Genome-Wide Linkage Study Meta-Analysis of Male Sexual Orientation.

Male sexual orientation is a scientifically and socially important trait shown by family and twin studies to be influenced by environmental and complex genetic factors. Individual genome-wide linkage studies (GWLS) have been conducted, but not jointly analyzed. Two main datasets account for > 90% of the published GWLS concordant sibling pairs on the trait and are jointly analyzed here: MGSOSO (Molecular Genetic Study of Sexual Orientation; 409 concordant sibling pairs in 384 families, Sanders et al. (2015)) and Hamer (155 concordant sibling pairs in 145 families, Mustanski et al. (2005)). We conducted multipoint linkage analyses with Merlin on the datasets separately since they were genotyped differently, integrated genetic marker positions, and combined the resultant LOD (logarithm of the odds) scores at each 1 cM grid position. We continue to find the strongest linkage support at pericentromeric chromosome 8 and chromosome Xq28. We also incorporated the remaining published GWLS dataset (on 55 families) by using meta-analytic approaches on published summary statistics. The meta-analysis has maximized the positional information from GWLS of currently available family resources and can help prioritize findings from genome-wide association studies (GWAS) and other approaches. Although increasing evidence highlights genetic contributions to male sexual orientation, our current understanding of contributory loci is still limited, consistent with the complexity of the trait. Further increasing genetic knowledge about male sexual orientation, especially via large GWAS, should help advance our understanding of the biology of this important trait.

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins.

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection. IMPORTANCE: There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160.

The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed. IMPORTANCE: It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies.

In vivo evaluation of safety and toxicity of a Lactobacillus jensenii producing modified cyanovirin-N in a rhesus macaque vaginal challenge model.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across the cervicovaginal mucosa in women is influenced by many factors including the microbiota and the presence of underlying inflammation. It is important that potential HIV preventative agents do not alter the mucosal environment in a way that enhances HIV acquisition. We examined the impact of a "live" microbicide on the vaginal mucosal environment in a rhesus macaque repeated vaginal simian-HIV (SHIVSF162P3) challenge model. The microbicide contained a human vaginal Lactobacillus jensenii expressing the HIV-1 entry inhibitor, modified Cyanovirin-N (mCV-N), and henceforth called LB-mCV-N. Macaques were colonized vaginally each week with LB-mCV-N and sampled six days after colonization for culturable bacteria, pH and cervical-vaginal cytokines during the duration of the six-week study. We show that macaques that retained the engineered LB-mCV-N strain in their vaginal microbiota, during SHIV challenge, had lower pH, when colonization levels were higher, and had no evidence of inflammatory cytokines. Indeed, Interleukin-13, a mediator of inflammation, was detected less often in LB-mCV-N colonized macaques than in controls and we found higher levels of Interleukin 1 receptor antagonist (IL-1RA) in LB-mCV-N colonized macaques during the SHIV challenge period. We noted an inverse correlation between levels of mucosal IL-1RA and peak plasma viral load, thus higher IL-1RA correlated with lower viral load in LB-mCV-N treated macaques. These data support the use of LB-mCV-N as a safe "live" microbicide and suggest that lactobacilli themselves may positively impact the mucosal environment.

Demonstration of vaginal colonization with GusA-expressing Lactobacillus jensenii following oral delivery in rhesus macaques.

The vaginal microbiome, which harbors beneficial Lactobacillus strains, is believed to be a major host defense mechanism for preventing infections of the urogenital tract. It has been suggested that the gastrointestinal tract serves as a reservoir for lactobacilli that colonize the vagina. Using rhesus macaques, we examined whether oral delivery of human vaginal Lactobacillus jensenii 1153-1646, a GusA-producing strain, would result in colonization of the rectum and the vagina. Lactobacilli were identified from the vagina tracts of three macaques on the basis of ß-glucuronidase enzyme production, 16S rRNA gene sequence and DNA homology using a repetitive sequence-based polymerase chain reaction.

Linking gene, brain, and behavior: DRD4, frontal asymmetry, and temperament.

Gene-environment interactions involving exogenous environmental factors are known to shape behavior and personality development. Although gene-environment interactions involving endogenous environmental factors are hypothesized to play an equally important role, this conceptual approach has not been empirically applied in the study of early-developing temperament in humans. Here we report evidence for a gene-endoenvironment (i.e., resting frontal brain electroencephalogram, EEG, asymmetry) interaction in predicting child temperament. The dopamine D4 receptor (DRD4) gene (long allele vs. short allele) moderated the relation between resting frontal EEG asymmetry (left vs. right) at 9 months and temperament at 48 months. Children who exhibited left frontal EEG asymmetry at 9 months and who possessed the DRD4 long allele were significantly more soothable at 48 months than other children. Among children with right frontal EEG asymmetry at 9 months, those with the DRD4 long allele had significantly more difficulties focusing and sustaining attention at 48 months than those with the DRD4 short allele. Resting frontal EEG asymmetry did not influence temperament in the absence of the DRD4 long allele. We discuss how the interaction of genetic and endoenvironmental factors may confer risk and protection for different behavioral styles in children.

A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development.

BACKGROUND: We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates. METHODS: Vaginal and rectal microflora in Chinese rhesus macaques (Macaca mulatta) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization. RESULTS: Chinese rhesus macaques harbored abundant vaginal Lactobacillus, with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals. CONCLUSIONS: The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus-based topical microbicides.

Evidence for a gene-gene interaction in predicting children's behavior problems: association of serotonin transporter short and dopamine receptor D4 long genotypes with internalizing and externalizing behaviors in typically developing 7-year-olds.

Recent work on the molecular genetics of complex traits in typical and atypical human development has focused primarily on associations of single genes with behavior. Disparate literature suggests that the presence of one or two copies of the short allele of the serotonin transporter (5-HTT) gene and the long allele (7-repeat allele) version of the dopamine receptor D4 (DRD4) gene predicts internalizing- and externalizing-related behaviors, respectively. Apparently for the first time in the extant literature, we report a gene-gene statistical interaction on behavior problems in a group of typically developing children at age 7. DNA was extracted from buccal cells collected from 108 children and genotyped for short and long alleles of the 5-HTT gene and the short (2-5 repeats) versus long (6-8 repeats) allele of the DRD4 gene. Mothers completed the Child Behavior Checklist. As predicted, children with one or two copies of the short allele of the 5-HTT gene and the long allele version of the DRD4 gene exhibited significantly more internalizing and externalizing behaviors at age 7 than children with other combinations of the 5-HTT and DRD4 short and long genotypes. As well, children with the 5-HTT long and DRD4 long genotypes had the lowest reported scores on internalizing and externalizing behaviors at age 7, suggesting that the presence of the 5-HTT long genotype may serve as a protective factor against these behaviors in children with the long DRD4 genotype. Implications of these findings for understanding cumulative biological risk and protective factors in childhood behavior problems and psychopathology are discussed.

Infection dynamics in HIV-specific CD4 T cells: does a CD4 T cell boost benefit the host or the virus?

Recent experimental data have shown that HIV-specific CD4 T cells provide a very important target for HIV replication. We use mathematical models to explore the effect of specific CD4 T cell infection on the dynamics of virus spread and immune responses. Infected CD4 T cells can provide antigen for their own stimulation. We show that such autocatalytic cell division can significantly enhance virus spread, and can also provide an additional reservoir for virus persistence during anti-viral drug therapy. In addition, the initial number of HIV-specific CD4 T cells is an important determinant of acute infection dynamics. A high initial number of HIV-specific CD4 T cells can lead to a sudden and fast drop of the population of HIV-specific CD4 T cells which results quickly in their extinction. On the other hand, a low initial number of HIV-specific CD4 T cells can lead to a prolonged persistence of HIV-specific CD4 T cell help at higher levels. The model suggests that boosting the population of HIV-specific CD4 T cells can increase the amount of virus-induced immune impairment, lead to less efficient anti-viral effector responses, and thus speed up disease progression, especially if effector responses such as CTL have not been sufficiently boosted at the same time.

Engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N.

Women are at significant risk of human immunodeficiency virus (HIV) infection, with the cervicovaginal mucosa serving as a major portal for virus entry. Female-initiated preventatives, including topical microbicides, are urgently needed to help curtail the HIV/AIDS pandemic. Here we report on the development of a novel, live microbicide that employs a natural vaginal strain of Lactobacillus jensenii engineered to deliver the potent HIV inhibitor cyanovirin-N (CV-N). To facilitate efficient expression of CV-N by this bacterium, the L. jensenii 1153 genome was sequenced, allowing identification of native regulatory elements and sites for the chromosomal integration of heterologous genes. A CV-N expression cassette was optimized and shown to produce high levels of structurally intact CV-N when expressed in L. jensenii. Lactobacillus-derived CV-N was capable of inhibiting CCR5-tropic HIV(BaL) infectivity in vitro with a 50% inhibitory concentration of 0.3 nM. The CV-N expression cassette was stably integrated as a single copy into the bacterial chromosome and resolved from extraneous plasmid DNA without adversely affecting the bacterial phenotype. This bacterial strain was capable of colonizing the vagina and producing full-length CV-N when administered intravaginally to mice during estrus phase. The CV-N-producing Lactobacillus was genetically stable when propagated in vitro and in vivo. This work represents a major step towards the development of an inexpensive yet durable protein-based microbicide to block the heterosexual transmission of HIV in women.

The association of DNA sequence variation at the MAOA genetic locus with quantitative behavioural traits in normal males.

Monoamine oxidase A (MAOA) catalyses the oxidative deamination of biogenic amines including neurotransmitters, mainly norepinephrine and serotonin in the brain and peripheral tissues. A nonsense mutation in the gene was shown to be involved in a rare X-linked behavioural syndrome, which includes impaired impulse control, aggression and borderline mental retardation (Brunner syndrome). Several recent studies have shown the association of genetic variation of a VNTR in the gene promoter with various pathological behavioural traits. In the present study the association of MAOA genetic variation with a large set of quantitative behavioural traits in normal individuals has been examined. DNA samples from 421 unrelated males were genotyped for 14 SNPs and for the promoter VNTR at the MAOA locus. An additional 16 SNPs were genotyped at apparently neutral loci across the X chromosome to serve as a genomic control for possible false positive associations due to population structure. Behavioural traits were measured using the NEO psychometric questionnaire, which is based on a 5-axis model of personality, and consists of 30 different quantitative traits. There was a robust association of the A2 ("straightforwardness") facet with common allelic variants at the promoter VNTR. Most of the tested traits were not associated with the VNTR despite reasonable power, thus demonstrating that the VNTR influence on quantitative behavioural traits in normal males may be very specific. In contrast, several traits of the C ("conscientiousness") axis were associated with less common SNP-defined haplotypes. Hence, it appears that common genetic variation at the VNTR contributes to the behavioural attribute of "straightforwardness", while rare haplotypes defined by SNPs downstream of the transcription start site may contribute to "conscientiousness". This study is used to address the validation, interpretation and limitation of genetic association studies of quantitative behavioural traits.

Extreme skewing of X chromosome inactivation in mothers of homosexual men.

Human sexual preference is a sexually dimorphic trait with a substantial genetic component. Linkage of male sexual orientation to markers on the X chromosome has been reported in some families. Here, we measured X chromosome inactivation ratios in 97 mothers of homosexual men and 103 age-matched control women without gay sons. The number of women with extreme skewing of X-inactivation was significantly higher in mothers of gay men (13/97=13%) compared to controls (4/103=4%) and increased in mothers with two or more gay sons (10/44=23%). Our findings support a role for the X chromosome in regulating sexual orientation in a subgroup of gay men.

Interfacial metal and antibody recognition.

The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca(2+), Ba(2+), or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with approximately 1.5 kcal/mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition.

Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide.

Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli, secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides were achieved. The genetically engineered Nissle 1917 were capable of colonizing mice for periods of weeks to months, predominantly in the colon and cecum, with lower concentrations of bacteria present in the rectum, vagina, and small intestine. Histological and immunocytochemical examination of the colon revealed bacterial growth and peptide secretion throughout the luminal mucosa and in association with epithelial surfaces. The use of genetically engineered live microbes as anti-HIV microbicides has important potential advantages in economy, efficacy, and durability.

Depression vulnerability, cigarette smoking, and the serotonin transporter gene.

People with current or past depression are more likely to have been smokers at some point in their lives. Smokers with depression histories are also less likely to quit. Attempts to understand this relationship are important insofar as they can help treatment efficacy for this group of smokers. Prior research indicates that different genetic variations affect the relationship between smoking and neuroticism. This study examined whether people with a short serotonin transporter genotype would likewise show a stronger relationship between depression vulnerability and smoking behavior than those with the long genotype. Although depression vulnerability was associated with smoking behaviors, genotype did not significantly affect this relationship. Discussion centered on possible reasons for varying results across conceptually similar studies.

A genomewide scan of male sexual orientation.

This is the first report of a full genome scan of sexual orientation in men. A sample of 456 individuals from 146 families with two or more gay brothers was genotyped with 403 microsatellite markers at 10-cM intervals. Given that previously reported evidence of maternal loading of transmission of sexual orientation could indicate epigenetic factors acting on autosomal genes, maximum likelihood estimations (mlod) scores were calculated separated for maternal, paternal, and combined transmission. The highest mlod score was 3.45 at a position near D7S798 in 7q36 with approximately equivalent maternal and paternal contributions. The second highest mlod score of 1.96 was located near D8S505 in 8p12, again with equal maternal and paternal contributions. A maternal origin effect was found near marker D10S217 in 10q26, with a mlod score of 1.81 for maternal meioses and no paternal contribution. We did not find linkage to Xq28 in the full sample, but given the previously reported evidence of linkage in this region, we conducted supplemental analyses to clarify these findings. First, we re-analyzed our previously reported data and found a mlod of 6.47. We then re-analyzed our current data, after limiting the sample to those families previously reported, and found a mlod of 1.99. These Xq28 findings are discussed in detail. The results of this first genome screen for normal variation in the behavioral trait of sexual orientation in males should encourage efforts to replicate these findings in new samples with denser linkage maps in the suggested regions.

Expression of latent HAART-persistent HIV type 1 induced by novel cellular activating agents.

The novel antitumor-promoting phorbol ester, prostratin, was evaluated for its ability to induce the expression of latent, highly active antiretroviral therapy (HAART)-persistent human immunodeficiency virus type I (HIV-1) from specific subsets of patients' peripheral blood cells. This evaluation was performed relative to the use of other cellular activating agents, such as OKT3, a monoclonal antibody against the human T cell receptor, interleukin-2 (IL-2), phytohemagglutinin (PHA), p24 antigen (HIV-1-specific capsid protein), and a molecular relative of prostratin, 12-deoxyphorbol 13-phenylacetate (DPP). Prostratin performed as efficiently as the other cellular activators at inducing the expression of latent HIV-1 from cells of patients on virally suppressive HAART. Of interest was the induction of a novel species of latent virus from the cells of an individual after exposure to the HIV-1-specific capsid protein, p24, relative to virus expression induced by several other cell activators. This suggests that a variety of agents may be available for animal model studies of lentiviral latency and clinical use to broadly induce the expression of latent, HAART-persistent HIV-1 in vivo with the goal of potential HIV-1 reservoir depletion or eradication.

Can HIV be Cured? Mechanisms of HIV persistence and strategies to combat it.

Stable remission is the ultimate goal of HIV therapy. A review of recent studies on the ability of HIV to persist despite highly active antiretroviral therapy (HAART) and immune stimulation suggests that achieving this goal will require four developments in basic and clinical science. First, more effective antiretroviral therapies, targeted at proteins other than reverse transcriptase and protease, in order to eliminate the cryptic replication that continues despite best available HAART. Second, agents that activate latent HIV gene expression in quiescent CD4 memory T cells, thereby exposing this viral reservoir to therapeutic intervention by a "shock and kill" strategy. Third, molecules such as immunotoxins that specifically recognize HIV-encoded membrane proteins and thereby potentiate the destruction of infected cells. Fourth, and still most distant, novel approaches such as genetically engineered cytotoxic T lymphocytes or anti-HIV microbes to suppress rekindling of infection by residual virus sequestered in anatomical and cellular reservoirs. Although each of these steps will be difficult to achieve, the many benefits of a cure for HIV make this a worthwhile pursuit.

A candidate gene study of CYP19 (aromatase) and male sexual orientation.

Aromatase cytochrome P450 (CYP19), which is necessary for the conversion of androgens to estrogens, plays an important role in the sexual differentiation of the brain. To investigate whether differences in the gene encoding the aromatase enzyme influence sexual orientation in men, we conducted linkage, association, and expression analyses in a large sample of homosexual brothers using microsatellite markers in and around CYP19. No linkage was detected, and a gene-specific relative risk of 1.5-fold could be excluded at a lod score of -2. Results of the TDT demonstrated no preferential transmission of any of the CYP19 alleles in this sample. Expression of aromatase mRNA by microarray analysis was not significantly different between heterosexual and homosexual men. These results suggest that variation in the gene for this subunit of the aromatase enzyme complex is not likely to be a major factor in the development of individual differences in male sexual orientation.

A potent anti-HIV immunotoxin blocks spreading infection by primary HIV type 1 isolates in multiple cell types.

Although several immunotoxins that selectively kill HIV-1-infected cells have been described, their clinical utility is limited by low potency against spreading viral infection. We show here that changing the carboxyterminal sequence of an anti-HIV-1 envelope immunotoxin to the consensus endoplasmic reticulum retention sequence KDEL substantially improves its ability to block infection of peripheral blood mononuclear cells by primary HIV-1 isolates without increasing nonspecific toxicity. Polychromatic flow cytometry of peripheral blood mononuclear cells (PBMC) infected with an HIV-1-GFP reporter virus demonstrated that the improved immunotoxin is active against a variety of primary cell types including memory T cells, NK-T cells, and monocyte/macrophages. The subnanomolar potency of this agent suggests that it could be clinically useful either as an adjuvant to highly active antiretroviral therapy (HAART) in drug-resistant patients or to reduce the reservoir of latently infected cells that is implicated in HIV-1 persistence.