RESUMO
High-grade cervical dysplasia caused by human papillomavirus (HPV) type 16 is a lesion that should be susceptible to an HPV-specific immune response; disease initiation and persistence is predicated on expression of two viral Ags, E6 and E7. In immune-competent subjects, at least 25% of HPV16(+) high-grade cervical dysplasia lesions undergo complete regression. However, in the peripheral blood, naturally occurring IFN-γ T cell responses to HPV E6 and E7 are weak, requiring ex vivo sensitization to detect, and are not sufficiently sensitive to predict regression. In this study, we present immunologic data directly assessing cervical lymphocytes from this cohort. We found that nearly all cervical tissue T cells express the mucosal homing receptor, α(4)ß(7) surface integrin. T cells isolated from dysplastic mucosa were skewed toward a central memory phenotype compared with normal mucosal resident T cells, and dysplastic lesions expressed transcripts for CCL19 and CCL21, raising the possibility that the tissue itself sustains a response that is not detectable in the blood. Moreover, lesion regression in the study window could retrospectively be predicted at study entry by the ability of CD8(+) T cells to gain access to lesional epithelium. Vascular endothelial expression of mucosal addressin cell adhesion molecule-1, the ligand that supports entry of α(4)ß(7)(+) T cells into tissues, colocalized tightly with the distribution of CD8 T cells and was not expressed in persistent dysplastic epithelium. These findings suggest that dysregulated expression of vascular adhesion molecules plays a role in immune evasion very early in the course of HPV disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Epiteliais/imunologia , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/imunologia , Displasia do Colo do Útero/imunologia , Neoplasias Vulvares/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Movimento Celular/imunologia , Estudos de Coortes , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Humanos , Integrina alfa4/biossíntese , Cadeias beta de Integrinas/biossíntese , Proteínas Oncogênicas Virais/biossíntese , Proteínas E7 de Papillomavirus/biossíntese , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Estudos Prospectivos , Proteínas Repressoras/biossíntese , Estudos Retrospectivos , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologia , Neoplasias Vulvares/patologia , Neoplasias Vulvares/virologiaRESUMO
Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.
Assuntos
Anaplasma phagocytophilum/fisiologia , Ehrlichiose/genética , Ehrlichiose/imunologia , Inativação Gênica/imunologia , Histona Desacetilases/genética , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Interpretação Estatística de Dados , Ehrlichiose/metabolismo , Ehrlichiose/microbiologia , Regulação da Expressão Gênica , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Patients with human granulocytic anaplasmosis present with fever, thrombocytopenia, leukopenia, and an elevated aspartate transaminase level. Clinical and histopathologic features of severe disease suggest macrophage activation. Twenty-nine patients with human granulocytic anaplasmosis had higher ferritin, interleukin-10, interleukin-12 p70, and interferon- gamma levels than did control subjects matched for age and sex; severity correlated with triglyceride, ferritin, and interleukin-12 p70 levels. Several severely affected patients had cases that fulfilled macrophage activation syndrome diagnostic criteria. Macrophage activation and excessive cytokine production may belie tissue injury associated with Ananplasma phagocytophilum infection.
Assuntos
Anaplasma phagocytophilum/isolamento & purificação , Anaplasmose/sangue , Anaplasmose/diagnóstico , Biomarcadores/sangue , Ativação de Macrófagos/fisiologia , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Anaplasmose/epidemiologia , Estudos de Casos e Controles , Citocinas/análise , DNA Bacteriano/análise , Feminino , Ferritinas/análise , Seguimentos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Probabilidade , Valores de Referência , Medição de Risco , Índice de Gravidade de Doença , Distribuição por Sexo , Estatísticas não Paramétricas , Triglicerídeos/análiseRESUMO
Msp2 is Anaplasma phagocytophilum's immunodominant protein. Antigenic variability with msp2 gene conversion may drive differential immunopathology with infection by bacteria of different in vitro passage intervals. We examined msp2 transcript variation and its relationship to histopathology, T-cell and antibody responses in mice infected with differentially passaged A. phagocytophilum. Hepatic inflammation peaked on day 2-4 with low passage bacteria and on day 4-7 with high passage bacteria infection. Nineteen msp2 variant transcripts were identified. The low and high passage inocula shared four, but differed in one and two msp2 transcript variants, respectively. After infection, three and two msp2 variants were only identified in low or high passage infected mice. However, per mouse, msp2 variant profiles were unique with no evident expression program. In low and high passage bacteria-infected mice, splenocytes proliferated to whole A. phagocytophilum at day 7-10, diminishing thereafter. Weak mitogenic responses to whole bacteria were detected in mock and infected mice at d0 and sporadically thereafter. Essentially no lymphoproliferation or IFN-gamma production resulted from stimulation by six Msp2 hypervariable region proteins, although antibodies were detected to all, including cross-reactions. Differential A. phagocytophilum Msp2 expression is unrelated to T-cell response and unlikely to induce the cellular immunopathology underlying disease manifestations.
Assuntos
Anaplasma phagocytophilum/imunologia , Variação Antigênica , Proteínas da Membrana Bacteriana Externa/imunologia , Ehrlichiose/microbiologia , Interferon gama/biossíntese , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/genética , Proliferação de Células , Modelos Animais de Doenças , Ehrlichiose/imunologia , Ehrlichiose/patologia , Histocitoquímica , Fígado/microbiologia , Fígado/patologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sequência de DNA , Baço/imunologia , Transcrição GênicaRESUMO
A pilot study was conducted to determine whether existing human or canine strains of Anaplasma phagocytophilum would reproduce clinical disease in experimentally inoculated dogs similar to dogs with naturally acquired granulocytic anaplasmosis. Six hounds were inoculated intravenously with one human and two canine strains of A. phagocytophilum that were propagated in vitro in HL-60 cells or in infected autologous neutrophils. Infected dogs were monitored for lethargy, anorexia, petechiae, lymphadenopathy, and fever. Dogs were assessed for complete blood count (CBC), serum chemistry, and serology (IFA and SNAP® 4Dx®); for A. phagocytophilum blood load by quantitative polymerase chain reaction; and for cytokine production. Prominent clinical signs were generalized lymphadenopathy and scleral injection; only one dog developed fever lasting 4 days. Notable laboratory alterations included sustained leukopenia and thrombocytopenia in all dogs. A. phagocytophilum morulae were noted in blood between days 10 and 11, although all dogs retained A. phagocytophilum DNA in blood through day 60. All dogs seroconverted by days 10-15 by IFA, and by days 17-30 by SNAP 4Dx; cytokine analyses revealed 10-fold increases in interleukin-2 and interleukin-18 in the neutrophil-propagated 98E4 strain-infected dog. All A. phagocytophilum strains produced infection, although canine 98E4 strain reproduced clinical signs, hematologic changes, and inflammatory cytokine elevations most consistent with granulocytic anaplasmosis when recognized clinically. Therefore, this strain should be considered for use in future studies of A. phagocytophilum canine infection models.
Assuntos
Anaplasma phagocytophilum/imunologia , Anaplasmose/microbiologia , Anticorpos Antibacterianos/sangue , Granulócitos/microbiologia , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/patogenicidade , Anaplasmose/imunologia , Animais , Carga Bacteriana , Sistema Livre de Células , Citocinas/sangue , DNA Bacteriano/sangue , Modelos Animais de Doenças , Cães , Células HL-60 , Humanos , Injeções Intravenosas , Doenças Linfáticas/microbiologia , Neutrófilos/microbiologia , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos EspecíficosRESUMO
Dogs are susceptible to different tickborne infections, including members of the Anaplasmataceae (Ehrlichia canis, E. ewingii, E. chaffeensis, Anaplasma phagocytophilum, A. platys), Borrelia burgdorferi, and Rickettsia rickettsii. These diseases can manifest with clinical signs including fever, anorexia, malaise, lameness, rash, and bleeding episodes; however, these signs are nonpathognomonic, and infections can occur in the absence of clinical signs. Hematologic abnormalities can include leukopenia, thrombocytopenia, hyperproteinemia and hypergammaglobulinemia. In biomedical research, diseases such as canine monocytic ehrlichiosis, Lyme disease, and Rocky Mountain spotted fever may cause morbidity among exposed dogs and confound research results. Random-source dogs are susceptible to these diseases because of their increased risk of arthropod exposure. Nonpurpose bred, randomly selected conditioned dogs (n = 21) were examined; blood samples were taken for hematology, biochemistry analysis, tickborne pathogen serology, and PCR. Of these, 2 dogs (10% of the population) presented with illness characterized by fever, malaise, lameness, or hemostatic abnormalities, and 15 (71%) had antibodies to one or more tickborne pathogens. No specific hematologic or biochemical differences were apparent between seronegative dogs and seropositive dogs reactive to all 3 pathogens. E. canis and B. burgdorferi PCR of tissues and blood were negative for all dogs. PCR amplification of several Ehrlichia and Anaplasma genes yielded no positive samples. From this cohort of dogs, serologic and molecular results indicate prior exposure without active infection or clinical disease. Exposure to and potential for infection with these bacteria and other pathogens may contribute to blood and tissue alterations that could confound experiments and lead to misinterpretation of data in canine models.
Assuntos
Anticorpos Antibacterianos/sangue , Borrelia burgdorferi/imunologia , Doenças do Cão/imunologia , Ehrlichia canis/imunologia , Rickettsia rickettsii/imunologia , Testes Sorológicos/veterinária , Animais , Cães , Ehrlichiose/imunologia , Ehrlichiose/veterinária , Doença de Lyme/imunologia , Doença de Lyme/veterinária , Estudos Retrospectivos , Febre Maculosa das Montanhas Rochosas/imunologia , Febre Maculosa das Montanhas Rochosas/veterináriaRESUMO
Borrelia burgdorferi and Anaplasma phagocytophilum coinfect and are transmitted by Ixodes species ticks. Clinical indicators suggest that A. phagocytophilum coinfection contributes to the severity, dissemination, and, possibly, sequelae of Lyme disease. Previous in vitro studies showed that spirochete penetration through human brain microvascular endothelial cells of the blood-brain barrier is facilitated by endothelial cell-derived matrix metalloproteases (MMPs). A. phagocytophilum-infected neutrophils continuously release MMPs and other vasoactive biomediators. We examined B. burgdorferi infection of brain microvascular barriers during A. phagocytophilum coinfection and showed that coinfection enhanced reductions in transendothelial electrical resistance and enhanced or synergistically increased production of MMPs (MMP-1, -3, -7, -8, and -9), cytokines (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), and chemokines (IL-8 and macrophage inflammatory protein 1alpha) known to affect vascular permeability and inflammatory responses.