RESUMO
The major goals of Kawasaki disease (KD) therapy are to reduce inflammation and prevent thrombosis in the coronary arteries (CA), but some children do not respond to currently available non-specific therapies. New treatments have been difficult to develop because the molecular pathogenesis is unknown. In order to identify dysregulated gene expression in KD CA, we performed high-throughput RNA sequencing on KD and control CA, validated potentially dysregulated genes by real-time reverse transcription-polymerase chain reaction (RT-PCR) and localized protein expression by immunohistochemistry. Signalling lymphocyte activation molecule CD84 was up-regulated 16-fold (P < 0·01) in acute KD CA (within 2 months of onset) and 32-fold (P < 0·01) in chronic CA (5 months to years after onset). CD84 was localized to inflammatory cells in KD tissues. Genes associated with cellular proliferation, motility and survival were also up-regulated in KD CA, and immune activation molecules MX2 and SP140 were up-regulated in chronic KD. CD84, which facilitates immune responses and stabilizes platelet aggregates, is markedly up-regulated in KD CA in patients with acute and chronic arterial disease. We provide the first molecular evidence of dysregulated inflammatory responses persisting for months to years in CA significantly damaged by KD.
Assuntos
Antígenos CD/metabolismo , Antígenos Nucleares/metabolismo , Plaquetas/imunologia , Síndrome de Linfonodos Mucocutâneos/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Fatores de Transcrição/metabolismo , Calcificação Vascular/imunologia , Doença Aguda , Antígenos CD/genética , Antígenos Nucleares/genética , Processos de Crescimento Celular/genética , Movimento Celular/genética , Sobrevivência Celular/genética , Doença Crônica , Vasos Coronários/patologia , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Lactente , Masculino , Síndrome de Linfonodos Mucocutâneos/sangue , Síndrome de Linfonodos Mucocutâneos/genética , Proteínas de Resistência a Myxovirus/genética , Agregação Plaquetária/genética , RNA Mensageiro/análise , Família de Moléculas de Sinalização da Ativação Linfocitária , Fatores de Transcrição/genética , Regulação para Cima , Calcificação Vascular/sangue , Calcificação Vascular/genéticaRESUMO
Microsporidia are ubiquitous obligate eukaryotic intracellular parasites that are now felt to be more akin to degenerate fungi than to protozoa. Microsporidia can be highly pathogenic, causing a broad range of symptoms in humans, especially individuals who are immunocompromised. The vast majority of human cases of microsporidiosis have been reported during the past 20 years, in patients with HIV/AIDS, while only relatively rare cases have been described in immunocompetent individuals. However, microsporidia infections are being increasingly reported in patients following solid-organ transplanation, where the main symptom has been diarrhea. The authors report the first case of pulmonary microsporidial infection in an allogeneic bone marrow transplant recipient in the United States and only the second case in the world. The patient, with a history of Hodgkin disease followed by acute myelogenous leukemia received a T-cell-depleted graft, but succumbed to respiratory failure 63 days post transplantation. An open lung biopsy, taken just before death, was originally thought to show toxoplasmosis. The correct diagnosis of microsporidiosis was made postmortem by light and electron microscopy. DNA polymerase chain reaction analysis confirmed the diagnosis and furthermore revealed it to be the dog strain of the microsporidia species Encephalitozoon cuniculi. Although to date rarely diagnosed, microsporidial infection should also be considered in the differential diagnosis of, e.g., unexplained pulmonary infection in bone marrow transplant patients.
Assuntos
Transplante de Medula Óssea , Encephalitozoon cuniculi/ultraestrutura , Encefalitozoonose/patologia , Leucemia Mieloide/terapia , Pneumopatias Parasitárias/patologia , Doença Aguda , Adulto , Animais , DNA de Protozoário/análise , DNA de Protozoário/genética , Encephalitozoon cuniculi/genética , Encefalitozoonose/parasitologia , Evolução Fatal , Feminino , Humanos , Pneumopatias Parasitárias/parasitologia , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Transplante HomólogoRESUMO
Microsporidia are obligate, intracellular protozoal parasites that can be pathogenic in immunocompromised individuals. The majority of cases of microsporidiosis have been documented in patients with HIV, and only a few case reports exist of infection in solid organ transplant patients. We report the first case of pulmonary microsporidial infection in an allogeneic bone marrow transplant recipient in the US. The patient was a recipient of a T-cell-depleted graft who succumbed to complications from respiratory failure 63 days post transplant. The diagnosis was made post mortem by electron microscopy and confirmed with PCR. Although rare, microsporidial infection should be considered in the differential diagnosis of unexplained pulmonary infection in bone marrow transplant patients.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Pneumopatias Parasitárias/etiologia , Microsporidiose/etiologia , Adulto , Evolução Fatal , Feminino , Humanos , Pneumopatias Parasitárias/diagnóstico , Depleção Linfocítica , Microscopia Eletrônica , Microsporidiose/diagnóstico , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/etiologia , Transplante HomólogoRESUMO
New HIV therapies are urgently needed to address the growing problem of drug resistance. In this article, we characterize the anti-HIV drug candidate 3-O-(3',3'-dimethylsuccinyl) betulinic acid (PA-457). We show that PA-457 potently inhibits replication of both WT and drug-resistant HIV-1 isolates and demonstrate that the compound acts by disrupting a late step in Gag processing involving conversion of the capsid precursor (p25) to mature capsid protein (p24). We find that virions from PA-457-treated cultures are noninfectious and exhibit an aberrant particle morphology characterized by a spherical, acentric core and a crescent-shaped, electron-dense shell lying just inside the viral membrane. To identify the determinants of compound activity we selected for PA-457-resistant virus in vitro. Consistent with the effect on Gag processing, we found that mutations conferring resistance to PA-457 map to the p25 to p24 cleavage site. PA-457 represents a unique class of anti-HIV compounds termed maturation inhibitors that exploit a previously unidentified viral target, providing additional opportunities for HIV drug discovery.
Assuntos
Fármacos Anti-HIV/farmacologia , Produtos do Gene gag/química , Succinatos/farmacologia , Triterpenos/farmacologia , Sítios de Ligação , Homólogo 5 da Proteína Cromobox , Desenho de Fármacos , Produtos do Gene gag/antagonistas & inibidores , Genótipo , Proteína do Núcleo p24 do HIV/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Microscopia Eletrônica , Modelos Químicos , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Testes de Precipitina , Succinatos/química , Triterpenos/química , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
The host response to infection or injury initiates a cascade of events involving recruitment of leukocytes and the release of multiple inflammatory mediators. One of these mediators, nitric oxide (NO), not only represents an important microbicidal agent in host defense, but also functions as a biological signaling and effector molecule in inflammation and immunity. However, overproduction of NO can be autotoxic and contribute to tissue damage and has been implicated in pathogenesis of tumors, and infectious, autoimmune and chronic degenerative diseases. NO is generated via constitutive and inducible nitric oxide synthases (iNOS) which catalyze the oxidation of a guanidino nitrogen associated with L-arginine. Whereas endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed, iNOS is transcriptionally induced by bacterial constituents and inflammatory mediators, including TNF alpha and IL-1. In an experimental model of bacterial component-induced joint inflammation and tissue degradation, functionally distinct roles of the constitutive NOS and iNOS were demonstrated. Following systemic delivery of an arthritogenic dose of streptococcal cell walls (SCW), these bacterial peptidoglycan-polysaccharide complexes disseminate and target the peripheral joints, liver and spleen of the treated animals. Following deposition of the SCW in the peripheral joints, an initial innate inflammatory response to the bacterial components progresses into an adaptive immune response with the recruitment and activation of mononuclear phagocytes and T lymphocytes. With the release of cytokines and inflammatory mediators, there is an upregulation of gene expression for iNOS, but not the constitutive nNOS or eNOS. Nonetheless, the constitutive NOS isoforms, regulated by calcium fluxes and interaction with calmodulin, may also enhance NO production. Increased release of NO was detected not only in the synovium, but also in the circulation, and plasma levels of nitrate plus nitrite, the stable products of NO reactions, correlated with disease progression. Following inhibition of NO production with nonspecific NOS inhibitors, such as N(G)-monomethyl-L-arginine, which target all three isoforms, there is a striking therapeutic benefit with reduced signs and symptoms of erosive arthritis. In contrast, selective targeting of iNOS with N-iminoethyl-L-lysine resulted in exacerbation of the synovial inflammation and degradation of joint structures. Based on these data, it appears that the constitutive isoforms of NOS contribute to the pathophysiology of the arthropathy, and that induced NOS and NO may function, in part, in a protective pathway. Moreover, the suppression of NO following treatment with TNF alpha antagonists results in reduced inflammation and the associated synovial pathology. Collectively, these data implicate discrete roles for the NOS isoforms in the emergence of local tissue pathology and underscore the need to define the specific pathways that are being targeted for interventional strategies.
Assuntos
Óxido Nítrico/metabolismo , Transtornos da Articulação Temporomandibular/imunologia , Transtornos da Articulação Temporomandibular/metabolismo , Animais , Humanos , Óxido Nítrico Sintase/metabolismo , Transtornos da Articulação Temporomandibular/patologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
A replication-competent rhabdovirus-based vector expressing human immunodeficiency virus type 1 (HIV-1) Gag protein was characterized on human cell lines and analyzed for the induction of a cellular immune response in mice. We previously described a rabies virus (RV) vaccine strain-based vector expressing HIV-1 gp160. The recombinant RV was able to induce strong humoral and cellular immune responses against the HIV-1 envelope protein in mice (M. J. Schnell et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000; J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001). Recent research suggests that the HIV-1 Gag protein is another important target for cell-mediated host immune defense. Here we show that HIV-1 Gag can efficiently be expressed by RV on both human and nonhuman cell lines. Infection of HeLa cells with recombinant RV expressing HIV-1 Gag resulted in efficient expression of HIV-1 precursor protein p55 as indicated by both immunostaining and Western blotting. Moreover, HIV-1 p24 antigen capture enzyme-linked immunosorbent assay and electron microscopy showed efficient release of HIV-1 virus-like particles in addition to bullet-shaped RV particles in the supernatants of the infected cells. To initially screen the immunogenicity of this new vaccine vector, BALB/c mice received a single vaccination with the recombinant RV expressing HIV-1 Gag. Immunized mice developed a vigorous CD8(+) cytotoxic T-lymphocyte response against HIV-1 Gag. In addition, 26.8% of CD8(+) T cells from mice immunized with RV expressing HIV-1 Gag produced gamma interferon after challenge with a recombinant vaccinia virus expressing HIV-1 Gag. These results further confirm and extend the potency of RV-based vectors as a potential HIV-1 vaccine.
Assuntos
Expressão Gênica , Produtos do Gene gag/genética , Vetores Genéticos , HIV-1/genética , Vírus da Raiva , Replicação Viral , Vacinas contra a AIDS , Animais , Citometria de Fluxo , Produtos do Gene gag/biossíntese , Produtos do Gene gag/imunologia , Proteína do Núcleo p24 do HIV/biossíntese , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/imunologia , HIV-1/imunologia , HIV-1/fisiologia , Células HeLa , Humanos , Imunização , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas , VírionRESUMO
The intestinal mucosa normally displays minimal inflammation despite the close proximity between mucosal macrophages and lumenal bacteria. Macrophages interact with bacteria and their products through CD14, a surface receptor involved in the response to LPS, and CD89, the receptor for IgA (FcalphaR). Here we show that resident macrophages isolated from normal human intestine lack CD14 and CD89. The absence of CD14 and CD89 was not due to the isolation procedure or mucosal cell products, but was evident at the transcriptional level, as the macrophages expressed neither CD14- nor CD89-specific mRNAs, but did express Toll-like receptor 2 and 4 transcripts. Consistent with their CD14(-) phenotype, lamina propria macrophages displayed markedly reduced LPS-induced cytokine production and LPS-enhanced phagocytosis. In addition, IgA-enhanced phagocytosis was sharply reduced in lamina propria macrophages. Thus, the absence of CD14 and CD89 on resident intestinal macrophages, due to down-regulated gene transcription, causes down-modulated LPS- and IgA-mediated functions and probably contributes to the low level of inflammation in normal human intestinal mucosa.
Assuntos
Antígenos CD/metabolismo , Proteínas de Drosophila , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Receptores Fc/metabolismo , Antígenos CD/genética , Sequência de Bases , Citocinas/biossíntese , Primers do DNA/genética , Regulação para Baixo , Humanos , Imunoglobulina A/metabolismo , Técnicas In Vitro , Jejuno/citologia , Jejuno/imunologia , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fagocitose/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Fc/genética , Transdução de Sinais , Receptor 2 Toll-Like , Receptores Toll-LikeRESUMO
Transforming growth factor (TGF)-beta1, a potent immunoregulatory molecule, was found to control the life and death decisions of T lymphocytes. Both thymic and peripheral T cell apoptosis was increased in mice lacking TGF-beta1 (TGF-beta1(-/-)) compared with wild-type littermates. Engagement of the T cell receptor enhanced this aberrant T cell apoptosis, as did signaling through either the death receptor Fas or the tumor necrosis factor alpha receptor in peripheral T cells. Strikingly, TGF-beta was localized within the mitochondria of normal T cells, and the absence of TGF-beta1 resulted in disruption of mitochondrial membrane potential (Deltapsi(m)), which marks the point of no return in a cell condemned to die. This TGF-beta-dependent regulation of viability appears dissociable from the TGF-beta1 membrane receptor-Smad3 signaling pathway, but associated with a mitochondrial antiapoptotic protein Bcl-XL. Thus, TGF-beta1 may protect T cells at multiple sites in the death pathway, particularly by maintaining the essential integrity of mitochondria. These findings may have broad implications not only for T cell selection and death in immune responses and in the generation of tolerance, but also for defining the mechanisms of programmed cell death in general.
Assuntos
Apoptose/fisiologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Antígenos CD/imunologia , Antígenos CD/fisiologia , Proteína Ligante Fas , Glicoproteínas de Membrana/imunologia , Camundongos , Microscopia Eletrônica , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo II do Fator de Necrose Tumoral , Transdução de Sinais , Timo/citologia , Timo/imunologia , Timo/ultraestrutura , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Receptor fas/imunologiaRESUMO
A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21(high)- and CD21(low)-expressing B cells, the CD21(low) fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21(high) fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21(low) B cell population but not in the CD21(high) fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.
Assuntos
Linfócitos B/imunologia , Infecções por HIV/imunologia , HIV-1/patogenicidade , Antígenos CD19/genética , Antígenos CD19/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/ultraestrutura , Linfócitos B/ultraestrutura , Sequência de Bases , Primers do DNA/genética , Expressão Gênica , Infecções por HIV/genética , Humanos , Hipergamaglobulinemia/etiologia , Imunofenotipagem , Ativação Linfocitária , Microscopia Eletrônica , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Viremia/genética , Viremia/imunologiaRESUMO
OBJECTIVE: To compare the primary diagnoses assigned by general surgical pathologists on a series of 103 consecutive colon biopsies from individuals infected with human immunodeficiency virus (HIV) with diagnoses rendered by a pathologist with extensive experience in gastrointestinal pathology in HIV/acquired immunodeficiency syndrome. DESIGN: New sections were cut from paraffin blocks of 103 consecutive colon biopsies taken during colonoscopies of 82 different HIV-infected patients; all new sections were stained with hematoxylin-eosin. These individuals either had negative stool studies or had failed to respond to therapy and had chronic large bowel symptoms, such as frequent small volume-type diarrhea, tenesmus, and/or bright red blood per rectum. Immunohistochemistry for cytomegalovirus (CMV) was performed on 18 of 22 specimens originally diagnosed with CMV colitis. RESULTS: The initial study yielded 70 (68%) negative or nonspecific diagnoses, 22 (21%) cases of CMV colitis, 5 (5%) Cryptosporidium diagnoses, 2 cases each of adenomatous polyps and Kaposi sarcoma, and 1 case each of spirochetosis and squamous cell carcinoma of the anorectum. Review of the recuts yielded 64 (62%) negative or nonspecific diagnoses, 12 (12%) new adenovirus infections (3 combined with CMV), and 11 (11%) lone CMV infections. Three attaching and effacing bacterial infections were diagnosed, 1 with adenovirus coinfection. A total of 4 spirochetosis cases were found on review. Seven (7%) of the biopsies showed at least 1 coinfection. Nine biopsies had features suggestive of inflammatory bowel disease. CONCLUSIONS: Colonoscopy with biopsy after negative stool studies or failure to respond to therapy yielded a high proportion of negative or nonspecific diagnoses. Adenovirus and enteropathogenic bacterial infections had been totally overlooked on initial examination. It takes particular experience to evaluate gastrointestinal biopsies from HIV-infected patients.
Assuntos
Síndrome da Imunodeficiência Adquirida/patologia , Colo/patologia , Colonoscopia , Síndrome da Imunodeficiência Adquirida/complicações , Polipose Adenomatosa do Colo/patologia , Infecções por Adenoviridae/patologia , Infecções Bacterianas/patologia , Biópsia , Colite/microbiologia , Colite/patologia , Colite/virologia , Doenças do Colo/parasitologia , Neoplasias do Colo/patologia , Criptosporidiose/patologia , Infecções por Citomegalovirus/patologia , Humanos , Sarcoma de Kaposi/patologiaRESUMO
Kaposi sarcoma-associated herpesvirus (KSHV) is associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman disease. Reactivation of KSHV in latently infected cells and subsequent plasma viremia occur before the development of KS. Intracellular signaling pathways involved in KSHV reactivation were studied. In latently infected PEL cells (BCBL-1), KSHV reactivation in single cells was determined by quantitative flow cytometry. Viral particle production was determined by electron microscope analyses and detection of minor capsid protein in culture supernatants. Agents that mobilized intracellular calcium (ionomycin, thapsigargin) induced expression of KSHV lytic cycle-associated proteins and led to increased virus production. Calcium-mediated virus reactivation was blocked by specific inhibitors of calcineurin-dependent signal transduction (cyclosporine, FK506). Similarly, calcium-mediated virus reactivation in KSHV-infected dermal microvascular endothelial cells was blocked by cyclosporine. Furthermore, retroviral transduction with plasmid DNA encoding VIVIT, a peptide specifically blocking calcineurin-NFAT interactions, inhibited calcium-dependent KSHV reactivation. By contrast, chemical induction of lytic-phase infection by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate was blocked by protein kinase C inhibitors, but not by calcineurin inhibitors. In summary, calcineurin-dependent signal transduction, an important signaling cascade in vivo, induces calcium-dependent KSHV replication, providing a possible target for the design of antiherpesvirus strategies in KSHV-infected patients.
Assuntos
Inibidores de Calcineurina , Sinalização do Cálcio/efeitos dos fármacos , Herpesvirus Humano 8/crescimento & desenvolvimento , Proteínas Nucleares , Ativação Viral/efeitos dos fármacos , Calcineurina/fisiologia , Sinalização do Cálcio/fisiologia , Capsídeo/análise , Células Cultivadas , Ciclosporina/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Endotélio Vascular/citologia , Endotélio Vascular/virologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Ionomicina/farmacologia , Maleimidas/farmacologia , Microscopia Eletrônica , Fatores de Transcrição NFATC , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Processamento de Proteína Pós-Traducional , Estaurosporina/farmacologia , Tacrolimo/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Transfecção , Proteínas Virais/biossínteseRESUMO
Langerhans cells (LC) represent dendritic cells (DC) within mucosal epithelium that are purported initial targets for HIV following sexual exposure to virus. Here, morphologic, phenotypic, functional and HIV infection experiments were performed using monocyte-derived DC cultured in the presence of GM-CSF, IL-4 and TGF-beta1 (G4T-DC), GM-CSF and IL-4 (G4-DC), and G4T-DC incubated for an additional 3 days with CD40 ligand (CD40L-DC). G4T-DC, which demonstrated characteristics of immature LC, could be productively infected by either R5- or X4-HIV strains. Infection levels, however, were markedly lower than those observed in immature G4-DC. Surprisingly, CD40L-DC, which demonstrated features of mature LC, could be productively infected with HIV at higher levels than immature G4T-DC. Productive HIV infection in these three DC populations correlated positively with cell surface expression of CD4, CCR5 and CXCR4. We suggest that low levels of HIV infection in LC-like G4T-DC indicate an inefficient mechanism by which HIV can initially infect individuals, perhaps explaining the relative difficulty in becoming infected during sexual exposure to virus. In addition, enhanced HIV infection in LC-like G4T-DC following CD40L treatment suggests a mechanism by which inflammatory CD40L(+) T cells, if present in mucosal tissue, could lead to increased HIV transmission rates.
Assuntos
Ligante de CD40/fisiologia , Células Dendríticas/virologia , HIV/fisiologia , Células de Langerhans/virologia , Fator de Crescimento Transformador beta/farmacologia , Replicação Viral , Adulto , Antígenos CD4/análise , Diferenciação Celular , Células Dendríticas/fisiologia , Endocitose , Humanos , Isoantígenos/imunologia , Células de Langerhans/imunologia , Células de Langerhans/ultraestrutura , Ativação Linfocitária , PinocitoseRESUMO
Macrophages play a key role in several critical aspects of HIV disease. They appear to be the first cells infected by HIV and perhaps the very source of HIV production when CD4+ cells are markedly depleted in the patient. Macrophages and microglial cells are the cells infected by HIV in the CNS. In tonsils and adenoids of HIV-infected patients, macrophages fuse into multinucleated giant cells that produce copious amounts of virus. Finally, opportunistic pathogens can cause an upregulation of HIV production by macrophages, often in the multinucleated form.
Assuntos
Infecções por HIV/virologia , HIV/imunologia , Macrófagos/virologia , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Tonsila Faríngea/imunologia , Tonsila Faríngea/virologia , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Células Gigantes/virologia , HIV/genética , HIV/crescimento & desenvolvimento , Infecções por HIV/imunologia , Humanos , Macaca , Macrófagos/imunologia , Tonsila Palatina/imunologia , Tonsila Palatina/virologia , Vírus da Imunodeficiência Símia/imunologia , Regulação para Cima , Replicação ViralRESUMO
Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1(Ba-L) infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08-4.77%). HIV-1-infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1-1 microg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1(Ba-L) (an R5 HIV-1 strain) more efficiently infected LC-T cell cocultures when compared with HIV-1(IIIB) (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1.
Assuntos
Fármacos Anti-HIV/farmacologia , Quimiocina CCL5/análogos & derivados , Células Epiteliais/virologia , Infecções por HIV/transmissão , HIV-1 , Células de Langerhans/virologia , Linfócitos T CD4-Positivos/virologia , Movimento Celular , Quimiocina CCL5/farmacologia , Técnicas de Cocultura , HumanosRESUMO
This study elucidates the morphology of HHV8 replication in human dermal endothelial cells and primary effusion lymphomas (PEL) and compares it to that seen in Kaposi sarcoma. Primary human dermal microvascular endothelial cells (DMVEC) exposed to the cell-filtered supernatant of the PEL JSC1 and PEL cell lines (KS-1, BCBL-1, BC-1, BC-3) were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or butyrate. Cells were fixed in neutral-buffered glutaraldehyde, gelled in cooled agar, and processed for TEM. There was a quantitative, but not a qualitative difference in viral expression associated with no treatment or exposure to TPA or butyrate of H HV8 in DMVEC and PEL. Two types of viral-induced intranuclear inclusions (INI) were visible at the light and ultrastructural levels. The more common INI had lighter staining material filling the nucleus, except for a rim of dense chromatin, and could be seen even before viral nucleocapsids (NC) were visible. The second type of INI resembled a target formed by condensation of electron-dense material surrounded by a lighter halo and marginated heterochromatin and containing NC. Collections of coalescing electron-dense granules resembling starbursts were often present in nuclei containing either type of INI. Next to appear in productively infected cells were mature enveloped particles that formed mostly by the budding of NC into cytoplasmic vacuoles. Mature particles were also seen free on the plasma membrane. Tufts of electron-dense intermediate filaments were associated with maturing particles. Mature virions lacked an electron-dense tegument. Viral production was ultimately associated with cell lysis. It appears that HHV8 propagate in DMVEC, with and without stimulation, and have a similar morphogenesis to that seen in PEL cell lines and Kaposi sarcoma lesions. Several unique features characterize cells productively infected by HHV8.
Assuntos
Endotélio Vascular/virologia , Herpesvirus Humano 8/crescimento & desenvolvimento , Linfoma Relacionado a AIDS/virologia , Derrame Pleural Maligno/virologia , Sarcoma de Kaposi/virologia , Pele/irrigação sanguínea , Butiratos/farmacologia , Endotélio Vascular/ultraestrutura , Herpesvirus Humano 8/efeitos dos fármacos , Herpesvirus Humano 8/ultraestrutura , Humanos , Linfoma Relacionado a AIDS/ultraestrutura , Microscopia Eletrônica , Morfogênese , Derrame Pleural Maligno/patologia , Sarcoma de Kaposi/ultraestrutura , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/ultraestrutura , Células Tumorais Cultivadas/virologia , Replicação ViralRESUMO
OBJECTIVE: To compare the architecture and HIV-1 RNA and Gag p24 protein expression in lymph nodes (LN) excised from individuals during chronic highly active antiretroviral therapy (HAART) with LN removed from the same patient after plasma virus rebound following the interruption of HAART. MATERIALS AND METHODS: Six HIV-1-infected patients on HAART, with CD4 cell counts greater than 350 cells/microl, and plasma HIV-1 RNA less than 50 copies/ml, underwent inguinal LN excision upon discontinuation of HAART, and again after rebound of plasma virus. Lymph nodes were evaluated by immunohistochemical staining for Gag p24 antigen and Ki67, in-situ hybridization for HIV-1 RNA and H3-histone, and transmission electron microscopy (TEM). RESULTS: LN at baseline were quiescent to mildly hyperplastic and generally contained more primary than secondary follicles. Only one LN had detectable follicular dendritic cell (FDC)-associated p24 antigen, none had HIV RNA. Few mononuclear cells (MNC) expressed RNA or p24 antigen. Plasma virus at the second biopsy ranged from 329 to 3.2 x 10(6) copies/ml. CD4 cell count decline ranged from 5 to 51% during drug hiatus, and was greatest in patients with highest viral rebound. Four of six of the second LN were more hyperplastic than the initial LN, two showed paracortical hyperplasia. MNC expression of HIV RNA in the second LN paralleled the level of plasma viremia. Increased Ki67 and H3-histone signal occurred in the second LN. CONCLUSION: Quiescent LN from individuals on HAART rapidly become hyperplastic and activated within 1-2 months after treatment interruption. As in acute HIV infection, virus expression by LN MNC parallels the rebound in plasma viremia and fall in CD4 cell count.
Assuntos
Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Linfonodos/virologia , Contagem de Linfócito CD4 , Proteína do Núcleo p24 do HIV/análise , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Hibridização In Situ , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Microscopia Eletrônica , RNA Viral/análise , RNA Viral/sangue , Carga Viral , Viremia/virologiaRESUMO
Immunodeficiency, the consequence of HIV-1 infection, predisposes the host to opportunistic infections. In turn, opportunistic pathogens influence target cell susceptibility to HIV-1 infection and replication. Although the advent of highly active antiretroviral therapy (HAART) has altered these sequelae, co-infections may prevail in some parts of the world and in failed HAART regimens. Moreover, immune activation as occurs in tonsil and non-infectious mucosal inflammatory lesions may also be associated with proximal sites of viral replication. These connections between enhancement of HIV-1 infection and activation/inflammation warrant further elucidation of the factors promoting permissiveness to HIV-1 infection. Using the opportunistic pathogen Mycobacterium avium as an in vitro model, we demonstrated that co-infection facilitated HIV-1 infection of monocyte-macrophages by multiple pathways. M. avium activated NF-kappaB, the downstream consequences of which included augmented expression of tumor necrosis factor alpha and CCR5 receptors, both permissive for sustaining HIV-1 infection. Pronounced viral replication in lymph nodes co-infected with M. avium and HIV-1 paralleled these in vitro findings. Furthermore, reduction in viral burden is associated with treatment of infected or inflamed tissues, underscoring the link between immune activation and viral replication.
Assuntos
Infecções por HIV/imunologia , HIV-1/fisiologia , Macrófagos/virologia , Infecções Oportunistas Relacionadas com a AIDS/complicações , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Complexo Mycobacterium avium , Infecção por Mycobacterium avium-intracellulare/complicações , Infecção por Mycobacterium avium-intracellulare/imunologia , Infecção por Mycobacterium avium-intracellulare/virologia , Replicação ViralRESUMO
Lymphoid tissue was examined to see whether in vivo cytopathic effects of human immunodeficiency virus (HIV) infection on lymphocytes could be detected. Transmission electron microscopy of mechanical suspensions prepared from lymph nodes showed both replication and phagocytosis of HIV particles by macrophages. Phagosomes contained cellular debris and virions, some of which were undergoing digestion. Neutrophils also contained HIV particles intermixed with cellular debris in phagosomes. Immunohistochemistry revealed whole Gag p24-positive lymphocytes and p24-positive cellular debris within the cytoplasm of paracortical macrophages. Lysing p24-positive lymphocytes were also seen. In the paracortex, p24-positive multinucleated lymphocytes with up to 5 nuclei were seen. In situ hybridization for HIV RNA in combination with immunohistochemistry for HAM56, a macrophage-specific marker, revealed colabeled cells. Thus, HIV-positive lymphocytes undergo lysis in lymphoid tissue. The cellular debris is phagocytized by macrophages, which themselves can replicate HIV. HIV-positive lymphocytes fuse in lymph nodes to form multinucleated cells.
Assuntos
Infecções por HIV/patologia , HIV-1/metabolismo , Linfócitos/virologia , Tecido Linfoide/virologia , Anticorpos Monoclonais/análise , Fusão Celular , Proteína do Núcleo p24 do HIV/biossíntese , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/genética , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Tecido Linfoide/imunologia , Tecido Linfoide/patologia , Macrófagos/fisiologia , Macrófagos/ultraestrutura , Macrófagos/virologia , Microscopia Eletrônica , Neutrófilos/metabolismo , Neutrófilos/virologia , Fagocitose , RNA Viral/análiseRESUMO
Although efavirenz-containing regimens effectively suppress plasma levels of human immunodeficiency virus (HIV) RNA, it is now clear that undetectable plasma viremia may not reflect a lack of viral replication. Because lymphoid tissue is an active site of HIV replication, the lymph node virus burden was analyzed in persons who received highly active antiretroviral therapy (HAART) containing either efavirenz or a protease inhibitor (PI). Testing with in situ hybridization revealed no detectable follicular dendritic cell-associated HIV RNA in either group, and only 2 of 8 persons in the efavirenz group and 1 of 4 in the PI group had detectable RNA in lymph node mononuclear cells (LNMC) when tested by use of nucleic acid sequencebased amplification. Low levels of replication-competent HIV were identified in both groups by use of quantitative coculture assays. There was no evidence of development of resistance to either regimen in virus isolated from LNMC. These data support the use of efavirenz as an alternative to a PI in initial HAART regimens.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/virologia , Linfonodos/virologia , Oxazinas/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Alcinos , Benzoxazinas , Técnicas de Cocultura , Estudos Transversais , Ciclopropanos , Genótipo , HIV/genética , HIV/fisiologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Humanos , Hibridização In Situ , Linfonodos/patologia , Inibidores de Proteases/uso terapêutico , RNA Viral/análise , Carga Viral , Replicação ViralRESUMO
Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory tract tissue, and are thought to be human lung carcinogens. Because Cr(VI) is mutagenic and carcinogenic at doses that evoke cell toxicity, the objective of these experiments was to examine the effect of Cr(VI) on the growth, survival, and mode of cell death in normal human lung fibroblasts (HLF cells). DNA adduct formation was monitored as a marker for bioavailability of genotoxic chromium. We also examined the modulation of these endpoints by vitamins C and E. Long-term Cr(VI) exposures were employed, which decreased clonogenic cell survival by 25% to 95% in a dose-dependent manner. The predominant cellular response to Cr(VI) was growth arrest. We found that Cr(VI) caused up to 20% of HLF cells to undergo apoptosis, and documented apoptotic morphology and the phagocytosis of apoptotic bodies by neighboring cells. P53 levels increased 4- to 6-fold in chromium-treated cells. In contrast with previous studies using CHO cells, the present study using HLFs found that pretreatment with either vitamin C or E did not exhibit a significant effect on Cr-induced apoptosis or clonogenic survival. In addition, pretreatment with vitamin C did not affect the p53 induction observed after chromium treatment. Neither vitamin had any effect on Cr-DNA adduct formation. These data indicate that although pretreatment with vitamin C or E alters the spectrum of cellular and/or genetic lesions induced by chromium(VI), neither vitamin altered the initiation or progression of apoptosis in diploid human lung cells.