Pre-Existing Cross-Reactive Antibodies to Avian Influenza H5N1 and 2009 Pandemic H1N1 in US Military Personnel

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.Outbreaks of 1997 avian influenza H5N1 and 2009 pandemic (p) H1N1 in humans have provided an opportunity to gain insight into cross-reactive immunity. The US military periodically collects and stores serum samples from service members linked to medical records.¹ We measured cross-reactive antibodies in stored serum to avian influenza H5N1 and 2009 pH1N1 from US military personnel and identified factors associated with presence of neutralizing antibodies.Two hundred archived serum samples were obtained from the US Department of Defense Serum Repository. They were representative of a wide cross-section of active military personnel at the times of collection, whereas specific geographic information was not available on the individual selected; the cohort represents the general US military population, which is deployed throughout the United States and globally. Fifty samples each were selected from four birth cohorts: (1) < 1949, (2) 1960–1965, (3) 1966–1971, and (4) 1972–1977. Within each cohort, 25 samples were collected in the year 2000 (before the introduction of intranasal live attenuated influenza vaccine [LAIV]), and 25 samples were collected in 2008 (where 51% of donors had received LAIV). It has been suggested that LAIV elicits cross-reactive immunity.^2,3 The samples were all collected before the outbreak of 2009 pH1N1, and there have not been any reported outbreaks of H5N1 in US military personnel.Assays used to measure antibodies included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay.⁴ Viral neutralization by antibodies against H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based (H5pp)⁵ and microneutralization assays, respectively. Electronic medical and vaccination records from the Defense Medical Surveillance System (DMSS), which captured records before the serum sample date, were linked to samples and compared with the in vitro results.¹The odds ratios (ORs) and 95% confidence intervals (95% CIs) of univariate and multivariate binary logistic regression analyses were used to determine the association between donor characteristics and positive antibody responses. A multiple logistic regression model was constructed, and it included independent variables with a P value of < 0.05 in univariate logistic regression. A P value of < 0.05 was considered to indicate statistical significance. SPSS 12.0 for Windows (SPSS Inc., Chicago, IL) was used to perform all statistical analysis.Cross-reactivity is summarized in 5 and 22.5% for the NI assay. H5pp and NI antibody titers to H5N1 were evenly distributed among birth cohorts and did not differ substantially based on history of vaccination or prior respiratory infections. Of those individuals with neutralizing antibodies to H5N1 (N = 28), 32.1% also had neutralizing antibodies to pH1N1, whereas 19.3% of those individuals with any H5N1-specific antibody response also had neutralizing antibodies to pH1N1 (

Evaluation of the safety and immunogenicity in rhesus monkeys of a recombinant malaria vaccine for Plasmodium vivax with a synthetic Toll-like receptor 4 agonist formulated in an emulsion

Plasmodium vivax is the major cause of malaria outside sub-Saharan Africa and inflicts debilitating morbidity and consequent economic impacts in developing countries. In order to produce a P. vivax vaccine for global use, we have previously reported the development of a novel chimeric recombinant protein, VMP001, based on the circumsporozoite protein (CSP) of P. vivax. Very few adjuvant formulations are currently available for human use. Our interest is to evaluate second-generation vaccine formulations to identify novel combinations of adjuvants capable of inducing strong, long-lasting immune responses. In this study rhesus monkeys were immunized intramuscularly three times with VMP001 in combination with a stable emulsion (SE) or a synthetic Toll-like receptor 4 (TLR4) agonist (glucopyranosyl lipid A [GLA]) in SE (GLA-SE). Sera and peripheral blood mononuclear cells (PBMCs) were tested for the presence of antigen-specific humoral and cellular responses, respectively. All groups of monkeys generated high titers of anti-P. vivax IgG antibodies, as detected by enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays. In addition, all groups generated a cellular immune response characterized by antigen-specific CD4(+) T cells secreting predominantly interleukin-2 (IL-2) and lesser amounts of tumor necrosis factor (TNF). We conclude that the combination of VMP001 and GLA-SE is safe and immunogenic in monkeys and may serve as a potential second-generation vaccine candidate against P. vivax malaria.     

CYP2C19 genetic polymorphism in Thai, Burmese and Karen populations

The genetic polymorphism of CYP2C19 was examined in three Southeast Asian populations. This study was conducted in 774 Thais, 127 Burmeses and 131 Karens. Genomic DNA was extracted from leucocytes and analyzed by the PCR-RFLP technique. Genotype analysis revealed that the allele frequencies of CYP2C19*1, CYP2C19*2 and CYP2C19*3 in the Thais were 0.68, 0.29 and 0.03, respectively, and those of the Burmese population were 0.66, 0.30 and 0.04, respectively. For Karens, the minority ethnic in Mynmar, the allele frequencies of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were 0.71, 0.28 and 0.01, respectively. The prevalence of PM estimated from genotype data among these three ethnic populations were 9.2%, 11.0%, and 8.4%, respectively. The PM phenotype and the frequencies of CYP2C19 defective alleles, particularly CYP2C19*3 among these three Southeast Asian ethnics appeared to be lower than other Asian populations. Lower prevalence of CYP2C19 PM suggests that these ethnics may have different capacity to metabolize drugs that are substrates of CYP2C19. Certain drug dosage regiments should be considered differently for Asian populations.     

Antioxidant effects of aqueous extracts from dried calyx of Hibiscus sabdariffa Linn. (Roselle) in vitro using rat low-density lipoprotein (LDL)

The present study quantitatively investigated the antioxidant effects of the aqueous extracts from dried calyx of Hibiscus sabdariffa LINN. (roselle) in vitro using rat low-density lipoprotein (LDL). Formations of the conjugated dienes and thiobarbituric acid reactive substances (TBARs) were monitored as markers of the early and later stages of the oxidation of LDL, respectively. Thus, we demonstrated that the dried calyx extracts of roselle exhibits strong antioxidant activity in Cu(2+)-mediated oxidation of LDL (p<0.05) in vitro. The inhibitory effect of the extracts on LDL oxidation was dose-dependent at concentrations ranging from 0.1 to 5 mg/ml. Moreover, 5 mg/ml of roselle inhibited TBARs-formation with greater potency than 100 microM of vitamin E. In conclusion, this study provides a quantitative insight into the potent antioxidant effect of roselle in vitro.     

Studies of human leprosy lesions in situ using suction-induced blisters: cell changes with IgM antibody to PGL-1 and interleukin-2 receptor in clinical subgroups of erythema nodosum leprosum.

To examine the immunopathogenesis of type 2 erythema nodosum leprosum (ENL) reactions in leprosy, we studied cellular and soluble immunologic components of skin lesions in 57 patients with reactions (19 acute ENL and 38 chronic ENL), 61 active patients without reactions, and 33 control patients whose leprosy had been treated and cured. Cells, IgM antibody to PGL-1 and Tac peptide levels were obtained from fluid aspirated from blisters induced by suction directly over representative skin lesions. During ENL reactions: a) the lesions in chronic ENL showed a decreased number of CD8+ (T-suppressor) cells and increased helper/suppressor ratio as compared to those in acute ENL and non-reactional leprosy; b) Tac peptide and IgM antibody to PGL-1 levels were elevated in the chronic ENL lesions; c) and systemic administration of corticosteroids appeared to cause a reduction in the intralesional CD4+ cell population and IgM antibody to PGL-1 but did not change CD8+ cell population and the levels of Tac peptide in the lesions. The elevated levels of Tac peptide were localized in the skin lesions while increased levels of IgM anti-PGL-1 seemed to be filtered from the peripheral blood. We conclude that spontaneous lymphocyte activation in situ, primarily of decreased CD8+ and relatively increased CD4+ cells, are important features of chronic, recurrent ENL reactions and may be an intermittent or cyclic phenomenon during the reaction. Understanding the mechanisms of these spontaneous changes in immunity in leprosy will enlarge our knowledge of reactions and of the underlying determinants of delayed type hypersensitivity and cell-mediated immunity in leprosy, which in turn will allow us to realize the potential for artificially manipulating these responses as proposed with vaccines or immunotherapy.     

Hypocholesterolemic and antioxidant effects of aqueous extracts from the dried calyx of Hibiscus sabdariffa L. in hypercholesterolemic rats

The present study was designed to investigate the hypolipidemic effects and antioxidant effects of Hibiscus sabdariffa L. (roselle) with regard to protection of LDL oxidation in vivo and ex vivo in rats made hypercholesterolemic by continuous cholesterol feeding. Administering the dried calyx extracts of roselle at doses of 500 and 1,000 mg/kg together with continuous cholesterol feeding to hypercholesterolemic rats for 6 weeks significantly decreased serum cholesterol level by 22% and 26%, respectively (p<0.001); serum triglycerides level by 33% and 28%, respectively (p<0.05); serum LDL level by 22% and 32%, respectively (p<0.05). However, serum HDL level was not affected. LDL was extracted from plasma of the hypercholesterolemic rats and the effects of the dried calyx extracts of roselle on the oxidation of LDL in vivo and ex vivo were examined. Six-week treatment with 250, 500 and 1,000 mg/kg of the extracts significantly decreased thiobarbituric acid reactive substances (TBARs) formation (p<0.05) while the formation of conjugated dienes during the oxidation of LDL induced by CuSO(4) was reduced, but not significantly different. These lines of evidence suggest that the aqueous extracts from the dried calyx of roselle possess both antioxidant effects against LDL oxidation and hypolipidemic effects in vivo. However, its mechanism(s) of action remains to be elucidated.     

HPV genotyping in neuroendocrine carcinoma of the uterine cervix in northern Thailand

Objective

To determine the distribution of HPV genotypes in cervical neuroendocrine carcinoma (NECA) in northern Thailand, and evaluate the correlation between HPV genotype and clinicopathologic features.

Methods

Samples from 111 women treated for cervical NECA at Chiang Mai University Hospital between 1992 and 2009 were tested for HPV genotype. Samples were formaldehyde-fixed, paraffin-embedded, and tested via nested PCR and dot blot hybridization.

Results

Ninety-seven of the 111 samples were adequate for DNA analysis. HPV DNA was detected in 93 samples, of which 76 (81.7%) were single, 14 (15.1%) were multiple, and 3 (3.2%) were untyped infections. HPV18 was the most common subtype (70 cases, 75.3%), followed by HPV16 (28 cases, 30.1%). Other genotypes included HPV58 (3.2%), HPV52 (2.1%), and HPV33 (1.1%). Collectively, HPV16 and/or HPV18 were found in 83 cases (89.3%). Women with HPV18 infection were significantly younger (42.0 years) than those with non-HPV18 infections (54.1 years) (P = 0.003). Associated adenocarcinoma in situ was more frequently seen among women with HPV18 infection (P = 0.034).

Conclusions

HPV18 infection was predominant in cervical NECA. Variations in HPV genotype may be related to the clinicopathologic features and pathogenetic pathways of NECA. Vaccination against HPV16 and HPV18 might provide protection against cervical NECA in almost 90% of cases.     

Coreceptor utilization of HIV type 1 subtype E viral isolates from Thai men with HIV type 1-infected and uninfected wives.

HIV-1 coreceptors CCR5 and CXCR4 play an important role in viral entry and pathogenesis. To better understand the role of viral tropism in HIV-1 transmission, we examined the coreceptor utilization of viral isolates obtained from men enrolled in a study of heterosexual transmission in northern Thailand. Viral isolates were obtained from HIV-1-positive males who had either HIV-1-infected spouses (RM; n = 5) or HIV-1-uninfected spouses (HM; n = 10). Viral isolates from 1 of the 5 RM males and 2 of the 10 HM males were CCR5 tropic, whereas isolates from 3 RM males and 6 of the HM male isolates were CXCR4 tropic. Of the nine X4-tropic isolates, seven also used at least one of the following coreceptors: CCR8, CCR1, CCR2b, or CX3CR1, and none employed CCR5 as an additional coreceptor. More importantly, three isolates, RM-15, HM-13, and HM-16 (one from a transmitter and two from nontransmitter), did not infect GHOST4.cl.34 cells expressing any of the known coreceptors. Further analysis using MAGI-plaque assays, which allow visualization of infected cells, revealed that RM-15 had low numbers of infected cells in MAGI-R5 and MAGI-X4 cultures, whereas HM-13 and HM-16 had high levels of plaques in MAGI-X4 cultures. Replication kinetics using activated lymphocytes revealed that these three isolates replicated in CCR5(+/+) as well as CCR5(-/-) peripheral blood mononuclear cells, suggesting that these isolates did not have an absolute requirement of CCR5 for viral entry. All three isolates were sensitive to the X4-antagonistic compounds T-22 and AMD3100. Analysis of the C2V3 region did not reveal any significant structural differences between any of the Thai subtype E isolates. Thus, there was no association between the pattern of coreceptor usage and transmissibility among these subtype E HIV-1 isolates.     

Investigation of human brain tumors for the presence of polyomavirus genome sequences by two independent laboratories

JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) may be associated with human brain tumors. These polyomaviruses have been shown to induce brain tumors in experimentally infected animals. Several studies have found polyomavirus genomic sequences in human brain tumor tissues by using polymerase chain reaction (PCR), while others have not. Inconsistencies in previous findings may be due in part to small sample sizes and differences in underlying patient populations, laboratory techniques and quality control measures. To assess the role of polyomaviruses in human brain tumors and address inconsistencies of previous reports, we investigated the prevalence of viral sequences in a series of 225 brain tumor tissue specimens in 2 independent laboratories. PCR followed by Southern hybridization was performed at the National Institute of Neurological Disorders and Stroke (NINDS). Real-time quantitative PCR was performed on the same tissues at Johns Hopkins University (JHU). Only those tumors with amplifiable DNA were tested further for polyomavirus sequences. Positive and negative control tissues were included, and all specimens were masked. Amplifiable DNA was detected in 225/225 (100%) tumors at NINDS, 9 (4%) of which contained polyomavirus sequences (3 JCV-positive, 3 BKV-positive and 3 SV40-positive). The JHU laboratory amplified DNA from 165/225 (73%) tumors, of which 1 tumor tested positive (for SV40). No tumors tested positive in both laboratories. Results for masked quality control tissues were concordant between laboratories. Nucleotide sequences for JCV, BKV and SV40 are rarely present in a large series of adult and pediatric brain tumors.