NK cell line modified to express a potent, DR5 specific variant of TRAIL, show enhanced cytotoxicity in ovarian cancer models.

Objective: Ovarian cancer is a lethal gynaecological malignancy with unsatisfactory 5 year survival rates of 30-50 %. Cell immunotherapy is a promising new cancer treatment where immune cells, such as Natural Killer (NK) cells, are administered to enable the patient to fight cancer through direct cytotoxicity. NK cells orchestrate an adaptive immune response by enabling the release of tumour antigens. NK cell cytotoxicity and effector responses are largely driven by TRAIL engagement. In this study we investigated the cytotoxic potential of a human NK cell line that were modified to express a potent DR5 specific TRAIL variant. We hypothesised that this modification would enhance NK cell cytotoxicity against TRAIL sensitive and resistant ovarian cancer cell lines in vitro. Methods: KHYG-1 human NK cells were modified with a TRAIL variant targeting DR5 (TRAILv-KHYG-1). Human ovarian cancer cell lines, OVCAR-3 and SKOV-3, were cultured with modified or non-modified NK cells at different effector:target (E:T) ratios for 4 or 16 h. Apoptosis was assessed by Annexin-APC and 7-AAD and measured using flow cytometry. Apoptotic cells were defined as annexin V 7-AAD double positive. Cytokine expression was measured by multiplex ELISA, and analysed by flow cytometry. Results: Modified and non-modified NK cells significantly reduced OVCAR-3 cell viability as compared to OVCAR-3 cells that were cultured alone after 4 and 16 h treatment. OVCAR-3 cell viability was reduced after treatment with 1:1 E:T ratio with TRAILv-KHYG-1 cells after 16 h. On the contrary, neither NK cell line had any effect of SKOV-3 cell viability despite SKOV-3 cells having more DR5 surface expression compared to OVCAR-3 cells. Conclusions: TRAILv-KHYG-1 cells significantly reduced OVCAR-3 cell viability as compared to non-modified NK cells. However, no significant reduction in viability was observed when SKOV-3 cell were cultured with either NK cells, despite having more DR5 surface expression compared to OVCAR-3 cells. These data indicate that mechanisms other than DR5 expression drive TRAIL resistance in ovarian cancer.

MEDICINE AND THE LAW.

Dolutegravir (DTG) is a pivotal antiretroviral medicine that has become the backbone of several HIV programmes, especially in sub-Saharan African countries. It has recently replaced efavirenz as the preferred third drug for people initiating antiretroviral therapy in South Africa (SA). Its tolerability, cost-effectiveness and favourable resistance profile have had a global influence on HIV management, including the recent revision of the World Health Organization antiretroviral guidelines. As with any medicine, however, informed decisions are important. Despite the several advantages DTG offers, additional data informing risks over benefits have emerged that warrant clinical attention before DTG is prescribed. This article aims to give the primary care provider an overview of the benefits and risks associated with the roll-out of DTG in SA.

Implementation of Public Health England infection prevention and control guidance in maternity units in response to the COVID-19 pandemic.

BACKGROUND: This study aimed to explore the successes and barriers to the implementation of Public Health England (PHE) infection prevention and control guidance in English maternity units during the COVID-19 pandemic. METHODS: Qualitative semi-structured interviews with obstetricians, midwives and neonatologists who worked in a maternity unit in England, UK, between March 2020 and July 2021. A thematic analysis was performed. RESULTS: Successes to the implementation of PHE guidance were related to existing infrastructure, training satisfaction, and organisational culture where subthemes considered the importance of a multidisciplinary approach, COVID-19 dedicated roles and hospital-wide communication. Barriers to implementation related to the applicability of the guidance with subthemes highlighting contradictions between updates, specialties and hospitals, undesirable timings and frequency of guidance updates, reductions in staff compliance and delayed implementation. Finally, the layout of some units made it difficult to implement various aspects of the guidance (e.g., social distancing), and many detailed issues related to information technology compatibility, a lack of availability and accessibility to appropriate personal protective equipment (PPE), and variations in testing arrangements between units. CONCLUSIONS: This research provides information on the experiences of healthcare professionals working on maternity units during the COVID-19 pandemic. Findings illustrate the importance of effective hospital-wide communication and the need for consistent, easily understood guidance. These results will be used to inform the content of an expert panel consensus meeting.

Maternity services' responses to the COVID-19 pandemic: how Public Health England guidance was implemented in practice.

INTRODUCTION: The rapidly evolving COVID-19 pandemic required systemic change in how healthcare was delivered to minimize virus transmission whilst maintaining safe service delivery. Deemed at 'moderate-high risk', maternity patients are an important patient group that require consideration. Public Health England (PHE) issued national guidance on how to adjust these services. AIM: To explore how maternity units in England implemented PHE guidance. METHODS: An online survey of 22 items was distributed to individuals that had worked on an England-based maternity unit during the COVID-19 pandemic. The questionnaire was designed and tested by the multidisciplinary research team. Data was collected from November 2020 to July 2021. FINDINGS: Forty-four participants across 33 maternity units responded. Ninety-three percent were able to test all women requiring an overnight stay for COVID-19. Only 27% reported birth partners were tested for COVID-19. Only 73% reported they were able to isolate all COVID-19-positive patients in single rooms. Eighty-four percent stated they were aware of current PHE guidance on personal protective equipment (PPE) and 82% felt 'confident' in donning/doffing of PPE. Priorities for the future include rapid testing and a focus on community service provision. CONCLUSIONS: PHE COVID-19 guidance was implemented differently in maternity units across England due to the varying resources available at each trust leading to variable ability to test and isolate patients as recommended. More specific, tailored guidance for infection control measures against COVID-19 is needed for maternity settings due to their unique position.

Slow Acceptance of Universal Antiretroviral Therapy (ART) Among Mothers Enrolled in IMPAACT PROMISE Studies Across the Globe.

The PROMISE trial enrolled asymptomatic HIV-infected pregnant and postpartum women not eligible for antiretroviral treatment (ART) per local guidelines and randomly assigned proven antiretroviral strategies to assess relative efficacy for perinatal prevention plus maternal/infant safety and maternal health. The START study subsequently demonstrated clear benefit in initiating ART regardless of CD4 count. Active PROMISE participants were informed of results and women not receiving ART were strongly recommended to immediately initiate treatment to optimize their own health. We recorded their decision and the primary reason given for accepting or rejecting the universal ART offer after receiving the START information. One-third of participants did not initiate ART after the initial session, wanting more time to consider. Six sessions were required to attain 95% uptake. The slow uptake of universal ART highlights the need to prepare individuals and sensitize communities regarding the personal and population benefits of the "Treat All" strategy.

Primary prevention of cardiovascular disease in South African women living with HIV.

The colliding epidemics of HIV and cardiovascular disease (CVD) are of great public health concern. People living with HIV (PLHIV) are more predisposed to CVD development as a result of a multitude of contributors. Women living with HIV (WLHIV) appear to be at a higher risk of developing CVD given a heightened immune activation and, in South Africa particularly, a higher body mass index compared with their male counterparts. The World Health Organization (WHO) has made recommendations for the provision of a CVD risk assessment for all PLHIV and has developed regional CVD prediction charts to identify PLHIV who may require primary prevention strategies by means of interventions such as the WHO Package of Essential Non-communicable Disease Interventions for primary health care in low-resource settings (WHO PEN). However, methods of risk prediction and risk reduction integrated strategies for atherosclerotic CVD in PLHIV such as the WHO PEN, particularly in women who may have sex-specific risk factors and culture-specific perceptions of body image, remain a major research gap in developing countries. Further research is crucial in guiding primary health care policy in South Africa.

Time trends in suicide rates by domestic gas or car exhaust gas inhalation in Japan, 1968-1994.

AIMS: A reduction in the carbon monoxide content of domestic gas and car exhaust gas has been associated with a decrease in gassing suicides in many western countries. In Japan, a reduction in the carbon monoxide content of domestic gas supply began in the early 1970s, and carbon monoxide emissions standards of new passenger cars were significantly strengthened in 1978. However, little is known about the impact of detoxification of these gases on gassing-related suicides in Japan. Therefore, we examined the changing patterns of suicide due to domestic gas or car exhaust gas inhalation by gender and age in Japan between 1968 and 1994. METHODS: Suicide mortality data were obtained from the Vital Statistics of Japan. In this study, age was divided into four groups: 15-24, 25-44, 45-64 and 65+ years. Method of suicide was divided into three groups: domestic gas, car exhaust gas and non-gases. We calculated method-specific age-standardised suicide rates by gender within each of the four age groups. We applied joinpoint regression to the data and quantified the observed changes. RESULTS: Suicide rates by domestic gas, regardless of gender and age, increased from 1968 to the mid-1970s and then decreased sharply. The proportion of all suicides accounted for by domestic gas was comparatively high in the mid-1970s among females aged 15-24 and 25-44 years, while for other gender-age-groups the proportion of domestic gas suicides remained small, even at the peak. For females aged 15-44 years, the decrease in domestic gas suicides appeared to cause a substantial decrease in overall suicides in this gender/age group. Car exhaust gas was a more common method for males, particularly those aged 25-64 years. Car exhaust gas suicide rates for males aged 25-64 years peaked in the mid-1980s, followed by a sharp decrease. CONCLUSIONS: A reduction in the carbon monoxide content of the domestic gas, which began in the early 1970s in Japan, was associated with a decrease in domestic gas suicides for both genders of all ages. Concerning females aged 15-44 years, a decrease in domestic gas suicides caused a substantial decrease in overall suicides in this gender/age group since the proportion of domestic gas suicides among all suicides combined was comparatively large. However, it remains uncertain whether the introduction of catalytic converters in the 1970s in Japan resulted in a reduction of suicides from car exhaust gas inhalation.

Algorithm that delivers an individualized rapid-acting insulin dose after morning resistance exercise counters post-exercise hyperglycaemia in people with Type 1 diabetes.

AIMS: To develop an algorithm that delivers an individualized dose of rapid-acting insulin after morning resistance exercise to counter post-exercise hyperglycaemia in individuals with Type 1 diabetes. METHODS: Eight people with Type 1 diabetes, aged 34 ± 7 years with HbA1c concentrations 72 ± 12 mmol/mol (8.7 ± 1.1%), attended our laboratory on two separate mornings after fasting, having taken their usual basal insulin the previous evening. These people performed a resistance exercise session comprising six exercises for two sets of 10 repetitions at 60% of the maximum amount of force that was generated in one maximal contraction (60% 1RM). In a randomized and counterbalanced order, the participants were administered an individualized dose of rapid-acting insulin (2 ± 1 units, range 0-4 units) immediately after resistance exercise (insulin session) by means of an algorithm or were not administered this (no-insulin session). Venous blood glucose concentrations were measured for 125 min after resistance exercise. Data (mean ± sem values) were analysed using anova (P ≤ 0.05). RESULTS: Participants had immediate post-resistance exercise hyperglycaemia (insulin session 13.0 ± 1.6 vs. no-insulin session 12.7 ± 1.5 mmol/l; P = 0.834). The decline in blood glucose concentration between peak and 125 min after exercise was greater in the insulin exercise session than in the no-insulin session (3.3 ± 1.0 vs. 1.3 ± 0.4 mmol/l: P = 0.015). There were no episodes of hypoglycaemia (blood glucose <3.9 mmol/l). CONCLUSIONS: Administration of rapid-acting insulin according to an individualized algorithm reduced the hyperglycaemia associated with morning resistance exercise without causing hypoglycaemia in the 2 h post-exercise period in people with Type 1 diabetes.

Time trends in method-specific suicide rates in Japan, 1990-2011.

BACKGROUND: Little is known about whether particular suicide methods have contributed differently to the recent unfavourable suicide mortality trends in Japan. Analysing such trends may shed light on the effect of potentially preventable factors, such as the impact of restricting access to certain popular suicide methods, on overall rates. Therefore, we assessed recent trends in method-specific suicide by gender and age in Japan. METHOD: Suicide mortality and population data between 1990 and 2011 were obtained from the Vital Statistics of Japan and used to calculate method-specific mortality rates. Suicide methods were divided into seven groups: overdose, gases, hanging, drowning, cutting, jumping and other means. Age was divided into four groups: 15-24, 25-44, 45-64 and 65+ years. We applied joinpoint regression to the data and quantified the observed changes. RESULTS: The results of the joinpoint regression analyses showed a sharp increase in overall suicide rates for males and females of all ages until the late 1990s. Suicide from hanging and jumping, in particular, contributed to this increase. After 2000, an increasing trend in overall suicide rates in both males and females aged 15-24 and 25-44 years was observed, with overdose, gases and hanging contributing to this increasing trend. CONCLUSIONS: Our findings revealed that different suicide methods varied in their contribution to the recent overall suicide transition in Japan. Regarding factors associated with the recent increase in suicides by overdose, gases, hanging and jumping, further research is needed in order to promote and implement effective means restriction strategies.

Similar magnitude of post-exercise hyperglycemia despite manipulating resistance exercise intensity in type 1 diabetes individuals.

The aim of this study was to compare the glycemic and glucoregulatory hormone responses to low- and moderate-intensity morning resistance exercise (RE) sessions in type 1 diabetes (T1DM). Following maximal strength assessments (1RM), eight T1DM (HbA1C :72 ± 12 mmol/mol, age:34 ± 7 years, body mass index:25.7 ± 1.6 kg/m(2) ) participants attended the research facility on two separate occasions, having fasted and taken their usual basal insulin but omitting rapid-acting insulin. Participants performed six exercises for two sets of 20 repetitions at 30%1RM during one session [low-intensity RE session (LOW)] and two sets of 10 repetitions at 60%1RM during another session [moderate-intensity RE session (MOD)], followed by 65-min recovery. Sessions were matched for total mass lifted (kg). Venous blood samples were taken before and after exercise. Data (mean ± SEM) were analyzed using analysis of variance (P ≤ 0.05). There were no hypoglycemic occurrences throughout the study. Blood glucose rose similarly between sessions during exercise (P = 0.382), remaining comparable between sessions throughout recovery (P > 0.05). There was no effect of RE intensity on metabolic acidosis (P > 0.05) or peak growth hormone responses (P = 0.644), but a tendency for greater catecholamine responses under LOW (individualized peak concentrations: adrenaline MOD 0.55 ± 0.13 vs LOW 1.04 ± 0.37 nmol/L, P = 0.155; noradrenaline MOD 4.59 ± 0.86 vs LOW 7.11 ± 1.82 nmol/L, P = 0.082). The magnitude of post-exercise hyperglycemia does not differ between equal volume low and moderate intensity RE sessions performed in the morning.

High HPV vaccination uptake rates for adolescent girls after regional governmental funding in Shiki City, Japan.

BACKGROUND: In Japan, the bivalent HPV vaccine was approved in October, 2009 and became available as a non-routine vaccine from December, 2009. While routine vaccinations are free, the cost and responsibility for non-routine vaccinations are left to the individual. In exceptional circumstances regional governments fund non-routine vaccinations. This was the case in Shiki City, Saitama Prefecture, where a high uptake rate for individual (non-school based) HPV vaccination was obtained. MATERIALS: On January 20, 2010, the mayor of Shiki City announced to the media his decision to vaccinate adolescent girls in Shiki City against HPV. A project team for HPV vaccination was set up in the city's Health Promotion Center. To gain mutual consent for HPV vaccination, senior health professionals, city officials, the head of the board of education, school principals and health-care teachers met several times. The cohort to be vaccinated was 1254 girls aged 12-15 years. Individual notifications were mailed to each girl on April 23, 2010, along with information about the HPV vaccine. CONCLUSIONS: As of April 10th, 2011, the uptake rate for girls aged 15 years old was 90.7% for the 1st dose. The vaccine registry is managed by the health care system of the city. The success of the HPV vaccination program and high uptake rates in Shiki City is a good model for the nationwide HPV vaccination program that started in February, 2011.

Evidence-based practice in school substance use prevention: fidelity of implementation under real-world conditions.

Fidelity of program implementation under real-world conditions is a critical issue in the dissemination of evidence-based school substance use prevention curricula. Program effects are diminished when programs are implemented with poor fidelity. We assessed five domains of fidelity--adherence, exposure (dosage), quality of delivery, participant responsiveness and program differentiation (lack of contamination from other programs)--in a subset of respondents (N = 342) from a national random sample of public schools with middle school grades (N = 1721). Respondents taught 1 of 10 evidence-based universal substance use prevention programs as their primary program during the 2004-05 school year. Their responses to survey questions about their recent implementation practices indicated that fidelity was high for quality of delivery and participant responsiveness, low for program differentiation and modest for adherence and exposure--the two core domains of fidelity. Results suggest the need for continued emphasis on fidelity in program materials, trainings and on-going technical support. Particular attention should be paid to supporting use of interactive delivery strategies.

An anchored linkage map for sugar beet based on AFLP, SNP and RAPD markers and QTL mapping of a new source of resistance to Beet necrotic yellow vein virus.

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is an important sugar-beet disease worldwide and can result in severe losses of root yield and sugar content. We have identified a major QTL for BNYVV resistance from a new source in a segregating population of 158 individuals. The QTL explained an estimated 78% of the observed phenotypic variation and the gene conferring the partial resistance is referred to as Rz4. AFLP was used in combination with bulked segregant analysis (BSA) to develop markers linked to the resistance phenotype. AFLP marker analysis was extended to produce a linkage map that was resolved into nine linkage groups. These were anchored to the nine sugar-beet chromosomes using previously published SNP markers. This represents the first anchored sugar-beet linkage map to be published with non-anonymous markers. The final linkage map comprised 233 markers covering 497.2 cM, with an average interval between markers of 2.1 cM. The Rz4 QTL and an Rz1 RAPD marker were mapped to chromosome III, the known location of the previously identified BNYVV resistance genes Rz1, Rz2 and Rz3. The availability to breeders of new resistance sources such as Rz4 increases the potential for breeding durable disease resistance.

Autocrine insulin action activates Akt and increases survival of isolated human islets.

AIMS/HYPOTHESIS: The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt. METHODS: In order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period. RESULTS: We demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation. CONCLUSIONS/INTERPRETATION: Our findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.

Morphogenetic plasticity of adult human pancreatic islets of Langerhans.

The aim of this study was to investigate the phenotypic plasticity of pancreatic islets of Langerhans. Quiescent adult human islets were induced to undergo a phenotypic switch to highly proliferative duct-like structures in a process characterized by a loss of expression of islet-specific hormones and transcription factors as well as a temporally related rise in the expression of markers of both duct epithelial and progenitor cells. Short-term treatment of these primitive duct-like structures with the neogenic factor islet neogenesis-associated protein (INGAP104-118) induced their reconversion back to islet-like structures in a PI3-kinase-dependent manner. These neoislets resembled freshly isolated human islets with respect to the presence and topological arrangement of the four endocrine cell types, islet gene expression and hormone production, insulin content and glucose-responsive insulin secretion. Our results suggest that adult human islets possess a remarkable degree of morphogenetic plasticity. This novel observation may have important implications for understanding pancreatic carcinogenesis and islet neogenesis.

Characterization of the flanking regions of Zea mays microsatellites reveals a large number of useful sequence polymorphisms.

Sequence characterization of the flanking regions of 52 sequence-tagged microsatellite loci and two gene fragments from 11 Zea mays inbred lines identified a total of 324 sequence polymorphisms. The sequence polymorphisms consisted of both single-nucleotide polymorphisms and insertions/deletions in a ratio of approximately two to one. The level of sequence variation within the flanking regions of microsatellites linked to expressed sequence tags was lower than microsatellites that were unlinked to expressed sequence tags. However, both types of microsatellites generated a similar number of sequence-based alleles across the 11 genotypes surveyed. In two out of 20 microsatellites examined in detail, evidence was found for size-based allele homoplasy. Conversion of the observed sequence polymorphisms into allele-specific oligonucleotides followed by covalent binding to glass slides allowed the sequence polymorphisms to be used in a simple hybridization-based genotyping procedure. This procedure enabled us to discriminate between different inbred lines and allowed variations within a single inbred to be identified. The sequence information presented in this report could be used as a starting point for other programmes in the further development of a non-gel based, multi-locus, multi-allele screen for large-scale maize genotyping.

A genetic linkage map of willow ( Salix viminalis) based on AFLP and microsatellite markers.

The genus Salix (willow) contains a number of species of great value as biomass crops. Efforts to breed varieties with improved biomass yields and resistances to pests and diseases are limited by the lack of knowledge on the genetic basis of the traits. We have used AFLP and microsatellite markers to construct a genetic map of willow from a full-sib cross of the diploid species Salix viminalis (2n = 38). In accordance with a double pseudo-testcross approach, separate parental maps were constructed and merged to produce a consensus map comprising 291 AFLP and 39 willow microsatellite markers. Nineteen poplar microsatellites were also tested in willow. Five of these amplified loci, of which two were mapped. Linkage groups of the consensus map that could be identified in the parental maps are presented here and spanned 1,256.5 cM with an average interval between markers of 4.4 cM.

Evaluation of a PCR/DNA probe colorimetric membrane assay for identification of Campylobacter spp. in human stool specimens.

DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the Campylobacter genus, Campylobacter jejuni, and Campylobacter coli in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for C. jejuni. The assay is rapid and simple and can be applied to stool specimens for the detection of Campylobacter.