RESUMO
Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer's patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer's patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer's patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer's patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunoglobulina A/farmacologia , Imunoglobulina G/farmacologia , Salmonella/efeitos dos fármacos , Administração Oral , África , Animais , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Hibridomas , Imunização Passiva , Imunoglobulina A Secretora , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados , Salmonella typhimurium/efeitos dos fármacosRESUMO
Increasing evidence supports early brain infection by human immunodeficiency virus (HIV). Definitive temporal studies determining when and within which brain cells viral DNA is present are lacking. This study utilized simian immunodeficiency virus (SIV)-infected macaques sacrificed at days 10, 21, 56, and 84 post inoculation. Laser-microdissection isolated pure perivascular macrophage, parenchymal microglia, and astrocyte populations. Nested polymerase chain reaction (PCR) and sequencing determined the presence and characteristics of SIV V3 and V1 env DNA from each population. At day 10, SIV DNA was detected in perivascular macrophage and astrocytes but not parenchymal microglia. gp41 expression was restricted to perivascular macrophage. At day 21, SIV DNA was not detected in any cell type. At day 56, SIV DNA was detectable in perivascular macrophage from one of two macaques, with no gp41 expression detected. At day 84 (morphologic and clinical encephalitis), SIV DNA was detected in all cell types, gp41 was only detected in perivascular macrophage and parenchymal microglia. The neurovirulent molecular clone, SIV/17E-Fr, was the only genotype identified in the brain cell populations. Early, productive brain SIV infection was transient and restricted to trafficking perivascular macrophage. During the nonencephalitic stage, there was a period of time when no SIV DNA could be detected in the brain cell populations. SIV was then seen to reenter the brain via infected perivascular macrophage, leading to productive infection of brain parenchymal macrophage/microglia with a terminal phase of encephalitis. These data challenge current notions of a HIV reservoir within latently infected, semipermanent brain cells and has significant implications for the timing and design of therapies to prevent HIV encephalitis (HIVE).
Assuntos
Encéfalo/virologia , Modelos Animais de Doenças , Encefalite Viral/virologia , Produtos do Gene env , Macaca nemestrina/virologia , Vírus da Imunodeficiência Símia/genética , Replicação Viral , Animais , Astrócitos/virologia , Encéfalo/patologia , Células Cultivadas , Encefalite Viral/patologia , HIV/fisiologia , Humanos , Macrófagos/virologia , Microglia/virologia , Especificidade de Órgãos , Fatores de TempoRESUMO
While it is well known that Staphylococcus aureus establishes chronic implant-associated osteomyelitis by generating and persisting in biofilm, research to elucidate pathogen, and host specific factors controlling this process has been limited due to the absence of a quantitative in vivo model. To address this, we developed a murine tibia implant model with ex vivo region of interest (ROI) imaging analysis by scanning electron microscopy (SEM). Implants were coated with Staphylococcus aureus strains (SH1000, UAMS-1, USA300LAC) with distinct in vitro biofilm phenotypes, were used to infect C57BL/6 or Balb/c mice. In contrast to their in vitro biofilm phenotype, results from all bacteria strains in vivo were similar, and demonstrated that biofilm on the implant is established within the first day, followed by a robust proliferation phase peaking on Day 3 in Balb/c mice, and persisting until Day 7 in C57BL/6 mice, as detected by SEM and bioluminescent imaging. Biofilm formation peaked at Day 14, covering â¼40% of the ROI coincident with massive agr-dependent bacterial emigration, as evidenced by large numbers of empty lacunae with few residual bacteria, which were largely culture negative (80%) and PCR positive (87.5%), supporting the clinical relevance of this implant model.
Assuntos
Biofilmes , Materiais Revestidos Biocompatíveis/química , Osteomielite/microbiologia , Próteses e Implantes/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/prevenção & controle , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Feminino , Teste de Materiais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Fenótipo , Reação em Cadeia da Polimerase , Especificidade da Espécie , Células-Tronco , Tíbia/efeitos dos fármacosRESUMO
Towards the development of a methicillin-resistant Staphylococcus aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus a placebo and a Gmd-deficient isogenic strain (ΔGmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis, and histomorphometry of abscess numbers (p < 0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies ΔGmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in ΔGmd cultures or 1C11 treated cultures of a protein A-deficient strain (ΔSpa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p < 0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of Staphylococcal abscess communities (SAC) and opsonophagocytosis of megaclusters. .
Assuntos
Anticorpos Monoclonais/administração & dosagem , Hexosaminidases/imunologia , Osteomielite/prevenção & controle , Fagocitose/imunologia , Infecções Relacionadas à Prótese/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Imunização Passiva , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Opsonizantes/toxicidade , Osteomielite/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/microbiologiaRESUMO
BACKGROUND: Staphylococcus aureus infections remain a major complication of orthopaedic surgery. Although serum C-reactive protein is useful for diagnosis, there are no specific tests for host immunity that can assess a patient's risk for serious infection. On the basis of the identification of glucosaminidase as a potentially protective antigen in animal models, we tested the hypotheses that anti-glucosaminidase IgG (immunoglobulin G) levels vary in sera of mice and orthopaedic patients with Staphylococcus aureus infections and that physical and neutralizing titers correlate. METHODS: In vitro ELISAs (enzyme-linked immunosorbent assays) were developed to quantify binding (physical) and enzyme-neutralizing (functional) anti-glucosaminidase IgG titers. The assays were validated with use of sera from naive, Staphylococcus aureus-challenged, and glucosaminidase-immunized mice. The physical, functional, and isotype titers of anti-glucosaminidase IgG were measured in sera from twenty-four patients with a confirmed Staphylococcus aureus infection following orthopaedic surgery and in sera from twenty noninfected patients. The specificity of the anti-glucosaminidase assay was evaluated by means of linear regression and receiver-operator characteristic curve analysis. RESULTS: In mice, the analytic range of the physical titer assay for anti-glucosaminidase IgG was determined to be 1 ng/mL to 1 µg/mL, and physical titers correlated with functional titers (p < 0.002). Although all patients had measurable anti-glucosaminidase IgG, the physical titers in the infected patients were significantly higher by a factor of two compared with those in the healthy controls (p = 0.015). The physical titers were significantly correlated with the functional titers (p < 0.0001). Receiver-operator characteristic curve analysis demonstrated a diagnostic specificity of 0.72 (p = 0.014) for the assay. The anti-glucosaminidase titer in almost every patient was dominated by the IgG1 isotype. CONCLUSIONS: Humoral immunity against glucosaminidase varied in mammals with Staphylococcus aureus osteomyelitis. Anti-glucosaminidase titers in sera were a potential biomarker of infection and have the potential to assess the quality of host immunity against Staphylococcus aureus. CLINICAL RELEVANCE: Staphylococcus aureus infections can be challenging to diagnose, and there is no diagnostic test for host immunity. We demonstrated a cost-effective assay for determining the anti-glucosaminidase titer, which can be readily combined with conventional serology to improve diagnosis and to assess host immunity against Staphylococcus aureus.
Assuntos
Hexosaminidases/antagonistas & inibidores , Hexosaminidases/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus , Animais , Artroplastia de Substituição , Biomarcadores/sangue , Feminino , Fraturas Ósseas/complicações , Fraturas Ósseas/microbiologia , Fraturas Ósseas/cirurgia , Humanos , Artropatias/complicações , Artropatias/microbiologia , Artropatias/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/complicaçõesRESUMO
Recently, methicillin-resistant Staphylococcus aureus (MRSA) has surpassed HIV as the most deadly pathogen in the United States, accounting for over 100,000 deaths per year. In orthopedics, MRSA osteomyelitis has become the greatest concern in patient care, despite the fact that improvements in surgical technique and aggressive antibiotic prophylaxis have decreased the infection rate for most procedures to less than 5%. This great concern is largely due to the very poor outcomes associated with MRSA osteomyelitis, which includes 30-50% failure rates for revision surgery. Thus, there is a need to develop additional therapeutic interventions such as passive immunization, particularly for immunocompromised patients and the elderly who are typically poor responders to active vaccines. Using a novel murine model of implant-associated osteomyelitis in which a stainless steel pin is coated with bioluminescent S. aureus and implanted transcortically through the tibial metaphysis, we discovered that mice protect themselves from this infection by mounting a specific IgG2b response against the peptidoglycan hydrolase, glucosaminidase (Gmd), an enzyme involved in cell wall digestion during binary fission. Since this subunit of S. aureus autolysin is essential for bacterial growth, and no genetic variation has been identified among clinical strains, we propose that monoclonal antibodies against this enzyme would have multiple mechanisms of action, including promotion of opsonophagocytosis and direct inhibition of enzyme function. Here we review the field of MRSA osteomyelitis and our research to date on the development of an anti-Gmd passive immunotherapy.
RESUMO
Despite the ability of combination antiretroviral treatment (cART) to reduce viral burden to nearly undetectable levels in cerebrospinal fluid and serum, HIV-1 associated neurocognitive disorders (HAND) continue to persist in as many as half the patients living with this disease. There is growing consensus that the actual substrate for HAND is destruction of normal synaptic architecture but the sequence of cellular events that leads to this outcome has never been resolved. To address whether central vs. peripheral myeloid lineage cells contribute to synaptic damage during acute neuroinflammation we injected a single dose of the HIV-1 transactivator of transcription protein (Tat) or control vehicle into hippocampus of wild-type or chimeric C57Bl/6 mice genetically marked to distinguish infiltrating and resident immune cells. Between 8-24 hr after injection of Tat, invading CD11b(+) and/or myeloperoxidase-positive leukocytes with granulocyte characteristics were found to engulf both microglia and synaptic structures, and microglia reciprocally engulfed invading leukocytes. By 24 hr, microglial processes were also seen ensheathing dendrites, followed by inclusion of synaptic elements in microglia 7 d after Tat injection, with a durable microgliosis lasting at least 28 d. Thus, central nervous system (CNS) exposure to Tat induces early activation of peripheral myeloid lineage cells with phagocytosis of synaptic elements and reciprocal microglial engulfment of peripheral leukocytes, and enduring microgliosis. Our data suggest that a single exposure to a foreign antigen such as HIV-1 Tat can lead to long-lasting disruption of normal neuroimmune homeostasis with deleterious consequences for synaptic architecture, and further suggest a possible mechanism for enduring neuroinflammation in the absence of productive viral replication in the CNS.
Assuntos
Comunicação Celular/efeitos dos fármacos , HIV-1/fisiologia , Microglia/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Animais , Receptor 1 de Quimiocina CX3C , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Granulócitos/virologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/virologia , Injeções , Camundongos , Microglia/imunologia , Microglia/patologia , Microglia/virologia , Células Mieloides/citologia , Células Mieloides/imunologia , Células Mieloides/virologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Receptores de Quimiocinas/genética , Sinapses/imunologia , Sinapses/patologia , Sinapses/virologia , Fatores de TempoRESUMO
Central nervous system (CNS) invasion during acute-stage HIV-infection has been demonstrated in a small number of individuals, but there is no evidence of neurological impairment at this stage and virus infection in brain appears to be controlled until late-stage disease. Using our reproducible SIV macaque model to examine the earliest stages of infection in the CNS, we identified immune responses that differentially regulate inflammation and virus replication in the brain compared to the peripheral blood and lymphoid tissues. SIV replication in brain macrophages and in brain of SIV-infected macaques was detected at 4 days post-inoculation (p.i.). This was accompanied by upregulation of innate immune responses, including IFNbeta, IFNbeta-induced gene MxA mRNA, and TNFalpha. Additionally, IL-10, the chemokine CCL2, and activation markers in macrophages, endothelial cells, and astrocytes were all increased in the brain at four days p.i. We observed synchronous control of virus replication, cytokine mRNA levels and inflammatory markers (MHC Class II, CD68 and GFAP) by 14 days p.i.; however, control failure was followed by development of CNS lesions in the brain. SIV infection was accompanied by induction of the dominant-negative isoform of C/EBPbeta, which regulates SIV, CCL2, and IL6 transcription, as well as inflammatory responses in macrophages and astrocytes. This synchronous response in the CNS is in part due to the effect of the C/EBPbeta on virus replication and cytokine expression in macrophage-lineage cells in contrast to CD4+ lymphocytes in peripheral blood and lymphoid tissues. Thus, we have identified a crucial period in the brain when virus replication and inflammation are controlled. As in HIV-infected individuals, though, this control is not sustained in the brain. Our results suggest that intervention with antiretroviral drugs or anti-inflammatory therapeutics with CNS penetration would sustain early control. These studies further suggest that interventions should target HIV-infected individuals with increased CCL2 levels or HIV RNA in the CNS.