PAI-1 gain-of-function genotype, factors increasing PAI-1 levels, and airway obstruction: The GALA II Cohort.

BACKGROUND: PAI-1 gain-of-function variants promote airway fibrosis and are associated with asthma and with worse lung function in subjects with asthma. OBJECTIVE: We sought to determine whether the association of a gain-of-function polymorphism in plasminogen activator inhibitor-1 (PAI-1) with airway obstruction is modified by asthma status, and whether any genotype effect persists after accounting for common exposures that increase PAI-1 level. METHODS: We studied 2070 Latino children (8-21y) with genotypic and pulmonary function data from the GALA II cohort. We estimated the relationship of the PAI-1 risk allele with FEV1/FVC by multivariate linear regression, stratified by asthma status. We examined the association of the polymorphism with asthma and airway obstruction within asthmatics via multivariate logistic regression. We replicated associations in the SAPPHIRE cohort of African Americans (n=1056). Secondary analysis included the effect of the at-risk polymorphism on postbronchodilator lung function. RESULTS: There was an interaction between asthma status and the PAI-1 polymorphism on FEV1 /FVC (P=.03). The gain-of-function variants, genotypes (AA/AG), were associated with lower FEV1 /FVC in subjects with asthma (ß=-1.25, CI: -2.14,-0.35, P=.006), but not in controls. Subjects with asthma and the AA/AG genotypes had a 5% decrease in FEV1 /FVC (P<.001). In asthmatics, the risk genotype (AA/AG) was associated with a 39% increase in risk of clinically relevant airway obstruction (OR=1.39, CI: 1.01, 1.92, P=.04). These associations persisted after exclusion of factors that increase PAI-1 including tobacco exposure and obesity. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease in the FEV1 /FVC ratio associated with the risk genotype was modified by asthma status. The genotype increased the odds of airway obstruction by 75% within asthmatics only. As exposures known to increase PAI-1 levels did not mitigate this association, PAI-1 may contribute to airway obstruction in the context of chronic asthmatic airway inflammation.

Novel genetic risk factors for asthma in African American children: Precision Medicine and the SAGE II Study.

Asthma, an inflammatory disorder of the airways, is the most common chronic disease of children worldwide. There are significant racial/ethnic disparities in asthma prevalence, morbidity, and mortality among US children. This trend is mirrored in obesity, which may share genetic and environmental risk factors with asthma. The majority of asthma biomedical research has been performed in populations of European decent. We sought to identify genetic risk factors for asthma in African American children. We also assessed the generalizability of genetic variants associated with asthma in European and Asian populations to African American children. Our study population consisted of 1227 (812 asthma cases, 415 controls) African American children with genome-wide single nucleotide polymorphism (SNP) data. Logistic regression was used to identify associations between SNP genotype and asthma status. We identified a novel variant in the PTCHD3 gene that is significantly associated with asthma (rs660498, p = 2.2 × 10(-7)) independent of obesity status. Approximately 5 % of previously reported asthma genetic associations identified in European populations replicated in African Americans. Our identification of novel variants associated with asthma in African American children, coupled with our inability to replicate the majority of findings reported in European Americans, underscores the necessity for including diverse populations in biomedical studies of asthma.

Maternal age and asthma in Latino populations.

BACKGROUND: Younger maternal age at birth is associated with increased risk of asthma in offspring in European descent populations, but has not been studied in Latino populations. OBJECTIVES: We sought to examine the relationship between maternal age at birth and prevalence of asthma in a nationwide study of Latino children. METHODS: We included 3473 Latino children aged 8-21 years (1696 subjects with physician-diagnosed asthma and 1777 healthy controls) from five US centres and Puerto Rico recruited from July 2008 through November 2011. We used multiple logistic regression models to examine the effect of maternal age at birth on asthma in offspring overall and in analyses stratified by ethnic subgroup (Mexican American, Puerto Rican and other Latino). Secondary analyses evaluated the effects of siblings, acculturation and income on this relationship. RESULTS: Maternal age < 20 years was significantly associated with decreased odds of asthma in offspring, independent of other risk factors (OR = 0.73, 95% CI: 0.57-0.93). In subgroup analyses, the protective effect of younger maternal age was observed only in Mexican Americans (OR = 0.53, 95% CI: 0.36, 0.79). In Puerto Ricans, older maternal age was associated with decreased odds of asthma (OR = 0.65, 95% CI: 0.44-0.97). In further stratified models, the protective effect of younger maternal age in Mexican Americans was seen only in children without older siblings (OR = 0.44, 95% CI: 0.23-0.81). CONCLUSION AND CLINICAL RELEVANCE: In contrast to European descent populations, younger maternal age was associated with decreased odds of asthma in offspring in Mexican American women. Asthma is common in urban minority populations but the factors underlying the varying prevalence among different Latino ethnicities in the United States is not well understood. Maternal age represents one factor that may help to explain this variability.

Outbreak of enterotoxigenic Escherichia coli O169 enteritis in schoolchildren associated with consumption of kimchi, Republic of Korea, 2012.

Enterotoxigenic Escherichia coli (ETEC) is now recognized as a common cause of foodborne outbreaks. This study aimed to describe the first ETEC O169 outbreak identified in Korea. In this outbreak, we identified 1642 cases from seven schools. Retrospective cohort studies were performed in two schools; and case-control studies were conducted in five schools. In two schools, radish kimchi was associated with illness; and in five other schools, radish or cabbage kimchi was found to have a higher risk among food items. Adjusted relative risk of kimchi was 5·87-7·21 in schools that underwent cohort studies; and adjusted odds ratio was 4·52-12·37 in schools that underwent case-control studies. ETEC O169 was isolated from 230 affected students, and was indistinguishable from the isolates detected from the kimchi product distributed by company X, a food company that produced and distributed kimchi to all seven schools. In this outbreak, we found that the risk of a kimchi-borne outbreak of ETEC O169 infection is present in Korea. We recommend continued monitoring regarding food safety in Korea, and strengthening surveillance regarding ETEC O169 infection through implementation of active laboratory surveillance to confirm its infection.

Neuroprotection using gene therapy to induce vascular endothelial growth factor-A expression.

Engineered zinc-finger protein (ZFP) transcription factors induce the expression of endogenous genes and can be remotely delivered using adenoviral vectors. One such factor, Ad-32Ep65-Flag (Ad-p65), targets and induces expression of vascular endothelial growth factor (VEGF; also called VEGF-A) splice variants in their normal biological stoichiometry. We show that Ad-p65 transfection of primary motor neurons results in VEGF variant expression and a significant increase in axon outgrowth in these cells. Given the neuroprotective effects of VEGF and its ability to increase neurite outgrowth, we examined the efficacy of Ad-p65 to enhance motor neuron regeneration in vivo using rats that have undergone recurrent laryngeal nerve (RLN)-crush injury. Injection of Ad-p65 after RLN crush accelerated the return of vocal fold mobility and the percentage of nerve-endplate contacts in the thyroarytenoid muscle. Overall, adenoviral delivery of an engineered ZFP transcription factor inducing VEGF-A splice variant expression enhances nerve regeneration. ZFP transcription factor gene therapy to increase expression of the full complement of VEGF-A splice variants is a promising avenue for the treatment of nerve injury and neurodegeneration.

Molecular characterization of hepatitis A virus isolated from acute gastroenteritis patients in the Seoul region of Korea.

Hepatitis A virus (HAV) is a major public health problem throughout the world. As a result of declining HAV endemic in Korea, an increasing number of children and adolescents have become susceptible to HAV infection. HAV is related with sanitation conditions of the environment and is transmitted via the fecal-oral route, either through person-to-person contact or by contaminated water and food. The present study has been carried out to determine the phylogenetic analysis and circulating patterns of HAV strains detected from hospitalized patients with acute gastroenteritis (AGE) in the Seoul region of Korea. In total, 2,782 stool specimens from hospitalized patients with AGE collected in October 2006 to September 2007 in Seoul were tested for HAV. A pair comparison of the nucleic acid sequence of a 159-bp base region at the putative VP1/2A junction of 85 Seoul isolates revealed that the most common HAV strain circulating in the region during 2006-2007 was subgenotype IA. HAV phylogenetic studies can provide important information on the genetic characteristics of HAV from AGE patients who may subsequently become the source of infection in Korea.

Caveolin-1 serves as a negative effector in senescent human gingival fibroblasts during Fusobacterium nucleatum infection.

It is well established that aging is associated with increased susceptibility to infectious diseases. Fusobacterium nucleatum is a well-known bacterial species that plays a central bridging role between early and late colonizers in the human oral cavity. Further, the ability of F. nucleatum to invade gingival fibroblasts (GFs) is critical to the development of periodontal diseases. However, the mechanisms underlying the age-related infection of GFs by F. nucleatum remain unknown. We used young (fourth passage) and senescent (22nd passage) GFs to investigate the mechanisms of F. nucleatum infection in aged GFs and first observed increased invasion of F. nucleatum in senescent GFs. We also found that the co-localization of caveolin-1 (Cav-1), a protein marker of aging, with F. nucleatum and the knockdown of Cav-1 in GFs reduced F. nucleatum invasion. Additionally, F. nucleatum infection triggered the production of reactive oxygen species (ROS) through activation of NADPH oxidase in GFs, but senescent GFs exhibited significantly lower levels of NADPH oxidase activity and ROS production compared with young GFs in both the uninfected and infected conditions. Also, senescent GFs exhibited a decline in proinflammatory cytokine production and extracellular signal regulated kinase (ERK) phosphorylation following F. nucleatum infection. Interestingly, the knockdown of Cav-1 in senescent GFs increased NADPH oxidase activity and caused the upregulation of interleukin-6 and interleukin-8 and the phosphorylation of ERK. Collectively, the increased expression of Cav-1 might play a critical role in F. nucleatum invasion and could hinder the host response in senescent GFs.

Host receptors in malaria merozoite invasion.

The clinical manifestations of Plasmodium falciparum malaria are directly linked to the blood stage of the parasite life cycle. At the blood stage, the circulating merozoites invade erythrocytes via a specific invasion pathway often identified with its dependence or independence on sialic acid residues of the host receptor. The invasion process involves multiple receptor-ligand interactions that mediate a complex series of events in a period of approximately 1 min. Although the mechanism by which merozoites invade erythrocytes is not fully understood, recent advances have put a new perspective on the importance of developing a multivalent blood stage-malaria vaccine. In this review, we highlight the role of currently identified host invasion receptors in blood-stage malaria.

Plasmodium falciparum erythrocyte membrane protein 1 is anchored to the actin-spectrin junction and knob-associated histidine-rich protein in the erythrocyte skeleton.

A distinctive pathological feature of Plasmodium falciparum malaria is the endothelial attachment of erythrocytes infected with mature asexual-stage parasites in microvessels of the major organs. Electron-dense protrusions described as knobs are displayed on the surface of parasitized erythrocytes and act as attachment points in cytoadherence. Parasite-encoded knob-associated histidine-rich protein (KAHRP) is a major component of knobs found on the cytoplasmic side of the host cell membrane. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of parasite-encoded cytoadherence receptors localized to knobs on the surface of parasitized erythrocytes. Despite its high antigenic diversity, PfEMP1 has a remarkably conserved cytoplasmic domain. We demonstrate in this study that the cytoplasmic domain of PfEMP1 (VAR(CD)) binds to host spectrin and actin and to full-length KAHRP in vitro. Apparent dissociation constants determined for VAR(CD)/F-actin and VAR(CD)/KAHRP interactions are 44.9+/-6.4 and 10. 7+/-2.2 nM, respectively. Further, we provide evidence that KAHRP polypeptides self-associate in solution to form structures similar to knobs and show binding of self-associated KAHRP clusters to spectrin-actin-protein 4.1 complexes. Findings in this study suggest that PfEMP1 is localized to the knob in P. falciparum-infected erythrocytes by binding to the host spectrin-actin junction and to self-associated KAHRP through its conserved cytoplasmic domain.

Mechanism of human placental aromatase: a new active site model.

Based on Akhtar's ferric peroxide mechanism and on recent studies in our own laboratory, we present a detailed proposal for aromatase action. This picture can account for the known stereochemical consequences at C-19 observed by others using isotopes of hydrogen and oxygen. The postulated process involves anchoring of the 19-hydroxymethyl and 19-oxo groups at the active site by a glutamate residue, which also serves to activate the 19-oxo group for attack by ferric peroxy species in the third oxidative step.

Neurodegeneration and early lethality in superoxide dismutase 2-deficient mice: a comprehensive analysis of the central and peripheral nervous systems.

The contribution of oxidative stress to diabetic complications including neuropathy is widely known. Mitochondrial and cellular damage are associated with the overproduction of reactive oxygen species and decreased levels or function of the cellular antioxidant mitochondrial manganese superoxide dismutase (SOD2). We hypothesized that targeted SOD2 deletion in the peripheral nervous system using cre-lox technology under control of the nestin promoter would accelerate neuropathy in a type 2 model of diabetes, the BKS.db/db mouse. SOD2-deficient mice, however, demonstrated severe gait deformities and seizures and died by 20 days of age. Examination of SOD2 expression levels revealed that SOD2 was lost in brain and reduced in the spinal cord, but appeared normal in dorsal root ganglia and peripheral nerves in SOD2-deficient mice. These findings indicate incomplete targeted knockout of SOD2. Morphological examination revealed cortical lesions similar to spongiform encephalopathy in the brain of SOD2-deficient mice. No lesions were evident in the spinal cord, but changes in myelin within the sciatic and sural nerves including a lack of cohesion between layers of compact myelin were observed. Together, these results indicate that targeted neuronal SOD2 knockout using the nestin promoter results in severe central nervous system degeneration and perinatal lethality in mice. A specific peripheral nervous system-targeting construct is required to examine the consequences of SOD2 knockout in diabetic neuropathy.

The development of a novel cooking method (alternate roasting with its own fat) for chicken to improve nutritional value.

The goal of this study was to develop a novel cooking method for fried meat products, to improve their nutritional value, and to provide superior taste and texture. We used the fat derived from each individual meat source during radiant heat roasting (alternate roasting with its own fat: AROF) without deep-fat frying (DFF), first without any air flow and subsequently with an exposure to air flow. We then compared these roasted chicken samples to breaded fried chicken samples that were deep-fat fried in 3 types of fat: soybean oil (SB), partially hydrogenated soybean oil (PSB), and lard. The final fat contents of both the skin and lean parts of the AROF samples of chicken were less than half of those of the DFF groups. The total trans-fatty acids (TFA) contents were significantly lower in the AROF samples compared to the DFF samples. The cholesterol levels of the samples did not show any significant differences among the tested groups, except for the sample fried in lard, which was significantly higher. Moreover, the sensory evaluation results showed that the crispy texture of the AROF samples was not significantly different from that of the DFF samples (P < 0.05); the AROF samples had higher scores for the characteristic fried flavor and for overall acceptability (P < 0.05). This study shows the potential value of products prepared by AROF, which can successfully replace DFF methods used for chicken and other meat products and improve their nutritional value.

Oxygen consumption of muscles from ob/ob and Ay/a strains of obese mice.

Oxygen consumption was measured in the in vitro preparations of soleus, extensor digitorum longus, gracilis, and diaphragm muscles from young ob/ob obese mice. In young Ay/a obese mice, similar measurements were made with in vitro preparations of soleus, gracilis, and diaphragm muscles. These young obese mice were heavier than their nonobese littermates. Muscle weights were similar in obese and nonobese mice. In the Ay/a obese mice, values of muscle oxygen consumption were similar to those found in the a/a nonobese littermates. This correlates well with other known information about the Ay/a variety of obesity. In the ob/ob obese mice, values of oxygen consumption in the gracilis and diaphragm muscle preparations were lower than those in nonobese littermates. These data indicate that some muscles in the ob/ob obese mice contribute to the depressed whole animal oxygen consumption by virtue of defective oxidative metabolism. The data also indicate that the tissue defects are very different in various types of obese animal models. This probably is also true for different types of human obesity.

Effect of triiodothyronine on glucose utilization in diaphragm of obese (ob/ob) mice.

Genetically obese-hyperglycemic (ob/ob) mice are hypothyroid, hyperinsulinemic, and insulin resistant. Because muscle plays an important role in glucose homeostasis, the role of triiodothyronine (T3) in regulation of insulin-sensitive glucose utilization by muscles of obese mice was examined. Four doses of T3, 5.0, 12.5, 25.0, and 50.0 micrograms/100 g body weight were injected, i.p., into obese and nonobese mice daily from 3 weeks until 6 weeks of age. Food consumption and body weight were decreased at lower doses of T3 and increased at higher doses of T3 in both obese and nonobese mice. By 6 weeks of age all doses of T3 treatment increased oxygen consumption in both genotypes. At 6 weeks of age, the diaphragms from the saline-injected nonobese mice had greater in-vitro insulin-stimulated glucose utilization than muscles from the saline-injected obese mice. Both anaerobic and aerobic glucose oxidation were increased by T3 treatment, but the obese had greater increases than those observed in muscles from nonobese mice. Muscles from obese mice increased insulin-stimulated glucose utilization by T3 treatment to that of the nonobese level, whereas there was no change in insulin-stimulated glucose utilization of nonobese mice. Muscle glycogen synthesis in obese and nonobese mice was decreased with very high T3 doses. A higher dose of T3 was required to increase glucose utilization in the obese muscles than in the nonobese muscles. The results suggest that a functional hypothyroidism or T3 resistance may be an early part of this particular obesity syndrome.

Early treatment of obese (ob/ob) mice with triiodothyronine increases oxygen consumption and temperature and decreases body fat content.

An early abnormality in the ob/ob mouse is a low circulating level of the thyroid hormone, triiodothyronine (T3). The possibility was explored that early T3 treatment of ob/ob mice will increase oxidative metabolism and lower body fat content. Doses of T3, ranging from 0.0 to 25.0 micrograms/100 g body wt were injected, ip, into ob/ob and non-ob/ob mice daily from 3 weeks until 6 weeks of age. Food intake was equalized across all groups to that consumed by non-ob/ob saline-treated group. At 6 weeks of age, body weight, serum concentrations of thyroxine (T4), T3, insulin and glucose, oxygen consumption, colonic temperature, and body composition were analyzed. T3 treatment decreased body weight, increased body oxygen consumption, increased colonic temperature, and decreased body fat without a significant change in body protein in ob/ob mice. T3 treatment also increased serum T3, and decreased serum T4, insulin, and glucose concentrations in ob/ob mice. Because total body protein did not change as a result of T3 treatment, the increased oxidative metabolism due to T3 treatment was probably via the change of metabolic activity of the total lean body mass or the specific metabolically active tissues in the ob/ob mice, such as brown adipose tissue, liver, or muscle.

Early treatment of obese (ob/ob) mice with triiodothyronine increases oxidative metabolism in muscle but not in brown adipose tissue or liver.

We explored the possibility that early replacement of low triiodothyronine (T3) may improve the low oxidative metabolism in metabolically important tissues of ob/ob mice. Triiodothyronine doses (2.5 to 25.0 micrograms/100 g body wt) were injected intraperitoneally into ob/ob and non-ob/ob mice daily from 3 wk until 6 wk of age. Untreated ob/ob and non-ob/ob mice were injected with saline (pH 9.1). Food intake was equalized across all groups. At 6 wk of age, the O2 consumption of muscle, white and brown adipose tissues, and hepatocytes was measured. The saline-treated ob/ob mice showed lower muscle weights, higher fat pad and liver weights, and larger fat cell sizes than saline-treated non-ob/ob mice. In ob/ob mice, tissue O2 consumption was the same in muscle, lower in brown and white adipose tissues, but higher in liver compared with values in non-ob/ob mice. Triiodothyronine treatment in ob/ob mice resulted in lower values for body weight, liver weight, hepatocyte number, liver protein, epididymal fat pad weight, and white adipocyte number and size than in saline-treated ob/ob mice. Triiodothyronine treatment increased soleus muscle, liver and brown adipose tissue O2 consumption in non-ob/ob mice. In ob/ob mice, triiodothyronine increased only soleus muscle O2 consumption and required higher doses than in non-ob/ob mice to achieve an effect. These data are consistent with the concept of tissue triiodothyronine resistance in ob/ob mice. Low triiodothyronine levels and tissue resistance to triiodothyronine might be important early defects in this obesity syndrome.

Erythrocyte membrane alterations in Plasmodium falciparum malaria sequestration.

Plasmodium falciparum malaria, the most lethal form of human malaria, claims at least 2 million lives worldwide each year. Recently, there has been a significant advance in our understanding of the molecular basis of P. falciparum sequestration, a distinctive pathologic feature that often leads to fatal human cerebral malaria. Parasite-derived VAR proteins (Plasmodium falciparum-infected erythrocyte membrane protein 1) have been cloned and identified as antigenically diverse cytoadherent receptors localized to the knob protrusions that act as attachment points in parasite sequestration. Evidence now supports the hypothesis that cryptic regions of band 3 protein are parasite-induced, host-derived erythrocyte receptors mediating parasite sequestration. Knob structures have been localized to spectrin-actin-protein 4.1 junctions in intact spread membrane skeletons. A recombinant domain of knob-associated histidine-rich protein, a major protein found in both membrane-intact and isolated knobs, has been shown to associate with filamentous actin and spectrin. Parasite- and host-derived erythrocyte membrane proteins involved in P. falciparum sequestration are discussed in this review.

Human homologue of the Drosophila discs large tumor suppressor binds to p56lck tyrosine kinase and Shaker type Kv1.3 potassium channel in T lymphocytes.

Human homologue of the Drosophila discs large tumor suppressor protein (hDlg) belongs to a newly discovered family of proteins termed MAGUKs that appear to have structural as well as signaling functions. Consistent with the multi-domain organization of MAGUKs, hDlg consists of three copies of the PDZ (PSD-95/Discs large/zO-1) domain, an SH3 motif, and a guanylate kinase-like domain. In addition, the hDlg contains an amino-terminal proline-rich domain that is absent in other MAGUKs. To explore the role of hDlg in cell signaling pathways, we used human T lymphocytes as a model system to investigate interaction of hDlg with known tyrosine kinases. In human T lymphocyte cell lines, binding properties of hDlg were studied by immunoprecipitation, immunoblotting, and immune complex kinase assays. Our results show that protein tyrosine kinase activity is associated with the immunoprecipitates of hDlg. Immunoblotting experiments revealed that the immunoprecipitates of hDlg contain p56lck, a member of the Src family of tyrosine kinases. The specificity of the interaction is demonstrated by the lack of p59fyn tyrosine kinase and phosphotidylinositol 3-kinase in the hDlg immunoprecipitates. Direct interaction between hDlg and p56lck is demonstrated using glutathione S-transferase fusion proteins of hDlg and recombinant p56lck expressed in the baculovirus-infected Sf9 cells. The p56lck binding site was localized within the amino-terminal segment of hDlg containing proline-rich domain. In addition, we show in vivo association of hDlg with Kv1.3 channel, which was expressed in T lymphocytes as an epitope-tagged protein using a vaccinia virus expression system. Taken together, these results provide the first evidence of a direct interaction between hDlg and p56lck tyrosine kinase and suggest a novel function of hDlg in coupling tyrosine kinase and voltage-gated potassium channel in T lymphocytes.

Effects of somatostatin and somatotropin on the in vitro testicular steroidogenesis in hamster.

Adult hamsters were exposed to short-photoperiod, and injected with either somatotropin (GH), somatostatin (GHRIH), or saline for eight weeks. Hamster testis fragments of similar size were incubated with or without hCG. No significant differences in the basal media testosterone and estradiol levels were observed among groups. Treatment with GH potentiated the hCG-dependent increase in media testosterone. Contrary to what was expected, treatment with GHRIH did not only not reduce the hCG-related elevation in media testosterone, but even produced a numerical increase of it. Treatment with GHRIH potentiated the hCG-dependent increase in media estradiol, whereas treatment with GH produced only a numerical increase of the response. Furthermore, the combined exposure to GHRIH and hCG appeared to cause an increase in the efficiency of testicular aromatase. Since previous data indicated that the combined deficiency of lactotropic and somatotropic actions severely impairs testicular steroidogenesis, treatment with GHRIH should have caused further steroidogenic impairment in hamsters exposed to short-photoperiod. Since this does not appear to be the case, it could be postulated that GHRIH has a direct stimulatory or at least a protective effect on testicular steroidogenesis.