. Our characterization of genomic structures in representative genomes of primates, rodents, carnivora, and macroscelidea indicates that both Type I and Type II MAGE genes have undergone lineage-specific evolution. The restricted expression pattern in germ cells of Type I MAGE orthologs is observed throughout evolutionary history. Unlike Type II MAGEs that have conserved promoter sequences, Type I MAGEs lack promoter conservation, suggesting that epigenetic regulation is a central mechanism for controlling their expression. Codon analysis shows that Type I but not Type II MAGE genes have been under positive selection. The combination of genomic and expression analysis suggests that Type 1 MAGE promoters and genes continue to evolve in the hominin lineage, perhaps towards functional diversification or acquiring additional specific functions, and that selection pressure at codon level is associated with expression spectrum.

Is there a difference in perioperative mortality between cemented and uncemented implants in hip fracture surgery?

INTRODUCTION: Although cemented implants have proven beneficial over uncemented implants for treatment of displaced sub-capital proximal femoral fractures, there are concerns regarding the haemodynamic consequence of using cemented implants in hip fracture patients. National Patient Safety Agency recently issued an alert regarding the use of cement in hip fracture surgery. We compared the incidence and pattern of 48 h perioperative mortality between patients receiving cemented and uncemented implants after hip fracture surgery. METHODS: Using data prospectively recorded in hospital care records, we retrospectively reviewed the case records of all patients who died in hospital following hip fracture surgery between January 2005 and April 2010. We recorded demographic variables, type of fracture, implant used, medical co-morbidity, seniority of operating surgeon and anaesthetist, perioperative haemodynamic status, time and cause of death. RESULTS: We identified 15 cases of perioperative death (PoD) over a 64-month period. PoD was 1% (15/1402). Eight of 15 deaths occurred following cemented hemiarthroplasty insertion. There were four cases of intra-operative death, two of them were following cemented hemiarthroplasty insertion. PoD following cemented hemiarthroplasty was 2.54% (8/314) and nil (0/168) following uncemented Austin-Moore hemiarthroplasty. Operations were performed by both trainees (six) and consultants (two). Both trainees (five) and consultants (three) anaesthetised the patients. None of the patients belonged to American Society of Anesthesiologists (ASA) I or II (ASA III 5 and IV 3). All patients had significant cardiovascular or pulmonary co-morbidity. Apart from the cases of immediate haemodynamic collapse and death, cemented implant insertion was followed by intra-operative haemodynamic instability in 2/15 and perioperative instability in 3/15 patients. Post-mortem was performed in 3/8 patients: 2/3 demonstrated pulmonary embolism (PE), 1/3 bronchopneumonia. Of the rest, 3/5 had suspected myocardial infarction (MI). CONCLUSION: There was 1% risk of perioperative death after hip fracture surgery. Risk of perioperative death was significantly higher following cemented implant insertion. Mortality risk was exacerbated in patients with pre-existing cardiovascular morbidity and was independent of the seniority of the surgeon or the anaesthetist.

Humanized Lewis-Y specific antibody based delivery of STAT3 siRNA.

The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Le(y)), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Le(y)-expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Le(y)-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a noncovalent construct based on (d-arginine)(9) (9r) modified hu3S193. Le(y)-specific binding and internalization of both the covalent and noncovalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the noncovalent system led to efficient STAT3 silencing in Le(y)-positive cancer cells (A431) but not in Le(y)-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified noncovalent construct induced ∼70% STAT3 knockdown at submicromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ∼50% inhibition of cell proliferation of Le(y)-positive cells. Noncovalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.