A phage-displayed single chain variable fragment that interacts with hepatitis B core antigen: library construction, selection and diagnosis.

BACKGROUND: Phage display is an alternative method for constructing and selecting antibodies with desired specificity towards an antigen. OBJECTIVES: To construct a library of single chain variable fragment (ScFv) towards hepatitis B core antigen (HBcAg). To isolate a ScFv phage clone that interacts with HBcAg and to develop a phage-ELISA for detecting the antigen. STUDY DESIGN: Mice were inoculated with HBcAg and RNA was extracted from their spleen cells. The genes encoding heavy (V(H)) and light (V(L)) chains were amplified, linked via PCR and cloned into a phagemid vector. Phage particles displaying ScFv were panned against HBcAg and a selected clone was characterized and employed as a diagnostic reagent for detecting HBcAg in serum samples. RESULTS: A phage clone that interacts with HBcAg was selected from the antibody library. The binding of the phage to HBcAg was inhibited by a cyclic peptide bearing the WSFFSNI sequence. A phage-ELISA was established using the recombinant phage and as low as 10ng of HBcAg can be detected by the assay. CONCLUSION: The ScFv displayed on the surface of filamentous phage is an alternative choice for diagnosis of HBcAg in serum samples.     

Lymphocyte-neutrophil interactions: opposite effects of interleukin-2 and tumour necrosis factor-beta (lymphotoxin) on human neutrophil adherence

Human neutrophil adherence was enhanced by recombinant human tumour necrosis factor-beta (TNF beta) but suppressed by recombinant human interleukin-2 (IL-2). The opposite effects of these two lymphokines were observed over a range of concentrations consistent with their other biological activities, occurred within 15 min of incubation, and were still evident after 60 min. Pretreatment of neutrophils with both IL-2 and TNF beta resulted in adherence values intermediate between the values obtained with the individual lymphokines. IL-2 suppressed the stimulatory effects of both the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine (FMLP) and the phorbol ester phorbol myristate acetate (PMA). The combination of TNF beta with either FMLP or PMA produced enhancement of neutrophil adherence which exceeded that of either agent alone. These effects of the lymphokines were not due to endotoxin contamination since their effects were sensitive to heating and insensitive to polymyxin B treatment. These experiments provide further evidence for the critical role of these lymphokines in the regulation of acute and chronic inflammatory processes.     

Expression of cytokine mRNA in lentivirus-induced arthritis.

Infection of goats with the lentivirus caprine arthritis encephalitis virus (CAEV) leads to persistent infection and development of chronic arthritis. We analyzed the expression of cytokines and viral RNA in the joints of goats at early time points after experimental infection with CAEV and in those of animals suffering from chronic arthritis as a result of natural infection. In situ hybridization experiments showed that the pattern of cytokine expression in caprine arthritis was similar to that found in rheumatoid arthritis (RA), with a few cells expressing the lymphocyte-derived cytokines interferon (IFN)-gamma and interleukin (IL)-2 and rather more cells expressing monocyte chemoattractant protein (MCP)-1, IL-6, and tumor necrosis factor (TNF)-alpha. IFN-gamma mRNA expression in experimentally infected joints peaked at day 12 and was mostly detected in areas containing viral RNA. At later time points, no IFN-gamma- or virus-expressing cells were found in inflamed joints but both were again detected in goats with severe arthritis. Interestingly, at the clinical stage of arthritis reflecting the chronic stage of infection, the inflammatory lesion was found to be immunologically compartmentalized. Humoral immune responses and cell-mediated immune responses appeared to concurrently occur in distinct areas of the synovial membrane.     

Steroids to reduce the impact on delirium (STRIDE): a double-blind,randomised, placebo-controlled feasibility trial of pre-operative dexamethasone in people with hip fracture

Neuro-inflammation may be important in the pathogenesis of postoperative delirium following hip fracture surgery. Studies have suggested a potential role for steroids in reducing postoperative delirium; however, the potential efficacy and safety of pre-operative high-dose dexamethasone in this specific population is largely unknown. Conducting such a study could be challenging, considering the multidisciplinary team involvement and the emergency nature of the surgery. The aim of this study was to assess feasibility and effectiveness of dexamethasone given as early as possible following hospital admission for hip fracture, to inform whether a full-scale trial is warranted. This single-centre, randomised, double-blind, placebo-controlled study randomly allocated 79 participants undergoing hip fracture surgery to dexamethasone 20 mg or placebo pre-operatively. Eligibility and recruitment rates, timing of the intervention and adverse events were recorded. Incidence and severity of postoperative delirium were assessed using the 4AT delirium screening tool and the Memorial Delirium Assessment Scale. Postoperative pain, length of stay and mortality were also assessed. The eligibility rate for inclusion was 178/527 (34%), and 57/178 (32%) of eligible patients presented to hospital when no researcher was available (e.g. after-hours, weekends, public holidays). Recruitment was limited mainly by ethical limitations (not including patients with impaired cognition) and lack of weekend staffing. Median (IQR [range]) time from emergency department admission to drug administration was 13.3 (5.9–17.6 [1.8–139.6]) hours. There was a significant difference in delirium severity scores, favouring the dexamethasone group: median (IQR [range]) 5 (3–6 [3–7]) vs. 9 (6–13 [5–14]) in the placebo group, with the probability of superiority effect size being 0.89, p = 0.010. Delirium incidence did not differ between groups: 6/40 (15%) in the dexamethasone group vs. 9/39 (23%) in the placebo group, relative risk (95%CI) 0.65 (0.22–1.65), p = 0.360). A larger randomised controlled trial is feasible and ideally this should include people with existing cognitive impairment, seven days-a-week cover and a multicentre design.     

Epidemiology of stillbirths based on different gestational thresholds at a tertiary hospital

INTRODUCTIONThe stillbirth rate (SBR) is an important public health indicator. We studied the distribution of maternal and fetal characteristics and time trends of the SBR at KK Women’s and Children’s Hospital (KKH), Singapore, from 2004 to 2016 based on various definitions of stillbirth.METHODSData was obtained from the Data Warehouse and Stillbirth Reporting System of KKH from 2004 to 2016. SBRs were calculated based on three definitions (fetal deaths at ≥ 20 weeks, 24 weeks or 28 weeks of gestation per 1,000 total births) and were described with maternal and fetal characteristics, and by year.RESULTSFrom 2004 to 2016, the SBR declined by 44.7%, 25.5% and 18.9% based on Definitions I, II and III, respectively. The SBR at KKH in 2016 was 5.2 (Definition I), 4.1 (Definition II) and 3.0 (Definition III) per 1,000 total births. The SBR was significantly higher in women aged ≥ 35 years, nulliparas and female fetuses. The number of live births at 24–27⁺⁶ weeks of gestation was more than four times higher than that of stillbirths (822 vs. 176). There were 104 (12.7%) neonatal deaths during this gestation period, giving a high survival rate of 87.3%.CONCLUSIONThe SBR in KKH is relatively lower than that in other developed countries. There is a need to consider revising our hospital and national definitions of the stillbirth lower boundary from 28 weeks to 24 weeks of gestation. This would allow us to make better comparisons with other developed countries, in line with improvements in healthcare.     

Factors affecting visual acuity after one year of follow up after repeated intravitreal ranibizumab for macular degeneration

Aim

Providing intravitreal ranibizumab therapy for neovascular age related macular degeneration (nARMD) is a source of increasing strain for many UK eye departments. Whilst most units attempt to adhere to the product licence of following up patients at four weekly intervals; delays in follow up appointments can and do occur. We aim to see if mean follow up intervals during the maintenance phase are correlated with visual outcomes at one year and perform a multivariate analysis of patient factors in a bit to understand the factors affecting visual acuity outcomes.

Method

A continuously updated prospective audit of patients receiving ranibizumab therapy at the Royal Gwent Hospital was accessed and a coefficient of determination and Spearman’s rank test undertaken to see whether mean follow up delays resulted in visual acuity penalties after nine months of maintenance. Multivariate analysis using ANOVA was then undertaken to examine in more detail the various factors affecting visual acuity outcomes.

Results

805 eyes of 708 patients were included in the study. Mean follow up intervals varied between 28.0 and 96.3 days over the first six treatments of the maintenance phase (mean 49.2 – SD 10.7) with a mean change in visual acuity from baseline of +7.1 letters at 12 weeks and +4.6 letters at 52 weeks. There was a negative correlation seen between visual acuity gains after nine months of the maintenance phase and increasing clinic follow up times although Spearman’s rank analysis demonstrated a correlation coefficient of only −0.078, which was not statistically significant. Variability in follow up appointments resulting in worse outcomes was however significant (p < 0.01), as was increasing age at presentation (p = 0.04). Smoking was found to decrease age of presentation by six years (74.2 years vs 80.0 years). The adjusted R² for the whole analysis was 0.44.

Conclusion

Wide variation in follow up intervals was associated with a worse visual acuity outcome although longer mean follow up interval was not. Smokers presented at a significantly younger age than non-smokers or ex-smokers. This was a large study with an adjusted R² of 0.44. The results are relevant to other macular degeneration service providers around the world.     

Relationship of Phenotype and Genotype in X-Linked Amelogenesis Imperfecta

X-linked amelogenesis imperfectas (AI) resulting from mutations in the amelogenin gene (AMELX) are phenotypically and genetically diverse. Amelogenin is the predominant matrix protein in developing enamel and is essential for normal enamel formation. To date, 12 allelic AMELX mutations have been described that purportedly result in markedly different expressed amelogenin protein products. We hypothesize that these AMELX gene mutations result in unique and functionally altered amelogenin proteins that are associated with distinct amelogenesis imperfecta phenotypes. The AMELX mutations and associated phenotypes fall generally into three categories. (1) Mutations (e.g., signal peptide mutations) causing a total of loss of amelogenin protein are associated with a primarily hypoplastic phenotype (though mineralization defects also can occur). (2) Missense mutations affecting the N-terminal region, especially those causing changes in the putative lectin-binding domain and TRAP (tyrosine rich amelogenin protein) region of the amelogenin molecule, result in a predominantly hypomineralization/hypomaturation AI phenotype with enamel that is discolored and has retained amelogenin. (3) Mutations causing loss of the amelogenin C terminus result in a phenotype characterized by hypoplasia. The consistent association of similar hypoplastic or hypomineralization/hypomaturation AI phenotypes with specific AMELX mutations may help identify distinct functional domains of the amelogenin molecule. The phenotype-genotype correlations in this study suggest there are important functional domains of the amelogenin molecule that are critical for the development of normal enamel structure, composition, and thickness.     

Ministry of Health Clinical Practice Guidelines: Anxiety Disorders

The Ministry of Health (MOH) has developed the clinical practice guidelines on Anxiety Disorders to provide doctors and patients in Singapore with evidence-based treatment for anxiety disorders. This article reproduces the introduction and executive summary (with recommendations from the guidelines) from the MOH clinical practice guidelines on anxiety disorders, for the information of SMJ readers. Chapters and page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Ministry of Health website: http://www.moh.gov.sg/content/moh_web/healthprofessionalsportal/doctors/guidelines/cpg_medical.html. The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.

1.1 Background information

Anxiety disorders are known to be one of the most prevalent of psychiatric conditions, yet they often remain under-diagnosed and under-treated. Their chronic, disabling symptoms cause considerable burden not only to sufferers but also to their families, and contribute to poorer quality of life and considerable economic burden on society.In many instances, there is a delay in seeking treatment and in some cases such delay may stretch up to nearly ten years. This may result from ignorance of the condition, fear of taking medications, and the stigma of receiving a psychiatric diagnosis, and or having to accept psychiatric treatment.The anxiety disorders include panic disorder with or without agoraphobia, social anxiety disorder, specific phobia, obsessive-compulsive disorder, generalised anxiety disorder, acute stress disorder and post-traumatic stress disorder. In the clinical evaluation of anxiety disorders, it is important to ascertain the type of anxiety disorder present. This would allow treatment to be targeted at the specific type of disorder.These guidelines are developed to provide practical, evidence-based recommendations to primary care physicians and specialists in psychiatry for the diagnosis and management of the anxiety disorders.The first edition of the guidelines was published in 2003. In this edition, we present data from newer research as well as older data not previously reported in the earlier guidelines.For example, we examine the efficacy of combining medications with psychological therapy over medications alone, or psychological therapy alone. In view of the majority of anxiety sufferers being female we have made recommendations for pharmacotherapy during pregnancy and breastfeeding. As these guidelines are intended for use in the Singapore context, we have omitted treatments that are currently not available in Singapore.

1.2 Aim

These guidelines are developed to facilitate the diagnosis and assessment of the anxiety disorders, and to ensure that their management is appropriate and effective.

1.3 Scope

These guidelines will cover the management of anxiety disorders in adults and address the issues of medication use during pregnancy and breastfeeding.

1.4 Target group

The content of the guidelines will be useful for all doctors treating patients with anxiety disorders. Efforts have been made to ensure that the guidelines are particularly useful for primary care physicians and specialists in psychiatry, including all those involved in the assessment and management of patients with anxiety disorders in the community. The doctor treating the patient is ultimately responsible for clinical decisions made after reviewing the individual patient’s history, clinical presentation and treatment options available.

1.5 Development of guidelines

These guidelines have been produced by a committee of psychiatrists, a clinical psychologist, pharmacist, patient representative, and family practitioners appointed by the Ministry of Health. They were developed by revising the existing guidelines, reviewing relevant literature, including overseas clinical practice guidelines, and by expert clinical consensus of professionals with experience in treating patients in the local setting.The following principles underlie the development of these guidelines:

• Treatment recommendations are supported by scientific evidence whenever possible (randomised controlled clinical trials represent the highest level of evidence) and expert clinical consensus is used when such data are lacking.
• Treatment should maximise therapeutic benefits and minimise side effects.

1.6 What’s new in the revised guidelines

This edition of the guidelines contains updated recommendations based on latest evidence, as well as detailed discussions and recommendations on the management of anxiety disorders in adult populations.The following represent changes to the revised guidelines

• An extensive review of the literature, including new evidence. This involved the re-writing and extensive revision of the chapters.
• Length of treatment, which provides answers to a pertinent question.
• Use of medications during pregnancy and breastfeeding. Given that females are more likely to be at risk of being diagnosed with anxiety disorders, this is an important subject.

We are aware that the Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5) was released in 2013. In DSM-5, post-traumatic stress disorder and obsessive-compulsive disorder have been removed and classified separately from the rest of the anxiety disorders. If we were to adhere strictly to DSM-5, this would entail omitting discussion on post-traumatic stress disorder and obsessive-compulsive disorder. As it is our aim to provide an update on the 2003 guidelines, post-traumatic stress disorder and obsessive-compulsive disorder have been included in this edition of the guidelines.In addition, anxiety conditions in children are included in DSM-5. Since the present guidelines are meant to address only adult anxiety disorders, guidelines on children’s anxiety conditions are not included here.Hence, for purposes of these guidelines, we will continue to use classifications based on the International Classification of Diseases-10 (ICD-10) and DSM-IV-TR criteria.

1.7 Review of guidelines

Evidence-based clinical practice guidelines are only as current as the evidence that supports them. Users must keep in mind that new evidence could supersede recommendations in these guidelines. The workgroup advises that these guidelines be scheduled for review five years after publication, or when new evidence appears that requires substantive changes to the present recommendations.