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Objective: To pilot test the effectiveness, feasibility, and acceptability of instituting a 15-pill quantity default in the electronic health record for new Schedule II opioid prescriptions., Design: A mixed-methods pilot study in two health systems, including pre-post analysis of prescribed opioid quantity and focus groups or interviews with prescribers and health system administrators., Methods: We implemented a 15-pill electronic health record default for new Schedule II opioids and assessed opioid quantity before and after implementation using electronic health record data on 6,390 opioid prescriptions from 448 prescribers. We then analyzed themes from focus groups and interviews with four staff members and six prescribers., Results: The proportion of opioid prescriptions for 15 pills increased at both sites after adding an electronic health record default, with one reaching statistical significance (from 4.1% to 7.2% at CHC, P = 0.280, and 15.9% to 37.2% at WVU, P < 0.001). The proportion of 15-pill prescriptions increased among high-prescribing departments and among most high- and low-frequency prescribers, except for low-frequency prescribers at CHC. Sites reported limited challenges in instituting the default, although ease of implementation varied by electronic health record vendor. Most prescribers were not aware of the default change and stated that they made prescribing decisions based on patient clinical characteristics rather than defaults., Conclusions: This pilot provides initial evidence that changing default settings can increase the number of prescriptions at the default level. This low-cost and relatively simple intervention could have an impact on opioid overprescribing. However, default settings should be selected carefully to avoid unintended consequences.

Significant ethnic and socio-economic disparities exist in infectious diseases (IDs) rates in New Zealand, so accurate measures of these characteristics are required. This study compared methods of ascribing ethnicity and socio-economic status. Children in the Growing Up in New Zealand longitudinal cohort were ascribed to self-prioritised, total response and single-combined ethnic groups. Socio-economic status was measured using household income, and both census-derived and survey-derived deprivation indices. Rates of ID hospitalisation were compared using linked administrative data. Self-prioritised ethnicity was simplest to use. Total response accounted for mixed ethnicity and allowed overlap between groups. Single-combined ethnicity required aggregation of small groups to maintain power but offered greater detail. Regardless of the method used, Māori and Pacific children, and children in the most socio-economically deprived households had a greater risk of ID hospitalisation. Risk differences between self-prioritised and total response methods were not significant for Māori and Pacific children but single-combined ethnicity revealed a diversity of risk within these groups. Household income was affected by non-random missing data. The census-derived deprivation index offered a high level of completeness with some risk of multicollinearity and concerns regarding the ecological fallacy. The survey-derived index required extra questions but was acceptable to participants and provided individualised data. Based on these results, the use of single-combined ethnicity and an individualised survey-derived index of deprivation are recommended where sample size and data structure allow it.

New Zealand children suffer from high rates of skin and soft tissue infection (SSTI). Staphylococcus aureus colonisation is known to increase the risk of nosocomial infection. We aimed to determine whether S. aureus colonisation also increased the risk of community-onset SSTI. This study, performed within the Growing Up in New Zealand cohort, used interview and administrative data, and bacterial culture results from the nose, throat, and skin swabs collected at 4½ years of age. Multivariable log-binomial regression was used to derive adjusted risk ratios. S. aureus was isolated from 2225/5126 (43.4%) children. SSTI affected 1509/5126 (29.4%) children before age five. S. aureus colonisation at any site was associated with SSTI (aRR = 1.09, 95%CI 1.01-1.19), particularly in the year prior to swab collection (aRR = 1.18, 95%CI 1.02-1.37). The strongest association was between skin colonisation and SSTI within the year prior to swab collection (aRR = 1.47, 95%CI 1.14-1.84). Socioeconomic and ethnic variables remained independent determinants of SSTI. S. aureus colonisation was associated with an increased risk of community-onset SSTI. Socioeconomic and ethnic factors and eczema had independent effects on SSTI risk. Interventions which reduce the prevalence of S. aureus colonisation may be expected to reduce the incidence of community-onset SSTI.

The lack of standardized diet for laboratory animals can have profound effects on animal health and lead to less reproducible research outcomes. Live diets are commonly used in zebrafish culture and, although they are a more natural feed than flake or pellet food, are also a potential source of pathogens and toxic compounds. Heavy metals are a group of such compounds, which can accumulate in fish leading to developmental abnormalities, reduced growth, and increased rates of mortality. Two to three weeks after feeding adult zebrafish a new lot of nonhatching decapsulated brine shrimp cysts (Decaps), embryos at the University of Minnesota Zebrafish Core Facility (ZCF) and the University of Utah Centralized Zebrafish Animal Resource (CZAR) began to exhibit an orange color in the yolk, and larval health began to decline. The concentration of chromium in the Decaps (69.6 mg/kg) was more than 30 times that of other zebrafish diets tested (up to 2.1 mg/kg) and is thought to be the cause of the observed symptoms. Within 3 weeks of removing the Decaps from the feeding regimen, the orange coloration in the yolks began to diminish, the morphological abnormalities began to subside, and larval survival rates began to increase. Thus, implementation of standardized zebrafish diets and regular feed-quality testing may help to prevent the introduction of contaminants to zebrafish research facilities.

Background: Increasing concerns about antibiotic resistance and microbiome disruption have stimulated interest in describing antibiotic consumption in young children. Young children are an age group for whom antibiotics are frequently prescribed., Objectives: To describe community antibiotic dispensing during the first 5 years of life in a large, socioeconomically and ethnically diverse cohort of children, and to determine how antibiotic dispensing varied between population subgroups., Methods: This study was performed within the Growing Up in New Zealand longitudinal cohort study ( www.growingup.co.nz ) with linkage to national administrative antibiotic dispensing data. Descriptive statistics and univariate and multivariable associations were determined., Results: The 5581 cohort children received 53 052 antibiotic courses, of which 54% were amoxicillin. By age 5 years, 97% of children had received one or more antibiotic courses, and each child had received a median of eight antibiotic courses (IQR 4-13). The mean incidence of antibiotic dispensing was 1.9 courses/child/year. Multivariable negative binomial regression showed that Māori and Pacific children received more antibiotic courses than European children, as did children in the most-deprived compared with the least-deprived areas. A distinct seasonal pattern was noted., Conclusions: This study provided a detailed description of antibiotic dispensing within a large and diverse child cohort. Antibiotic exposure was near universal by age 5 years. The predominance of amoxicillin use and the seasonal pattern suggest much antibiotic use may have been for self-limiting respiratory infections. There is a need for safe and effective interventions to improve antibiotic prescribing practices for New Zealand children., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aim: Infectious disease (ID) hospitalisation rates are increasing in New Zealand (NZ), especially in pre-school children, and Māori and Pacific people. We aimed to identify risk factors for ID hospitalisation in infancy within a birth cohort of NZ children, and to identify differences in risk factors between ethnic groups., Methods: We investigated an established cohort of 6846 NZ children, born in 2009-2010, with linkage to a national data set of hospitalisations. We used multivariable logistic regression to obtain odds ratios (OR) for factors associated with ID hospitalisation in the first year of life, firstly for all children, and then separately for Māori or Pacific children., Results: In the whole cohort, factors associated with ID hospitalisation were Māori (OR: 1.49, 95% CI: 1.17-1.89) or Pacific (2.51; 2.00-3.15) versus European maternal ethnicity, male gender (1.32; 1.13-1.55), low birthweight (1.94, 1.39-2.66), exclusive breastfeeding for <4 months (1.22, 1.04-1.43), maternal experience of health-care racism (1.60, 1.19-2.12), household deprivation (most vs. least deprived quintile of households (1.50, 1.12-2.02)), day-care attendance (1.43, 1.12-1.81) and maternal smoking (1.55, 1.26-1.91). Factors associated with ID hospitalisation for Māori infants were high household deprivation (2.16, 1.06-5.02) and maternal smoking (1.48, 1.02-2.14); and for Pacific infants were delayed immunisation (1.72, 1.23-2.38), maternal experience of health-care racism (2.20, 1.29-3.70) and maternal smoking (1.59, 1.10-2.29)., Conclusions: Māori and Pacific children in NZ experience a high burden of ID hospitalisation. Some risk factors, for example maternal smoking, are shared, while others are ethnic-specific. Interventions aimed at preventing ID hospitalisations should address both shared and ethnic-specific factors., (© 2016 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

By most measures, the University of Utah Centralized Zebrafish Animal Resource is a successful zebrafish core facility: we house ∼4000-5000 tanks for over 16 research groups; provide services and equipment for ∼150 users; are currently undergoing an expansion by 3000 tanks; and have been praised by institutional and national regulatory agencies for the cleanliness and efficiency of our facility. In recent years, we have implemented new programs to improve the overall health of our colony and believe we have seen a reduction in apparently sick fish. However, there are still deficiencies in our monitoring and pathogen control programs. Our histopathology sample sizes have been insufficient to estimate prevalence, but our sentinel tank program reveals the presence of Pseudoloma neurophilia and myxozoan, presumably Myxidium streisinger, in our facility. As we develop protocols to further reduce the burden of disease, we are focused on defining our baseline, establishing goals, and implementing methods to monitor our progress. The data generated by this approach will allow us to evaluate and implement the most cost-effective protocols to improve fish health.

Background: Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide-binding protein (G-protein)-coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia., Methods: We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells., Results: The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2-associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2-associated mutations increased cell sensitivity., Conclusions: Gα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.).

Multiple sclerosis (MS) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 Americans. GBV-C or hepatitis G is a flavivirus that is found in the serum of 1-2% of blood donors. It was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. Fifty frozen specimens from the brains of deceased persons affected by MS were obtained along with 15 normal control brain specimens. RNA was extracted and ribosomal RNAs were depleted before sequencing on the Illumina GAII. These 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in GenBank organized into 4080 taxa. An individual read successfully aligned to the viral database was considered to be a "hit". Normalized MS specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. Seventeen MS and 11 control brain extracts were sequenced, yielding 4-10 million sequences ("reads") each. Over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 MS samples. Sequences resembling other viruses previously implicated in the pathogenesis of MS were not significantly enriched in any of the diseased brain specimens. Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. GBV-C in this brain specimen was confirmed by specific amplification in this single MS brain specimen, but not in the 30 other MS brain samples available. The entire 9.4 kb sequence of this GBV-C isolate is reported here. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. The first isolation of GBV-C from human brain is reported here.

Background: Herpes simplex virus type 1 (HSV-1) infects >70% of the United States population. We identified a 3-megabase region on human chromosome 21 containing 6 candidate genes associated with herpes simplex labialis (HSL, "cold sores")., Methods: We conducted single nucleotide polymorphism (SNP) scans of the chromosome 21 region to define which of 6 possible candidate genes were associated with cold sore frequency. We obtained the annual HSL frequency for 355 HSV-1 seropositive individuals and determined the individual genotypes by SNPlex for linkage analysis and parental transmission disequilibrium testing (ParenTDT)., Results: Two-point linkage analysis showed positive linkage between cold sore frequency and 2 SNPs within the C21orf91 region, 1 of which is nonsynonymous. ParenTDT analysis revealed a strong association between another C21orf91 SNP, predicted to lie in the 3' untranslated region, and frequent HSL (P = .0047). C21orf 91 is a predicted open reading frame of unknown function that encodes a cytosolic protein., Conclusions: We evaluated candidate genes in the cold sore susceptibility region using fine mapping with 45 SNP markers. 2 complementary techniques identified C21orf91 as a gene of interest for susceptibility to HSL. We propose that C21orf91 be designated the Cold Sore Susceptibility Gene-1 (CSSG1).

Background: Severe malaria (SM) syndromes caused by Plasmodium falciparum infection result in major morbidity and mortality each year. However, only a fraction of P. falciparum infections develop into SM, implicating host genetic factors as important determinants of disease outcome. Previous studies indicate that tumour necrosis factor (TNF) and lymphotoxin alpha (LTα) may be important for the development of cerebral malaria (CM) and other SM syndromes., Methods: An extensive analysis was conducted of single nucleotide polymorphisms (SNPs) in the TNF, LTA and LTB genes in highland Papuan children and adults, a population historically unexposed to malaria that has migrated to a malaria endemic region. Generated P-values for SNPs spanning the LTA/TNF/LTB locus were corrected for multiple testing of all the SNPs and haplotype blocks within the region tested through 10,000 permutations. A global P-value of < 0.05 was considered statistically significant., Results: No associations between SNPs in the TNF/LTA/LTB locus and susceptibility to SM in highland Papuan children and adults were found., Conclusions: These results support the notion that unique selective pressure on the TNF/LTA/LTB locus in different populations has influenced the contribution of the gene products from this region to SM susceptibility.

Background: Age and host genetics are important determinants of malaria severity. Lymphotoxin-alpha (LTalpha) has been associated with the development of cerebral malaria (CM) and other severe malaria (SM) syndromes. Mutations in genes regulating LTalpha production contribute to other acute vascular diseases and may contribute to malaria pathogenesis., Methods: We tested the association between rs7291467, a single-nucleotide polymorphism (SNP) in the LTalpha-related gene encoding galectin-2 (LGALS2), disease severity, and function in a case-control study of ethnic Highland Papuan adults and children with SM (n = 380) and asymptomatic malaria-exposed controls (n = 356) originating from a non-malaria-endemic region but residing in a lowland malaria-endemic area of Papua, Indonesia., Results: The LGALS2 SNP showed a significant association with susceptibility to SM (including CM), in children (odds ratio, 2.02 [95% confidence interval, 1.14-3.57]) but not in adults. In SM, the C allele at rs7291467 was associated with enhanced galectin-2 transcript levels. In a separate group of Tanzanian children originating from a malaria-endemic region, we found preservation of the major ancestral LGALS2 allele and no association with susceptibility to CM., Conclusions: Results suggest differences in the inflammatory contribution to the development of SM between children and adults in the same population and potential differences between individuals originating from malaria-endemic and non-malaria-endemic areas.

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.

Ethanol (EtOH), isopropyl alcohol (IPA), and propylene glycol (PG) increase topical drug delivery, but are sometimes associated with erythema. A potential genetic basis for alcohol-associated erythema was investigated as the function of polymorphisms in coding and non-coding regions of class IB alcohol dehydrogenase (ADHIB) and evaluated for altered gene expression in vitro and metabolic activity in vivo via altered skin blood flow (Doppler velocimeter) and erythema (reflectance colorimeter a*) following topical challenge to 5 M EtOH, IPA, PG, and butanol (ButOH). Promoter polymorphisms G-887A and C-739T and exon G143A form eight ADHIB haplotypes with different frequencies in Caucasians vs Asians and exhibit variable gene expression and metabolic activity. Polymorphisms C-739T and G-887A independently alter gene expression, which is further increased by IPA and PG, but not EtOH or ButOH. EtOH and ButOH increase erythema as a function of skin blood flow. IPA increases skin blood flow without erythema and PG increased erythema with decreased skin blood flow, all as a function of ADHIB haplotype. PG-induced erythema was uniquely associated with tumor necrosis factor-alpha expression. Thus, erythema following alcohol exposure is alcohol type specific, has a pharmacogenetic basis related to ADHIB haplotype and can be functionally evaluated via Doppler velocimetry and reflectance colorimetry in vivo.

Background: Most of the United States population is infected with either herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, or both. Reactivations of HSV-1 infection cause herpes simplex labialis (HSL; cold sores or fever blisters), which is the most common recurring viral infection in humans., Methods: To investigate the possibility of a human genetic component conferring resistance or susceptibility to cold sores (i.e., a HSL susceptibility gene), we conducted a genetic linkage analysis that included serotyping and phenotyping 421 individuals from 39 families enrolled in the Utah Genetic Reference Project., Results: Linkage analysis identified a 2.5-Mb nonrecombinant region of interest on the long arm of human chromosome 21, with a multipoint logarithm of odds score of 3.9 noted near marker abmc65 (D21S409). Nonparametric linkage analysis of the data also provided strong evidence for linkage (P = .0005). This region of human chromosome 21 contains 6 candidate genes for herpes susceptibility., Conclusions: The development of frequent cold sores is associated with a region on the long arm of human chromosome 21. This region contains several candidate genes that could influence the frequency of outbreaks of HSL.

Cerebral malaria is associated with decreased production of nitric oxide and decreased levels of its precursor, l-arginine. Abnormal amino acid metabolism may thus be an important factor in malaria pathogenesis. We sought to determine if other amino acid abnormalities are associated with disease severity in falciparum malaria. Subjects were enrolled in Dar es Salaam, Tanzania (children) (n = 126), and Papua, Indonesia (adults) (n = 156), in two separate studies. Plasma samples were collected from subjects with WHO-defined cerebral malaria (children), all forms of severe malaria (adults), and uncomplicated malaria (children and adults). Healthy children and adults without fever or illness served as controls. Plasma amino acids were measured using reverse-phase high-performance liquid chromatography with fluorescence detection. Several plasma amino acids were significantly lower in the clinical malaria groups than in healthy controls. Despite the differences, phenylalanine was the only amino acid with mean levels outside the normal range (40 to 84 microM) and was markedly elevated in children with cerebral malaria (median [95% confidence interval], 163 [134 to 193] microM; P < 0.0001) and adults with all forms of severe malaria (median [95% confidence interval], 129 [111 to 155] microM; P < 0.0001). In adults who survived severe malaria, phenylalanine levels returned to normal, with clinical improvement (P = 0.0002). Maintenance of plasma phenylalanine homeostasis is disrupted in severe malaria, leading to significant hyperphenylalaninemia. This is likely a result of an acquired abnormality in the function of the liver enzyme phenylalanine hydroxylase. Determination of the mechanism of this abnormality may contribute to the understanding of neurological complications in malaria.

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by parathyroid tumours, which are frequently carcinomas, and ossifying jaw fibromas. In addition, some patients may develop renal tumours and cysts. The gene causing HPT-JT, which is referred to as HRPT2 and is located on chromosome 1q31.2, encodes a 531 amino acid protein called PARAFIBROMIN. To date 42 mutations, of which 22 are germline, have been reported and 97% of these are inactivating and consistent with a tumour suppressor role for HRPT2. We have investigated another four HPT-JT families for germline mutations, searched for additional clinical phenotypes, and examined for a genotype-phenotype correlation. Mutations were found in two families. One family had a novel deletional-insertion at codon 669, and the other had a 2 bp insertion at codon 679, which has been reported in four other unrelated patients. These five unrelated patients and their families with the same mutation were not found to develop the same tumours, thereby indicating an absence of a genotype-phenotype correlation. An analysis of 33 HPT-JT kindreds revealed that affected women in 13 HPT-JT families suffered from menorrhagia in their second to fourth decades. This often required hysterectomy, which revealed the presence of uterine tumours. This resulted in a significantly reduced maternal transmission of the disease. Thus, the results of our analysis expand the spectrum of HPT-JT-associated tumours to include uterine tumours, and these may account for the decreased reproductive fitness in females from HPT-JT families.

Polymorphisms in the inducible nitric oxide synthase gene (NOS2) promoter have been associated with clinical outcome from malaria. These include a CCTTT repeat (CCTTTn) 2.5 kilobases upstream from the NOS2 transcription start site, and two single nucleotide substitutions: G-->C at position -954 (G-954C), and C-->T at position -1173 (C-1173T). Although hypothesized to influence NO production in vivo, the functional relevance of (CCTTT)n and G-954C is uncertain because disease association studies have yielded inconsistent results. This study found no association between CCTTT repeat number and levels of plasma NO metabolites or peripheral blood mononuclear cell NOS activity in a cohort of asymptomatic malaria-exposed coastal Papua New Guineans 1-60 years old. This suggests that (CCTTT)n does not independently influence NOS2 transcription in vivo. Neither the G-954C nor the C-1173T polymorphisms were identified in this population, indicating the variability and complexity of selection for NOS2 promoter polymorphisms in different malaria-endemic populations.

Nitric oxide (NO) production and mononuclear cell NO synthase 2 (NOS2) expression are high in healthy Tanzanian children but low in those with cerebral malaria. Factors that downregulate NOS2 also diminish factors involved in cellular uptake and biosynthesis of L-arginine, the substrate for NO synthesis. We therefore postulated that L-arginine concentrations would be low in individuals with cerebral malaria. We measured concentrations of L-arginine in cryopreserved plasma samples from Tanzanian children with and without malaria. L-arginine concentrations were low in individuals with cerebral malaria (mean 46 micromol/L, SD 14), intermediate in those with uncomplicated malaria (70 micromol/L, 20), and within the normal range in healthy controls (122 micromol/L, 22; p<0.0001). Analysis by logistic regression showed that hypoargininaemia was significantly associated with cerebral malaria case-fatality. Hypoargininaemia may contribute to limited NO production in children with cerebral malaria and to severe disease.

Objective: Systemic lupus erythematosus (SLE) is an autoimmune disease in which morbidity and mortality are higher in African-Americans. The etiology of this racial disparity is unknown. A genetic predisposition to enhanced nitric oxide (NO) production may predispose African-Americans to develop SLE and may increase disease severity. We have demonstrated a correlation between NO production and disease activity in SLE. Two polymorphisms in the inducible NO synthase (NOS2) promoter region (G-954C and CCTTT microsatellite repeat polymorphisms) are associated with improved outcome in some African patients with malaria. This study was designed to determine if these polymorphisms are associated with SLE., Methods: We assessed the frequency of both the G-954C and CCTTT microsatellite repeat NOS2 promoter polymorphisms in a cohort of patients with SLE and age, sex, and race matched controls in North Carolina and South Carolina., Results: Both polymorphisms were more frequent among African-American female SLE patients when compared with controls (p = 0.04 for the G-954C polymorphism and p = 0.03 for the CCTTT-8 repeat polymorphism). Further, the G-954C and CCTTT-8 repeat polymorphisms were in linkage disequilibrium (D cent = 0.89, p = 0.0001) among African-American female SLE patients., Conclusion: Altered genetic control of NOS2 transcription may be a risk factor for SLE among African-American females. The extent of linkage disequilibrium between the G-954C and CCTTT-8 repeat NOS2 promoter polymorphisms suggests that they were co-inherited.

We report here the identification of a gene associated with the hyperparathyroidism-jaw tumor (HPT-JT) syndrome. A single locus associated with HPT-JT (HRPT2) was previously mapped to chromosomal region 1q25-q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT-JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT-JT and in development of some sporadic parathyroid tumors.

Background: Nitric oxide (NO) is a mediator of immunity to malaria, and genetic polymorphisms in the promoter of the inducible NO synthase gene (NOS2) could modulate production of NO. We postulated that NOS2 promoter polymorphisms would affect resistance to severe malaria., Methods: We assessed genomic DNA from healthy children and from those diagnosed with malaria from Tanzania (n=47 and n=138, respectively) and Kenya (n=1106) for polymorphisms by single-stranded conformational polymorphism (SSCP) analysis and sequencing. We also measured in-vivo NO production in Tanzanian children., Findings: We identified a novel single nucleotide polymorphism, -1173 C-->T, in the NOS2 promoter that was significantly associated with protection from symptomatic malaria (odds ratio 0.12, 95% CI 0.03-0.48, p=0.0006) in 179 Tanzanian children, and significantly associated with protection from severe malarial anaemia (adjusted relative risk 0.25, 95% CI 0.09-0.66, p=0.0005) in 1106 Kenyan children studied over 5 years. The risk of parasitaemia was not significantly different in wild-type or -1173 C-->T individuals. -1173 C-->T protection in Tanzanians was independent of the previously recognised NOS2-954 G-->C polymorphism. The (CCTTT)(n) NOS2 polymorphism (Tanzania and Kenya) was not associated with severe malaria outcomes. -1173 C-->T was associated with increased fasting urine and plasma NO metabolite concentrations in Tanzanian children, suggesting that the polymorphism was functional in vivo. Interpretation The NOS2 promoter -1173 C-->T single nucleotide polymorphism is associated with protection against cerebral malaria and severe malarial anaemia. Increased NO production in individuals with the -1173 C-->T polymorphism lends support to a protective role for NO against these syndromes. Targeted interventions to increase NO delivery or production could provide novel preventive and therapeutic strategies against these major causes of mortality in African children.

Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type 1 diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population.

Nitric oxide (NO) plays an important role in host resistance to infection with a variety of organisms. Two recent reports from Gabon and Gambia identified associations of malaria disease severity with the inducible NO synthase (NOS2) promoter G-954C and short allele (<11 repeats) pentanucleotide microsatellite polymorphisms, respectively. It was postulated that there would be a correlation of these polymorphisms with malaria disease severity and with measures of NO production in our cohort of Tanzanian children with malaria. In Tanzanian children, 15% were heterozygous or homozygous for the G-954C polymorphism, and 13% had the short-allele microsatellite polymorphism. There was no significant correlation of either polymorphism with disease severity or with measures of NO production and NOS2 expression. Black and white Americans differed significantly in the frequencies of these polymorphisms. The various association of these gene polymorphisms with malaria severity in different populations underscores the complexity of host resistance to malaria.

Hyperparathyroidism-jaw tumor syndrome (HPT-JT) is an autosomal dominant disease characterized by the development of multiple parathyroid adenomas and multiple fibro-osseous tumors of the maxilla and mandible. Some families have had affected members with involvement of the kidneys, variously reported as Wilms tumors, nephroblastomas, and hamartomas. The HPT-JT gene (HRPT2) maps to chromosome 1q25-q31. We describe further investigation of two HPT-JT families (K3304 and K3349) identified through the literature. These two expanded families and two previously reported families were investigated jointly for linkage with 21 new, closely linked markers. Multipoint linkage analysis resulted in a maximum LOD score of 7.83 (at recombination fraction 0) for markers D1S2848-D1S191. Recombination events in these families reduced the HRPT2 region to approximately 14.7 cM. In addition, two of these four study families (i.e., K3304 and K11687) share a 2.2-cM length of their (expanded) affected haplotype, indicating a possible common origin. Combining the linkage data and shared-haplotype data, we propose a 0.7-cM candidate region for HRPT2.

The q21 region of chromosome 17 contains the gene BRCA1, which is involved in familial early-onset breast and ovarian cancers. A physical map of a region that extends from a distal boundary of the BRCA1 region, D17S78, to GP2B has been constructed. The map consists of 30 STSs, including 2 new short tandem repeat polymorphic markers. The contig is composed of a mixture of 7 YACs, 5 P1 plasmids, and 14 cosmids and was ordered by STS-content mapping.

BRCA1, a breast and ovarian cancer susceptibility locus, has been isolated and maps to 17q21. A physical map of the BRCA1 region which extended from the proximal boundary at D17S776 to the distal boundary at D17S78 was constructed and consists of 51 sequence tagged sites (STSs) from P1 and YAC ends, nine new short-tandem repeat (STR) polymorphic markers, and eight identified genes. The contig, which spans the estimated 2.3 Mb region, contains 29 P1s, 11 YACs, two BACs, and one cosmid. Based on key recombinants in two linked families, BRCA1 was further localized to a region bounded by D17S1321 on the proximal side and D17S1325 on the distal side. Within this estimated 600 kb region, the contig was composed completely of P1s and BACs ordered by STS-content mapping and confirmed by DNA restriction fragment fingerprinting.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.

Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine.

Utilizing first-strand cDNA from different stages, a gene family was identified that is expressed in bloodstream form trypanosomes but not in cultured procyclic forms. This family of 50-100 genes, termed bloodstream-specific 1 (BS1), shares a chromosomal distribution pattern similar to the variant surface glycoprotein (VSG) genes and the expression-site-associated genes (ESAGs). The BS1 genes are expressed in several variants of Trypanosoma brucei brucei and in Trypanosoma brucei gambiense. Sequence analysis of five members of this gene family reveals the recently described ESAG 6 and ESAG 7 genes as well as the ESAG X gene to be members of this family. We have been unable to localize the BS1 gene product in the cell but show that chronically infected rabbit serum recognizes recombinant BS1 protein arguing for expression in vivo. Finally we note that the derived protein sequence for the BS1 genes suggests an evolutionary relationship with at least one variant surface glycoprotein gene, and hence these studies may provide clues to understanding the molecular origins of antigenic variation in trypanosomes.