Vitamin D supplementation and adverse skeletal and non-skeletal outcomes in individuals at increased cardiovascular risk: Results from the International Polycap Study (TIPS)-3 randomized controlled trial.

BACKGROUND AND AIMS: Vitamin D has mostly been tested in Western populations. We examined the effect of high dose vitamin D in a population drawn predominantly from outside of Western countries. METHODS AND RESULTS: This randomized trial tested vitamin D 60,000 IU monthly in 5670 participants without vascular disease but at increased CV risk. The primary outcome was fracture. The secondary outcome was the composite of CV death, myocardial infarction stroke, cancer, fracture or fall. Death was a pre-specified outcome. Mean age was 63.9 years, and 3005 (53.0%) were female. 3034 (53.5%) participants resided in South Asia, 1904 (33.6%) in South East Asia, 480 (8.5%) in South America, and 252 (4.4%) in other regions. Mean follow-up was 4.6 years. A fracture occurred in 20 participants (0.2 per 100 person years) assigned to vitamin D, and 19 (0.1 per 100 person years) assigned to placebo (HR 1.06, 95% CI 0.57-1.99, p-value = 0.86). The secondary outcome occurred in 222 participants (1.8 per 100 person years) assigned to vitamin D, and 198 (1.6 per 100 person years) assigned to placebo (HR 1.13, 95% CI 0.93-1.37, p = 0.22). 172 (1.3 per 100 person years) participants assigned to vitamin D died, compared with 135 (1.0 per 100 person years) assigned to placebo (HR 1.29, 95% CI 1.03-1.61, p = 0.03). CONCLUSION: In a population predominantly from South Asia, South East Asia and South America, high-dose vitamin D did not reduce adverse skeletal or non-skeletal outcomes. Higher mortality was observed in the vitamin D group. REGISTRATION NUMBER: NCT01646437.

Development of two salmonella-based oral vaccines against human respiratory syncytial virus.

Human respiratory syncytial virus is the most common cause of bronchiolitis and other respiratory infections in infants and the elderly worldwide. We have developed two new oral vaccines using Salmonella typhi TY21a to carry and express the immunogenic epitopes of RSV fusion (F) and attachment (G) glycoproteins on its surface, separately. To evaluate the efficacy of the designed vaccines, BALB/c mice were orally immunized and then infected with RSV. Immune response analyses showed that cellmediated, mucosal and humoral immunity in the vaccinated mice were significantly enhanced compared to the control group. Both vaccines generated a balanced Th1/Th2 immune response which is crucial for efficiency of vaccines against RSV. Furthermore, histopathological examination proved that these vaccines were safe as they did not cause any Th2-associated adverse effects in the lungs of RSV-infected mice. The findings of this research suggest that Salmonella-F and Salmonella-G vaccine candidates may have strong potential to prevent RSV infection.

Segregation and genetic linkage analyses of river catfish, Mystus nemurus, based on microsatellite markers.

The river catfish Mystus nemurus is an important fresh water species for aquaculture in Malaysia. We report the first genetic linkage map of M. nemurus based on segregation analysis and a linkage map using newly developed microsatellite markers of M. nemurus. A total of 70 of the newly developed polymorphic DNA microsatellite markers were analyzed on pedigrees generated using a pseudo-testcross strategy from 2 mapping families. In the first mapping family, 100 offspring were produced from randomly selected dams of the same populations; dams of the second family were selected from 2 different populations, and this family had 50 offspring. Thirty-one of the 70 markers segregated according to the Mendelian segregation ratio. Linkage analysis revealed that 17 microsatellite markers belonging to 7 linkage groups were obtained at a logarithm of the odds score of 1.2 spanning 584 cM by the Kosambi mapping function, whereas the other 14 remained unlinked. The results from this study will act as primer to a more extensive genetic mapping study aimed towards identifying genetic loci involved in determining economically important traits.

Immunogenicity of a truncated enterovirus 71 VP1 protein fused to a Newcastle disease virus nucleocapsid protein fragment in mice.

Enterovirus 71 (EV71) is one of the viruses that cause hand, foot and mouth disease. Its viral capsid protein 1 (VP1), which contains many neutralization epitopes, is an ideal target for vaccine development. Recently, we reported the induction of a strong immune response in rabbits to a truncated VP1 fragment (Nt-VP1t) displayed on a recombinant Newcastle disease virus (NDV) capsid protein. Protective efficacy of this vaccine, however, can only be tested in mice, since all EV71 animal models thus far were developed in mouse systems. In this study, we evaluated the type of immune responses against the protein developed by adult BALB/c mice. Nt-VP1t protein induced high levels of VP1 IgG antibody production in mice. Purified VP1 antigen stimulated activation, proliferation and differentiation of splenocytes harvested from these mice. They also produced significant levels of IFN-γ, a Th1-related cytokine. Taken together, Nt-VP1t protein is a potent immunogen in adult mice and our findings provide the data needed for testing of its protective efficacy in mouse models of EV71 infections.

Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus).

AIMS: To evaluate a live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 (Lac-D1ae) and/or D4 (Lac-D4ae) in protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus). METHODS AND RESULTS: The polymerase chain reaction (PCR)-amplified 250- and 750-bp sequences coding for domains D1 and D4 of aerolysin were individually cloned into pNZ8048 and electrotransformed into L. lactis. The recombinant vaccine candidates were then either orally fed or injected intraperitoneally into tilapia. The development of antibodies in sampled fish compared to control groups implied that the recombinant epitopes expressed in L. lactis were able to elicit an immunogenic response in tilapia. Interestingly, the lower doses of both Lac-D1ae and Lac-D4ae gave higher antibody levels over the study period. Fish immunized with Lac-D1ae and Lac-D4ae together showed the highest level of protection, and the mortality was reduced significantly compared to control strains in both modes of vaccination. CONCLUSIONS: The recombinant L. lactis strain expressing D1 and D4 produced aerolysin-specific serum IgM in tilapia. Both D1 and D4 promoted 55-82% relative per cent survival (RPS) against Aeromonas infection through intraperitoneal injection, whereas the RPS following oral feeding of the vaccine was 70-100%. SIGNIFICANCE AND IMPACT OF THE STUDY: The D1 and D4 regions of the aerolysin protein have been successfully identified as immunogenic regions that can elicit antibody production in tilapia and protect against challenge with Aer. hydrophila. A promising oral vaccine using L. lactis harbouring the D1 and D4 regions has been developed to control Aer. hydrophila.

Surface display of respiratory syncytial virus glycoproteins in Lactococcus lactis NZ9000.

AIMS: A system for displaying heterologous respiratory syncytial virus (RSV) glycoproteins on the surface of Lactococcus lactis NZ9000 was developed. METHODS AND RESULTS: Fusion of the USP45 signal peptide and the cA (C terminus of the peptidoglycan-binding) domains of AcmA, a major autolysin from L. lactis, to the N- and C-terminal of the target proteins, respectively, was carried out. The target protein was the major immunogenic domain of either the F (40.17-kDa) or G (11.49-kDa) glycoprotein domains of the RSV. Whole-cell ELISA readings obtained after 24 h of induction showed an increase in protein expression as the cA domain repeats increased, for the G glycoprotein of RSV. On the other hand, the F glycoprotein indicated decreasing expression levels as the number of cA domain repeats increased. The difference in the expression levels of the F and G domains may be attributed to the different sizes of the antigenic domains. CONCLUSIONS: The size and properties of the target proteins are vital in determining the amount of antigenic domains being displayed on the surface of live cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The system demonstrated here can aid in the utilization of the generally regarded as safe (GRAS) bacteria L. lactis, as a vaccine delivery vehicle to surface display the antigenic proteins of RSV.

Glycosylation is not necessary for recognition of the fusion glycoprotein domain of the human respiratory syncytial virus by a polyclonal antibody.

Human respiratory syncytial virus (HRSV) is a leading pathogen causing lower respiratory tract infections in infants and young children worldwide. In line with the development of an effective vaccine against HRSV, a domain of the fusion (F) glycoprotein of HRSV was produced and its immunogenicity and antigenic properties, namely the effect of deficient glycosylation was examined. A His-tagged recombinant F (rF) protein was expressed in Escherichia coli, solubilized with 8 mol/l urea, purified by the Ni-NTA affinity chromatography and used for the raising of a polyclonal antibody in rabbits. The non-glycosylated rF protein proved to be a strong immunogen that induced a polyclonal antibody that was able to recognize also the glycosylated F1 subunit of native HRSV. The other way around, a polyclonal antibody prepared against the native HRSV was able to react with the rF protein. These results indicated that glycosylation was not necessary for the F domain aa 212-574 in order to be recognized by the specific polyclonal antibody.

Immunogenic properties of recombinant ectodomain of Newcastle disease virus hemagglutinin-neuraminidase protein expressed in Escherichia coli.

Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a vital role in the viral infectivity, host immunity, and disease diagnosis. A portion of the HN gene encoding the ectodomain (nt 142-1739) was cloned and expressed in Escherichia coli yielding an insoluble HN protein and a soluble NusA-HN protein containing N-utilization substance A (NusA) fusion component. Both recombinant proteins were purified and used for immunization of chickens. The recombinant HN protein induced higher antibody titers as compared to the recombinant NusA-HN protein. These antibodies were able to react in immunoblot analysis with the corresponding recombinant proteins as well as with the HN protein of NDV.

Isolation of trinucleotide microsatellite markers for Mystus nemurus.

Twelve single locus trinucleotide microsatellite markers were developed to characterize the Asian river catfish, Mystus nemurus, an important food fish in South East Asia. They were obtained by using a rapid method namely the 5' anchored PCR enrichment protocol. The specific primers were designed to flank the repeat sequences and these were subsequently used to characterize 90 unrelated fish from Malaysia. The number of alleles per locus ranged from 2 (MnVj2-281) to 12 (MnBp8-4-43b) while the levels of heterozygosity ranged from 0.0444 (MnVj2-1-19) to 0.7458 (MnVj2-291).

Eleven novel polymorphic microsatellite DNA markers from the green-lipped mussel Perna viridis.

We report on the characterization of 11 polymorphic microsatellite loci in P. viridis, the first set of such markers developed and characterized for this species. The number of alleles per locus ranged from 2 to 7, whereas the observed heterozygosity ranged from 0.0447 to 0.4837. These markers should prove useful as powerful genetic markers for this species.

Goniothalamin induces apoptosis in vascular smooth muscle cells.

Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.

Improved protection from velogenic Newcastle disease virus challenge following multiple immunizations with plasmid DNA encoding for F and HN genes.

Specific-pathogen free (SPF) chickens were inoculated with the plasmid constructs encoding the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus (NDV), either individually or in combination and challenged with velogenic NDV. The antibody level against NDV was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, administration of the plasmid constructs with Freund's adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculated twice with the plasmid constructs emulsified with Freund's adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3%) chickens vaccinated with pEGFP-HN, five of ten (50.0%) chickens vaccinated with pEGFP-F and nine of ten (90.0%) chickens vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chickens were vaccinated with linearized pEGFP-F. The results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce higher level of antibodies for protection against velogenic NDV in chickens.

ALLHAT in perspective: implications to clinical practice and clinical trials.

ALLHAT study is the biggest randomized clinical trial in hypertension ever conducted. Its objective was to ompare the efficacy of newer (calcium channel blocker amlodipine and angiotensin-converting enzyme inhibitor inopril) to the older (diuretic chlorthalidone) antihypertensive agents in the treatment of patients with hypertension. After enrolling 42,000 patients who were followed for an average of 4.9 years, ALLHAT did not find significant differences in the primary end-points between these antihypertenive agents. ALLHAT however found significant differences in the secondary end-points such as heart failure and strokes between chlorthalidone and amlodipine or lisinopril. Based on these and on economic reasons, the investigators unequivocally recommended diuretics as the first line therapy for hypertension. Since its publication, ALLHAT has been much discussed, debated A and opined. The choice of drugs for study, the study design, the conduct of the study and the conclusions drawn by the investigators had all been criticised or controversial. Yet ALLHAT has been widely quoted, commented upon or referred to and it has been instrumental in initiating the JNC VII Guidelines. Thus a thorough understanding of ALLHAT is necessary for clinical practice and in designing and evaluating clinical trials in the future. Moving Points: in Medicine will capture the essence of ALLHAT, discusses its implications to clinical trials and explores its possible impact on the practice of medicine in this country.

Obesity in Malaysia.

This study was undertaken to assess the recent data on Malaysian adult body weights and associations of ethnic differences in overweight and obesity with comorbid risk factors, and to examine measures of energy intake, energy expenditure, basal metabolic rate (BMR) and physical activity changes in urban and rural populations of normal weight. Three studies were included (1) a summary of a national health morbidity survey conducted in 1996 on nearly 29 000 adults > or =20 years of age; (2) a study comparing energy intake, BMR and physical activity levels (PALs) in 409 ethnically diverse, healthy adults drawn from a population of 1165 rural and urban subjects 18-60 years of age; and (3) an examination of the prevalence of obesity and comorbid risk factors that predict coronary heart disease and type 2 diabetes in 609 rural Malaysians aged 30-65 years. Overweight and obesity were calculated using body mass index (BMI) measures and World Health Organization (WHO) criteria. Energy intake was assessed using 3-d food records, BMR and PALs were assessed with Douglas bags and activity diaries, while hypertension, hyperlipidaemia and glucose intolerance were specified using standard criteria. The National Health Morbidity Survey data revealed that in adults, 20.7% were overweight and 5.8% obese (0.3% of whom had BMI values of >40.0 kg m(-2)); the prevalence of obesity was clearly greater in women than in men. In women, obesity rates were higher in Indian and Malay women than in Chinese women, while in men the Chinese recorded the highest obesity prevalences followed by the Malay and Indians. Studies on normal healthy subjects indicated that the energy intake of Indians was significantly lower than that of other ethnic groups. In women, Malays recorded a significantly higher energy intake than the other groups. Urban male subjects consumed significantly more energy than their rural counterparts, but this was not the case in women. In both men and women, fat intakes (%) were significantly higher in Chinese and urban subjects. Men were moderately active with the exception of the Dayaks. Chinese women were considerably less active than Chinese men. Chinese and Dayak women were less active than Malay and Indian women. In both men and women, Indians recorded the highest PALs. Hence, current nutrition and health surveys reveal that Malaysians are already affected by western health problems. The escalation of obesity, once thought to be an urban phenomenon, has now spread to the rural population at an alarming rate. As Malaysia proceeds rapidly towards a developed economy status, the health of its population will probably continue to deteriorate. Therefore, a national strategy needs to be developed to tackle both dietary and activity contributors to the excess weight gain of the Malaysian population.

Soluble intercellular adhesion molecule-1 and interleukin-6 levels reflect endothelial dysfunction in patients with primary hypercholesterolaemia treated with atorvastatin.

Adhesion molecules and cytokines are involved in the pathogenesis of intimal injury in atherosclerosis but their relationship with endothelial function remains unclear. The objectives of this study were to examine the effects of atorvastatin on soluble adhesion molecules, interleukin-6 (IL-6) and brachial artery endothelial-dependent flow mediated dilatation (FMD) in patients with familial (FH) and non-familial hypercholesterolaemia (NFH). A total of 74 patients (27 FH and 47 NFH) were recruited. Fasting lipid profiles, soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular-cellular adhesion molecule-1 (sVCAM-1), E-selectin, IL-6 and FMD were measured at baseline, 2 weeks, 3 and 9 months post-atorvastatin treatment (FH--80 mg/day, NFH--10 mg/day). In both groups, compared to baseline, sICAM-1 levels were significantly reduced at 2 weeks, further reduced at 3 months and maintained at 9 months (P<0.0001). The IL-6 levels were significantly reduced at 3 months and 9 months compared to baseline for FH (P<0.005) and NFH (P<0.0001). In both groups, the FMD at 2 weeks was higher than baseline (P<0.005), with progressive improvement up to 9 months. FMD was negatively correlated with sICAM-1 and IL-6. In conclusion, both low and high doses of atorvastatin lead to early progressive improvement in endothelial function in patients with primary hypercholesterolaemia. sICAM-1 and IL-6 levels reflect endothelial dysfunction in these patients.

PCR cycle sequencing protocol for population mitochondrial cytochrome b analysis.

Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.

Nipah virus glycoprotein: production in baculovirus and application in diagnosis.

A method for serological diagnosis of Nipah virus (NiV) is described. DNA encoding truncated G protein of NiV was cloned into the pFastBac HT vector, and the fusion protein to His-tag was expressed in insect cells by recombinant baculovirus. The resulting His-G recombinant fusion protein was purified by affinity chromatography and used as the coating antigen for serological testing by indirect enzyme-linked immunosorbant assay (ELISA). When tested against a panel of swine serum samples, the recombinant G protein-based ELISA successfully discriminated all 40 samples previously determined to be serum neutralizing test (SNT) positive from 11 SNT negatives samples. The data show that the recombinant G protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. The main advantage of the recombinant G protein-based NiV ELISA compared to an ELISA using whole virus antigen is the use of a single antigenic protein instead of inactivated whole virus which is required to be prepared under high risk and cost. This test is suitable for routine diagnosis of NiV and also for epidemiological surveys as it allows highly reliable testing of a large number of sera rapidly.

Mapping of three antigenic sites on the haemagglutinin-neuraminidase protein of Newcastle disease virus.

Nine neutralizing monoclonal antibodies (MAbs), each of which react with the haemagglutinin-neuraminidase (HN) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV), have been used in competitive binding assays to delineate three non-overlapping antigenic sites A, B and C. Epitopes within these sites have been identified on the basis of cross-reactivity of MAb-resistant mutants against the panel of MAbs, determined by plaque assays and Western blotting. Site A contains three non-overlapping epitopes (A1, A2 and A3). A1 is the only linear epitope; all remaining epitopes are conformational. MAbs which react with epitopes A2 and A3 inhibit neuraminidase activity (NA) when assayed with neuraminlactose. Site B contains three partially overlapping epitopes (B1, B2 and B3) and site C is represented by a single epitope (C1). HN gene sequence analysis of MAb-resistant mutants showed that they each had only single amino acid substitutions which range from amino acid residues 347-460 for site A, 284-325 for site B, and at 481 for the C1 epitope. The apparent molecular mass of the HN glycoprotein of one mutant was increased from 72 to 75 kDa. This correlates well with the creation of an additional potential glycosylation site in this mutant from Asn-Ser-Pro(325) to Asn-Ser-Ser(325).

Importance of lead selection in QT interval measurement.

The influence of lead selection on QT estimation in the 12-lead electrocardiogram was assessed in 63 patients (21 control subjects, 21 with anterior myocardial infarction, 21 with inferior myocardial infarction). QT estimates varied between leads. The variation was greater in patients with myocardial infarction than in control subjects (mean dispersion of QT: control subjects, 48 +/- 18 ms [+/- standard deviation]; anterior myocardial infarction, 70 +/- 30 ms; inferior myocardial infarction, 73 +/-32 ms). The maximum QT in any lead (QTmax) was determined and the deviation of each lead from this maximum value calculated. In all 3 groups, anteroseptal leads (V2 or V3) provided the closest approximation to QTmax. Interlead variability was found to be mainly due to variation in timing of the end of the T wave, rather than the onset of the QRS complex. The variability due to leads was considerably greater than the variability due to cycles, observers or measurement error. Implementation of a variety of current lead selection practices resulted in widely divergent estimates of QT interval. It is concluded that there is a need for standardization of lead selection practice for QT measurement. If measurements are confined to one or a few leads, anteroseptal leads provide the closest approximation to QTmax.