RESUMO
BACKGROUND: Bacterial vaginosis (BV) is characterized by low abundance of Lactobacillus species, high pH, and immune cell infiltration and has been associated with an increased risk of human papillomavirus (HPV) infection. We molecularly assessed the cervicovaginal microbiota over time in human immunodeficiency virus (HIV)-infected and HIV-uninfected women to more comprehensively study the HPV-microbiota relationship, controlling for immune status. METHODS: 16S ribosomal RNA gene amplicon pyrosequencing and HPV DNA testing were conducted annually in serial cervicovaginal lavage specimens obtained over 8-10 years from African American women from Chicago, of whom 22 were HIV uninfected, 22 were HIV infected with a stable CD4+ T-cell count of > 500 cells/mm3, and 20 were HIV infected with progressive immunosuppression. Vaginal pH was serially measured. RESULTS: The relative abundances of Lactobacillus crispatus and other Lactobacillus species were inversely associated with vaginal pH (all P < .001). High (vs low) L. crispatus relative abundance was associated with decreased HPV detection (odds ratio, 0.48; 95% confidence interval, .24-.96; Ptrend = .03) after adjustment for repeated observation and multiple covariates, including pH and study group. However, there were no associations between HPV and the relative abundance of Lactobacillus species as a group, nor with Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii individually. CONCLUSIONS: L. crispatus may have a beneficial effect on the burden of HPV in both HIV-infected and HIV-uninfected women (independent of pH).
Assuntos
Colo do Útero/microbiologia , Colo do Útero/virologia , Infecções por HIV/etiologia , Microbiota/genética , Papillomaviridae/genética , Vagina/microbiologia , Vagina/virologia , Adulto , Contagem de Linfócito CD4/métodos , Linfócitos T CD4-Positivos/imunologia , Colo do Útero/imunologia , Estudos de Coortes , DNA Viral/genética , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Lactobacillus/imunologia , Lactobacillus/fisiologia , Microbiota/imunologia , Papillomaviridae/imunologia , RNA Ribossômico 16S/genética , Vagina/imunologia , Vaginose Bacteriana/complicações , Vaginose Bacteriana/imunologia , Vaginose Bacteriana/microbiologia , Vaginose Bacteriana/virologiaRESUMO
BACKGROUND: Colonization of the female lower genital tract with Lactobacillus provides protection against STIs and adverse pregnancy outcomes. Growth of genital Lactobacillus is postulated to depend on epithelial cell-produced glycogen. However, the amount of cell-free glycogen in genital fluid available for utilization by Lactobacillus is not known. METHODS: Eighty-five genital fluid samples from 7 pre-menopausal women taken over 4-6 weeks were obtained using the Instead SoftCup® (EvoFem, Inc., San Diego, CA, USA) by consented donors. Cell-free glycogen and glucose in genital fluids and estrogen and progesterone in blood were quantified. FINDINGS: Glycogen ranged from 0.1-32 µg/µl. There were significant differences between women in glycogen over the observation period. There was a strong negative correlation between glycogen and vaginal pH (r = -0.542, p<0.0001). In multivariable analysis, free glycogen levels were significantly negatively associated with both vaginal pH and progesterone (p < 0.001 and p = 0.004, respectively). Estrogen, glucose, age, sexual intercourse 24 hours prior to visit, and days after the initial visit were not significantly associated with free glycogen levels. CONCLUSION: Cell-free glycogen concentrations can be very high, up to 3% of genital fluid, and are strongly associated with acidic vaginal pH. However, the fluctuations in glycogen levels in individuals and differences between individuals do not appear to be associated with estrogen.
Assuntos
Líquidos Corporais/metabolismo , Estrogênios/metabolismo , Glicogênio/metabolismo , Progesterona/metabolismo , Vagina/metabolismo , Adulto , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lactobacillus/metabolismo , Pessoa de Meia-Idade , Pré-Menopausa/metabolismoRESUMO
Two conserved epitopes, located in the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41, are recognized by two HIV-1 broadly neutralizing antibodies 2F5 and 4E10, and are promising targets for vaccine design in efforts to elicit anti-HIV-1 broadly neutralizing antibodies. Since most HIV-1 infections initiate at mucosal surfaces, induction of mucosal neutralizing antibodies is necessary and of utmost importance to counteract HIV-1 infection. Here, we utilized a mucosal vaccine vector, bovine papillomavirus (BPV) virus-like particles (VLPs), as a platform to present HIV-1 neutralizing epitopes by inserting the extended 2F5 or 4E10 epitope or the MPER domain into D-E loop of BPV L1 respectively. The chimeric VLPs presenting MPER domain resembled the HIV-1 natural epitopes better than the chimeric VLPs presenting single epitopes. Oral immunization of mice with the chimeric VLPs displaying the 2F5 epitope or MPER domain elicited epitope-specific serum IgGs and mucosal secretory IgAs. The induced antibodies specifically recognized the native conformation of MPER in the context of HIV-1 envelope protein. The antibodies induced by chimeric VLPs presenting MPER domain are able to partially neutralize HIV-1 viruses from clade B and clade C.
Assuntos
Vacinas contra a AIDS/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/análise , Anticorpos Anti-HIV/sangue , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Administração Oral , Animais , Bovinos , Portadores de Fármacos , Epitopos/genética , Feminino , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Imunidade nas Mucosas , Imunoglobulina A Secretora/análise , Imunoglobulina A Secretora/sangue , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Papillomaviridae/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.
Assuntos
Ebolavirus/fisiologia , Infecções por Filoviridae/metabolismo , Lectina de Ligação a Manose/metabolismo , Receptores Mitogênicos/metabolismo , Internalização do Vírus , Animais , Chlorocebus aethiops , Proteínas do Sistema Complemento/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Glicoproteínas de Membrana/metabolismo , Pinocitose , Células Vero , Proteínas do Envelope Viral/metabolismoRESUMO
Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.
Assuntos
Antivirais/farmacologia , Ebolavirus/metabolismo , Lectinas/química , Lectina de Ligação a Manose/química , Calreticulina/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Proteínas do Sistema Complemento/química , Desenho de Fármacos , Humanos , Cinética , Microscopia de Força Atômica/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Ressonância de Plasmônio de Superfície/métodos , FicolinasRESUMO
BACKGROUND: HIV infection of cells varies greatly between individuals, with multiple steps in the replication cycle potentially contributing to the variability. Although entry and post-entry variability of HIV infection levels in cells has been demonstrated, variability in HIV binding has not been examined. In this study, we examined variability of HIV binding to peripheral blood mononuclear cells (PBMC) from different donors. RESULTS: HIV binding to PBMC varied up to 3.9-fold between individuals and was independent of CD4. Replication of HIV in donor PBMC required CD4 and paralleled virus binding trends of donor PBMC. To assess the stability of virus binding phenotypes over time, HIV was bound to donors with low- and high-binding phenotypes. The binding phenotypes were maintained when tested weekly over a 4-week period for 3 of 4 donors, while one high-binding donor decreased to lower binding on the 4th week. The low- and high-binding phenotypes were also preserved across different HIV strains. Experiments performed to determine if there was an association between HIV binding levels and specific cell subset levels within PBMC showed no correlation, suggesting that HIV binds to multiple cell subsets. CONCLUSION: These results show that differences exist in HIV binding to donor PBMC. Our data also show that HIV binding to donor PBMC is CD4-independent and can change over time, suggesting that virus binding variability is due to differences in the expression of changeable cell-surface host factors. Taken together, this study highlights the impact of cell-surface factors in HIV binding to, and infection of, PBMC which likely represents an important step in HIV infection in vivo.
Assuntos
Doadores de Sangue , Infecções por HIV/virologia , HIV/fisiologia , Leucócitos Mononucleares/virologia , Ligação Viral , HumanosRESUMO
Infections and inflammation in the genital tract can influence HIV expression or HIV susceptibility. The goal of this study was to determine if significant relationships exist between cytokines and HIV in genital tract secretions from 57 HIV-seropositive Rwandan women. Genital tract secretions were obtained by cervicovaginal lavage (CVL). Ten different cytokines in CVL were measured by multiplex cytometric bead arrays. HIV RNA in CVL and plasma were measured by quantitative PCR. In univariate analysis, genital tract HIV RNA was significantly associated with plasma HIV RNA and several of the cytokines, while in multivariate analysis, genital tract HIV RNA was significantly associated only with plasma HIV RNA and IL-6. This association of IL-6 with HIV RNA levels suggests that IL-6 is an indicator for conditions that induce HIV expression and that IL-6 may contribute to induction of HIV expression in the genital tract.
Assuntos
Doenças dos Genitais Femininos/imunologia , Doenças dos Genitais Femininos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV/isolamento & purificação , Interleucina-6/análise , RNA Viral/análise , Adulto , Feminino , Citometria de Fluxo , HIV/genética , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Ruanda , Ducha Vaginal , Carga ViralRESUMO
Bacterial vaginosis (BV) is associated with preterm labor, pelvic inflammatory disease (PID) and increased HIV acquisition, although the pathways that mediate these pathological effects have not been elucidated. To determine the presence of Toll-like receptor (TLR)-ligands and their specificity in BV, genital tract fluids were collected from women with and without BV by cervicovaginal lavage (CVL). The CVL samples were evaluated for their ability to stimulate secretion of proinflammatory cytokines and to activate NFkappaB and the HIV long terminal repeat (LTR), indicators of TLR activation, in human monocytic cells. Stimulation with BV CVLs induced higher levels of IL-8 and TNFalpha secretion, as well as higher levels of HIV LTR and NFkappaB activation, than CVLs from women with normal healthy bacterial flora. To identify which TLRs were important in BV, 293 cells expressing specific TLRs were exposed to CVL samples. BV CVLs induced higher IL-8 secretion by cells expressing TLR2 than CVLs from women without BV. Surprisingly, BV CVLs did not stimulate cells expressing TLR4/MD2, although these cells responded to purified lipopolysaccharide (LPS), a TLR4 ligand. BV CVLs, in cells expressing TLR2, also activated the HIV LTR. Thus, these studies show that soluble factor(s) present in the lower genital tract of women with BV activate cells via TLR2, identifying a pathway through which BV may mediate adverse effects.
Assuntos
Receptor 2 Toll-Like/fisiologia , Vaginose Bacteriana/imunologia , Adulto , Linhagem Celular , Feminino , Genitália Feminina/imunologia , Repetição Terminal Longa de HIV , Humanos , Interleucina-8/biossíntese , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Human cytomegalovirus (HCMV) and human immunodeficiency virus type-1 (HIV-1) infect the female genital tract. A human cervical explant model was developed to study single and dual infection by these viruses in the genital compartment. An HCMV strain expressing green fluorescent protein, and two clinical HCMV strains produced peak viral DNA copies at 14 to 21 days post-infection. Peak levels of HIV-1(Ba-L) p24 antigen occurred at 7 days post-infection. HIV-1(Ba-L) appeared to enhance HCMV in co-infected tissues. Singly and dually infected explants produced increased levels of cytokines IL-6, IL-8, and GRO-alpha in culture supernatants. Immunohistochemical and flow cytometric analysis showed HCMV infection of leukocytes with the phenotype CD45+/CD1a+/CD14+/HLA-DR+ but not stromal or endothelial cells. Cells expressing both GFP and HIV-1 p24 antigen were detected in co-infected tissues. The cervical explants provide an ex vivo human model for examining mechanisms of virus-virus interaction and pathogenesis in clinically relevant tissue.
Assuntos
Colo do Útero/virologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/crescimento & desenvolvimento , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Quimiocina CXCL1/análise , Meios de Cultura/química , Células Endoteliais/virologia , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteína do Núcleo p24 do HIV/biossíntese , Humanos , Interleucina-6/análise , Interleucina-8/análise , Leucócitos/virologia , Técnicas de Cultura de Órgãos , Células Estromais/virologiaRESUMO
Approximately half of the molecular mass of gp120, the receptor-binding envelope protein of human immunodeficiency virus (HIV), consists of N-linked glycans. Nearly half of these glycans are of the high mannose type. These high mannose glycans furnish a rich forest of mannose residues on the virus surface making HIV a prime target for interaction with mannose-specific lectins of the immune system. This review focuses on the known interactions between gp120 and immune system lectins some of which HIV appears to exploit. The effect of variation in glycosylation of gp120, especially with respect to clades of HIV, on binding of immune system lectins is highlighted.
Assuntos
HIV/metabolismo , Sistema Imunitário/virologia , Lectinas/química , Polissacarídeos/química , Animais , Proteínas de Bactérias/química , Proteínas de Transporte/química , Moléculas de Adesão Celular/química , Glicosilação , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/imunologia , Humanos , Lectinas Tipo C/química , Estrutura Terciária de Proteína , Receptores de Superfície Celular/químicaRESUMO
Strategies that prevent initial HIV infection of cells are greatly needed. In this study, we determined the requirement of divalent cations for HIV infection of and attachment to peripheral blood mononuclear cells (PBMC), which contain several types of HIV-infectable cells-CD4(+) T cells, monocytes and dendritic cells. EDTA, added only during PBMC exposure to HIV, reduced infection by an average of 92%. The reduction of infection by EDTA was accompanied by a reduction in HIV binding to PBMC; R5, X4 and dual-tropic HIV binding to PBMC were inhibited by >85%. EGTA similarly reduced HIV binding to PBMC, while addition of Ca(2+) or Mn(2+), but not Mg(2+), fully restored binding. Virus attachment was inhibited in a dose-dependent manner by trypsin treatment of PBMC, indicating protein involvement in HIV binding. In contrast, mannan or soluble ICAM-1 did not inhibit HIV binding to PBMC. These data indicate that a Ca(2+)-dependent cell-surface protein(s) is responsible for the majority of HIV attachment to and infection of PBMC. Further studies of this are likely to reveal novel strategies to prevent infection of PBMC.
Assuntos
Cálcio/metabolismo , HIV/fisiologia , Leucócitos Mononucleares/virologia , Ligação Viral , Internalização do Vírus , Antivirais/farmacologia , Células Cultivadas , Ácido Edético/farmacologia , Proteína do Núcleo p24 do HIV/biossíntese , Humanos , Molécula 1 de Adesão Intercelular/farmacologia , Magnésio/farmacologia , Manganês/farmacologia , Mananas/farmacologia , Tripsina/farmacologiaRESUMO
Bacterial vaginosis (BV) is a clinical syndrome presenting with a malodorous vaginal discharge and increased vaginal pH. Diagnosis has been based on clinical Amsel criteria and direct Gram stain of vaginal secretions. Human immunodeficiency virus (HIV)-infected participants in the Women's Interagency HIV Study contributed cervicovaginal lavage (CVL) samples. Lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage samples were quantified by PCR. Gynecologic evaluation included Nugent score and Amsel criterion assessment. We compared the gold standard Nugent score to Amsel criteria and quantitative bacterial PCR for diagnosing BV in 203 CVL samples from women with Nugent scores of 7 to 10 (BV group) and 203 samples from women with BV Nugent scores of 0 to 3 ("No-BV" group). Only 75 of the 203 CVL samples from women with Nugent scores of 7 to 10 met positive Amsel criteria. Increasing levels of G. vaginalis and M. hominis and decreasing levels of lactobacilli were significantly associated with BV by Nugent score. Of the group with Nugent scores of 7 to 10, 83% and 81% had log(10) G. vaginalis counts and log(10) M. hominis counts greater than 6.81 and 4.82, respectively, while only 30% and 31% of the group with Nugent scores of 0 to 3 were above these thresholds, respectively. There was significant overlap in the log(10) lactobacillus counts between the two groups. Utilizing all three log(10) bacterial counts (G. vaginalis, M. hominis, and lactobacilli) in our model improved the sensitivity and specificity to 83% and 78%, respectively, in comparison with Nugent score. In this cohort, Amsel criteria were poorly predictive of BV. PCR quantification of G. vaginalis and M. hominis from CVL is significantly more sensitive than Amsel criteria for diagnosing BV.
Assuntos
Gardnerella vaginalis/isolamento & purificação , Infecções por HIV/complicações , Lactobacillus/isolamento & purificação , Mycoplasma hominis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Vaginose Bacteriana/diagnóstico , DNA Bacteriano/análise , Feminino , Gardnerella vaginalis/genética , Violeta Genciana , HIV , Infecções por HIV/virologia , Humanos , Lactobacillus/genética , Mycoplasma hominis/genética , Fenazinas , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Esfregaço Vaginal/métodos , Vaginose Bacteriana/microbiologiaRESUMO
Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.
Assuntos
Moléculas de Adesão Celular/metabolismo , Ebolavirus/metabolismo , Produtos do Gene env/metabolismo , Lectinas Tipo C/metabolismo , Lectina de Ligação a Manose/metabolismo , Marburgvirus/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas do Envelope Viral/metabolismo , Ligação Competitiva , Linhagem Celular , Proteínas do Sistema Complemento/metabolismo , Ebolavirus/patogenicidade , Humanos , Marburgvirus/patogenicidade , Testes de NeutralizaçãoRESUMO
We measured levels of six cytokines and human immunodeficiency virus (HIV) RNA in saliva from HIV-seropositive individuals and compared salivary cytokine levels in HIV-seropositives and seronegatives. All of the six tested cytokines were detected in saliva although interleukin-1beta, interferon-gamma and interleukin-10 were detected more frequently (90%, 68% and 61% of samples, respectively) than interleukin-6, tumor necrosis factor-alpha and tumor necrosis factor-alpha receptor II (2-17%). There was no significant association between cytokine levels in saliva and plasma suggesting that cytokines were produced locally. Interferon-gamma levels were significantly higher in saliva from HIV-seropositives when compared to seronegatives while interleukin-10 levels were lower in seropositive saliva. Interleukin-10 levels were higher in individuals with low CD4 counts in the seropositive group. HIV RNA was detected in 29% of saliva samples from seropositives and there was a significant correlation between saliva and plasma HIV RNA levels. However, HIV RNA levels in saliva were not significantly associated with any of the saliva or plasma cytokine levels or with CD4 cell numbers. This study shows no association between inflammatory cytokine levels and HIV levels in saliva and suggests that saliva HIV levels are more influenced by blood HIV RNA levels than oral inflammation.
Assuntos
Citocinas/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , RNA Viral/metabolismo , Saliva/imunologia , Saliva/virologia , Contagem de Linfócito CD4 , Citocinas/sangue , Soronegatividade para HIV/imunologia , Soropositividade para HIV/imunologia , Soropositividade para HIV/virologia , Humanos , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , RNA Viral/sangue , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Bacterial vaginosis (BV) is associated with complications of pregnancy and increased susceptibility to human immunodeficiency virus (HIV) sexual transmission. METHODS: The ability of genital mucosal fluids from women with BV and of microbial flora associated with BV to induce tumor necrosis factor (TNF)- alpha secretion and Toll-like receptor (TLR) 2 and TLR4 mRNA expression was assessed. RESULTS: Primary peripheral-blood mononuclear cells and THP-1 monocytic cells secreted TNF- alpha in response to cervicovaginal lavage (CVL) samples from women with BV. Mycoplasma hominis and Gardnerella vaginalis also stimulated TNF- alpha secretion. Strikingly, CVL samples from women with BV induced up to 60-fold increases in TLR4 mRNA expression, compared with CVL samples from women without BV and with bacteria not associated with BV. Anti-TNF- alpha antibody blocked increases in TLR4 mRNA expression induced by CVL samples from women with BV, indicating that TNF- alpha plays a critical role in induction of TLR4. Both TLR2 and TLR4 mRNA expression were approximately 60-fold higher in cells isolated from the lumen of the genital tract than in cervical mucosal tissue, but lumen TLR mRNA levels did not change significantly after BV treatment. CONCLUSIONS: These experiments show that genital mucosal fluids and certain bacteria from women with BV stimulate TNF- alpha secretion and TLR4 mRNA expression, suggesting mechanisms whereby BV affects pregnancy and HIV transmission.
Assuntos
Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vaginose Bacteriana/metabolismo , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Humanos , Imunidade nas Mucosas , Mucosa/metabolismo , RNA Mensageiro/metabolismo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Vaginose Bacteriana/imunologiaRESUMO
Bacterial vaginosis (BV) has been associated with HIV sexual transmission and increased levels of genital tract HIV RNA. We postulated that BV induces the appearance of substances in the genital tract that stimulate HIV expression locally. To test this, we measured HIV RNA levels in genital mucosal fluid from women with or without BV (defined by Nugent score) and compared them with the ability of those fluids to stimulate HIV expression in the chronically HIV-infected monocytic line U1. The U1 activity was significantly higher in women with BV (median = 1320 pg/ml p24) than in women with normal flora (median = 103 pg/ml p24, p = 0.0001). However, levels of the U1 activity were not significantly associated with levels in the genital tract of HIV RNA. Levels of the U1 activity were also not associated with levels of Gardnerella vaginalis or Mycoplasma hominis in genital fluids, suggesting these bacteria were not the source of the activity. Thus, while these data show a strong association of U1 stimulatory activity with BV, no influence of the U1 activity on genital tract HIV expression was observed.
Assuntos
Genitália Feminina/virologia , Infecções por HIV/complicações , HIV/fisiologia , Monócitos/virologia , RNA Viral/análise , Vaginose Bacteriana/complicações , Linhagem Celular , Contagem de Colônia Microbiana , Feminino , Gardnerella vaginalis/isolamento & purificação , Genitália Feminina/microbiologia , HIV/genética , Proteína do Núcleo p24 do HIV/análise , Humanos , Mycoplasma hominis/isolamento & purificação , Carga ViralRESUMO
BACKGROUND: Bacterial vaginosis (BV) is associated with human immunodeficiency virus (HIV) acquisition. We examined the association between BV and BV-associated bacteria and expression of HIV in the female genital tract. METHODS: HIV RNA, lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage (CVL) samples were quantified by polymerase chain reaction (PCR). Gynecologic evaluation included Nugent score assessment, Amsel criteria assessment, detection of other genital-tract infections, and dysplasia grading. CD4 cell count, plasma HIV RNA level, and antiretroviral history were obtained. RESULTS: A total of 203 CVL samples from women with Nugent scores of 7-10 (BV group) and 203 samples from women with Nugent scores of 0-3 (no-BV group) were matched by plasma HIV RNA level and analyzed. After controlling for plasma HIV RNA level and Nugent score in univariate analyses, we found that G. vaginalis and M. hominis bacterial counts, Candida vaginitis, and herpes simplex virus (HSV) were positively associated with CVL HIV RNA levels. In multivariate analysis, only lactobacilli bacterial counts (P=.006; inverse association), M. hominis bacterial counts (P=.0001; positive association), Candida vaginitis (P=.007), and HSV (P=.03) were significantly associated with CVL HIV RNA levels. CONCLUSION: Bacteria associated with BV increase genital-tract HIV RNA levels. Quantitative bacterial counts for lactobacilli and M. hominis are better correlates of CVL HIV RNA than are Nugent score or Amsel criteria. Since plasma virus and CD4 cell levels did not differ between the BV and no-BV groups, these data suggest that the bacterial flora associated with BV influence genital-tract HIV shedding.
Assuntos
Infecções por HIV/virologia , HIV/isolamento & purificação , Lactobacillus/isolamento & purificação , Vagina/microbiologia , Vagina/virologia , Vaginose Bacteriana/microbiologia , Adulto , Idoso , Contagem de Linfócito CD4 , Candida/isolamento & purificação , Candidíase Vulvovaginal/microbiologia , Contagem de Colônia Microbiana , Feminino , Gardnerella vaginalis/isolamento & purificação , Humanos , Pessoa de Meia-Idade , Infecções por Mycoplasma/microbiologia , Mycoplasma hominis/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Viral/análise , Análise de Regressão , Simplexvirus/isolamento & purificação , Ducha Vaginal , Carga ViralRESUMO
The envelope protein (gp120/gp41) of HIV-1 is highly glycosylated with about half of the molecular mass of gp120 consisting of N-linked carbohydrates. While glycosylation of HIV gp120/gp41 provides a formidable barrier for development of strong antibody responses to the virus, it also provides a potential site of attack by the innate immune system through the C-type lectin mannose binding lectin (MBL) (also called mannan binding lectin or mannan binding protein). A number of studies have clearly shown that MBL binds to HIV. Binding of MBL to HIV is dependent on the high-mannose glycans on gp120 while host cell glycans incorporated into virions do not contribute substantially to this interaction. It is notable that MBL, due to its specificity for the types of glycans that are abundant on gp120, has been shown to interact with all tested HIV strains. While direct neutralization of HIV produced in T cell lines by MBL has been reported, neutralization is relatively low for HIV primary isolates. However, drugs that alter processing of carbohydrates enhance neutralization of HIV primary isolates by MBL. Complement activation on gp120 and opsonization of HIV due to MBL binding have also been observed but these immune mechanisms have not been studied in detail. MBL has also been shown to block the interaction between HIV and DC-SIGN. Clinical studies show that levels of MBL, an acute-phase protein, increase during HIV disease. The effects of MBL on HIV disease progression and transmission are equivocal with some studies showing positive effects and other showing no effect or negative effects. Because of apparently universal reactivity with HIV strains, MBL clearly represents an important mechanism for recognition of HIV by the immune system. However, further studies are needed to define the in vivo contribution of MBL to clearance and destruction of HIV, the reasons for low neutralization by MBL and ways that MBL anti-viral effects can be augmented.
Assuntos
HIV/imunologia , Lectinas de Ligação a Manose/metabolismo , Moléculas de Adesão Celular/imunologia , Ativação do Complemento , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Humanos , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/uso terapêutico , Receptores de Superfície Celular/imunologiaRESUMO
Resting neutrophils bind human immunodeficiency virus type 1 (HIV-1) and efficiently transfer infection to lymphocytes. The present study shows that a brief activation by inflammatory stimuli increases the neutrophil binding levels of both R5 and X4 isolates of HIV-1 at least twofold. The binding occurs independently of CD4, gp120, and incubation temperature and is observed with HIV-1 propagated either in lymphocytes or in HEK293 cells. Significantly, HIV-1 bound to the activated neutrophils accelerates the infection of activated lymphocytes compared to free HIV-1 or to HIV-1 bound to resting neutrophils. It is proposed that these events may contribute to the increased risk of HIV-1 transmission at sites of mucosal infection.
Assuntos
HIV-1/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/virologia , Antígenos CD4/metabolismo , Células Cultivadas , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Lipopolissacarídeos/imunologia , N-Formilmetionina Leucil-Fenilalanina/imunologia , Fito-Hemaglutininas/imunologia , Receptores de HIV/metabolismo , Temperatura , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Human immunodeficiency virus type 1 (HIV-1) envelope-specific neutralizing antibodies are generated late after initial infection, and the neutralizing antibody response is weak in the infected individuals. Administration of neutralizing antibodies such as 2F5 to HIV-1-infected individuals resulted in reductions in viral loads. Because HIV-1 is transmitted mainly via mucosa and because HIV-specific neutralizing antibodies reduce HIV-1 in infected individuals, a vaccine that can induce both mucosal and systemic HIV-1-specific neutralizing antibodies may be used to prevent and to treat HIV-1 infection. In this study, we made a bovine papillomavirus (BPV) L1-HIV-1 gp41 fusion protein in which ELDKWA of gp41 was inserted into the N terminus of BPV L1 (amino acids 130 to 136). Expression of the fusion protein in insect cells led to the assembly of chimeric virus-like particles (CVLPs). The CVLPs had sizes similar to those of BPV particles and were able to bind to the cell surface and penetrate the cell membrane. Oral immunization of mice with CVLPs induced gp41-specific serum immunoglobulin G (IgG) and intestinal secretory IgA. However, intramuscular immunization with the CVLPs resulted in similar amounts of gp41-specific IgG but low levels of secretory IgA. The antibodies specifically recognized the fixed HIV-1 gp41 on the cell surface. Importantly, the sera and fecal extracts from mice orally immunized with the CVLPs neutralized HIV-1(MN) in vitro. Thus, BPV-HIV-1 gp41 CVLPs may be used to prevent and to treat HIV-1 infection.