Expression of beta defensin genes in frozen thawed and cultured immortalised human corneal epithelial cell line

Human beta defensins (hBD) are important host defense molecules at the ocular surface. In addition to their antimicrobial activities, hBD may also act as regulatory factors in recruiting and activating immune cells. Only hBD-1 – hBD-4 have been well characterised. To date, a complete profile of the beta defensin genes (DEFB) expression in immortalised human corneal epithelial cell line (HCE-2) has not been established. Therefore, this study is aimed to explore a spectrum of DEFB expression in HCE-2. Total RNAs were extracted from frozen thawed HCE-2 and cultured HCE-2 to ensure the gene expression were identical. The RNAs were reverse transcribed into cDNAs. The expression of 10 DEFB (DEFB1, DEFB4A, DEFB103, DEFB104, DEFB105, DEFB106, DEFB109, DEFB123, DEFB126 and DEFB127) were analysed using polymerase chain reaction (PCR) and gel electrophoresis. DEFB1 and DEFB103 were the only hBD mRNAs found constitutively expressed in frozen thawed HCE-2 and cultured HCE-2. It was also interesting to note that PCR enhancer was needed to amplify the genes in cultured HCE-2. Our findings suggest that corneal epithelium constantly produce hBD-1 and hBD-3, which presumably provide the baseline defense against infection. Further investigation on the expression of these genes when HCE-2 stimulated with proinflammatory cytokines would help in better understanding of the ocular surface defense mechanism

A case–control study of childhood leukaemia and paternal occupational contact level in rural Sweden

In a national case–control study in Sweden, we investigated whether in rural areas (where susceptible individuals are more prevalent than in urban areas) leukaemia risk was higher among the young children of fathers with many work contacts, as the infective hypothesis has predicted. A total of 1935 cases diagnosed in 1958–1998 together with 7736 age-matched (within 1 year) population controls (of whom 970 and 3880 respectively were aged 0–4) were linked to paternal occupational details as recorded in the census closest to the year of birth. Applying the two classifications of occupational contact level used in a study of rural Scotland, the odds ratios for children aged 0–4 years in the highest contact category (which includes teachers) in the most rural Swedish counties were 3.47 (95% CI 1.54, 7.85) and 1.59 (1.07, 2.38) respectively, relative to the medium and low (reference) category; no such excess was found in urban or intermediate counties. There was also a significant positive trend at ages 0–4 in the rural counties across the three levels of increasing occupational contact (P for trend 0.02 and 0.03, respectively), but again not in the urban or intermediate counties. No such effect or trend was found at ages 5–14 in any of the three county groupings. The findings confirm those of a recent study in rural Scotland, and also suggest that unusual population mixing (as occurred in Scotland as a result of the North Sea oil industry) is not a necessary requirement for the effect, since comparable mixing has not been a feature of rural Sweden

Validation of Resilience Assessment Tool 25 (RAT-25)

Being resilience in any adversities or crises is crucial especially after the COVID-19 outbreak among the adolescents. Resilience is an ability to adapt psychologically in order to learn bouncing back. The process to balance the protective factors and risk factors in improving adolescents’ mental health and wellbeing at school. Recent studies have revealed that identification of resilience domains such Basics, Belonging, Learning, Coping and Core Self influence the resiliency of individuals. There are limited resilience assessments. Hence, this study fills up the limitations by revising the current Resilience Assessment Tool 43 (RAT-43) to a shorter version as known Resilience Assessment Tool 25 (RAT-25) by considering the psychological need in adolescents in responding the items. RAT-25’s validity and reliability testing were performed with the recruitment from 13-year-olds, lower secondary students in Malaysia. The Exploratory Factor Analysis and Confirmatory Factor Analysis were performed to examine the construct validity particularly the factorial structure and reliability of this instrument. Results from the EFA yielded 5-factor structure. Similarly, results from the CFA using Structural Equation Modelling (AMOS) also validated the five-factor structure of RAT (B, B, L, C, CS). Findings also indicated that the measured items are reliable. The findings established that all five resilience domains are useful for assessing adolescents’ resilience. The implications for psychometric assessment were evident in terms of giving empirical evidence to corroborate theory-based constructs and also validating items' quality to appropriately represent the measurement

Polymorphisms in Toll-like receptor genes influence antibody responses to cytomegalovirus glycoprotein B vaccine

<p>Abstract</p> <p>Background</p> <p>Congenital Cytomegalovirus (CMV) infection is an important medical problem that has yet no current solution. A clinical trial of CMV glycoprotein B (gB) vaccine in young women showed promising efficacy. Improved understanding of the basis for prevention of CMV infection is essential for developing improved vaccines.</p> <p>Results</p> <p>We genotyped 142 women previously vaccinated with three doses of CMV gB for single nucleotide polymorphisms (SNPs) in TLR 1-4, 6, 7, 9, and 10, and their associated intracellular signaling genes. SNPs in the platelet-derived growth factor receptor (PDGFRA) and integrins were also selected based on their role in binding gB. Specific SNPs in TLR7 and IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) were associated with antibody responses to gB vaccine. Homozygous carriers of the minor allele at four SNPs in TLR7 showed higher vaccination-induced antibody responses to gB compared to heterozygotes or homozygotes for the common allele. SNP rs1953090 in IKBKE was associated with changes in antibody level from second to third dose of vaccine; homozygotes for the minor allele exhibited lower antibody responses while homozygotes for the major allele showed increased responses over time.</p> <p>Conclusions</p> <p>These data contribute to our understanding of the immunogenetic mechanisms underlying variations in the immune response to CMV vaccine.</p

Design and Analysis of Rhesus Cytomegalovirus IL-10 Mutants as a Model for Novel Vaccines against Human Cytomegalovirus

Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV's ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection

Washing our hands of the congenital cytomegalovirus disease epidemic

BACKGROUND: Each year in the United States, an estimated 40,000 children are born with congenital cytomegalovirus (CMV) infection, causing an estimated 400 deaths and leaving approximately 8000 children with permanent disabilities such as hearing or vision loss, or mental retardation. More children are affected by serious CMV-related disabilities than by several better-known childhood maladies, including Down syndrome, fetal alcohol syndrome, and spina bifida. DISCUSSION: Congenital CMV is a prime target for prevention not only because of its substantial disease burden but also because the biology and epidemiology of CMV suggest that there are ways to reduce viral transmission. Because exposure to the saliva or urine of young children is a major cause of CMV infection among pregnant women, it is likely that good personal hygiene, especially hand-washing, can reduce the risk of CMV acquisition. Experts agree that such measures are likely to be efficacious (i.e., they will work if consistently followed) and the American College of Obstetricians and Gynecologists recommends that physicians counsel pregnant women about preventing CMV acquisition through careful attention to hygiene. However, because of concerns about effectiveness (i.e., Will women consistently follow hygienic practices as the result of interventions?), the medical and public health communities appear reluctant to embrace primary CMV prevention via improved hygienic practices, and educational interventions are rare. Current data on the effectiveness of such measures in preventing CMV infection are promising, but limited. There is strong evidence, however, that educational interventions can prevent other infectious diseases with similar transmission modes, suggesting that effective interventions can also be found for CMV. Until a CMV vaccine becomes available, effective educational interventions are needed to inform women about congenital CMV prevention. SUMMARY: Perhaps no single cause of birth defects and developmental disabilities in the United States currently provides greater opportunity for improved outcomes in more children than congenital CMV. Given the present state of knowledge, women deserve to be informed about how they can reduce their risk of CMV infection during pregnancy, and trials are needed to identify effective educational interventions

Host Immune Responses to a Viral Immune Modulating Protein: Immunogenicity of Viral Interleukin-10 in Rhesus Cytomegalovirus-Infected Rhesus Macaques

, consistent with a central role for rhcmvIL-10 during acute virus-host interactions. Since cmvIL-10 and rhcmvIL-10 are extremely divergent from the cIL-10 of their respective hosts, vaccine-mediated neutralization of their function could inhibit establishment of viral persistence without inhibition of cIL-10.As a prelude to evaluating cmvIL-10-based vaccines in humans, the rhesus macaque model of HCMV was used to interrogate peripheral and mucosal immune responses to rhcmvIL-10 in RhCMV-infected animals. ELISA were used to detect rhcmvIL-10-binding antibodies in plasma and saliva, and an IL-12-based bioassay was used to quantify plasma antibodies that neutralized rhcmvIL-10 function. rhcmvIL-10 is highly immunogenic during RhCMV infection, stimulating high avidity rhcmvIL-10-binding antibodies in the plasma of all infected animals. Most infected animals also exhibited plasma antibodies that partially neutralized rhcmvIL-10 function but did not cross-neutralize the function of rhesus cIL-10. Notably, minimally detectable rhcmvIL-10-binding antibodies were detected in saliva.This study demonstrates that rhcmvIL-10, as a surrogate for cmvIL-10, is a viable vaccine candidate because (1) it is highly immunogenic during natural RhCMV infection, and (2) neutralizing antibodies to rhcmvIL-10 do not cross-react with rhesus cIL-10. Exceedingly low rhcmvIL-10 antibodies in saliva further suggest that the oral mucosa, which is critical in RhCMV natural history, is associated with suboptimal anti-rhcmvIL-10 antibody responses

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified