Orientadores: Raquel Franco Leal, Maria Lourdes Setsuko Ayrizono / Dissertação (Mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T16:08:44Z (GMT). No. of bitstreams: 1 Paiva_NielceMariade_M.pdf: 3729371 bytes, checksum: 4077b1681124ea203fae5904b5fd8254 (MD5) Previous issue date: 2011 / Resumo: A retocolectomia total com anastomose do reservatório ileal (RI) ao canal anal é a cirurgia de escolha para doentes com retocolite ulcerativa inespecífica (RCUI) refratária ao tratamento clínico e para a polipose adenomatosa familiar (PAF). Entretanto, a ileíte primária do reservatório é uma das complicações mais comuns após a cirurgia do RI em pacientes com RCUI, sendo rara na PAF; e somente ocorre após o fechamento da ileostomia de proteção. Neste sentido, há necessidade de estudos que avaliem a forma como as bactérias, por meio de receptores específicos, possam participar no processo inflamatório do RI. Desta forma, foi analisada a expressão dos Toll-like receptors (TLR) em mucosa do RI endoscópica e histologicamente normal em pacientes operados por RCUI e PAF, a fim de se detectar alguma anormalidade nesta via em indivíduos assintomáticos, que poderia contribuir com processo inflamatório no RI. Casuística e Método: Doze pacientes (seis com RCUI e seis com PAF) submetidos à retocolectomia total e confecção do RI em "J", foram estudados após a reconstrução do trânsito intestinal. Foram obtidas biópsias da mucosa do RI por meio de endoscopia do mesmo. O grupo controle foi constituído por seis doentes com íleo-colonoscopia normal. As biópsias foram congeladas em nitrogênio líquido e as expressões de TLR-2, TLR-4 (receptores de reconhecimento de antígenos bacterianos) e p- JNK (fator de sinalização nuclear) foram avaliadas por meio de imunoblot de extrato protéico total. A ausência de ileíte do RI foi determinada por parâmetros clínicos, histológicos e endoscópicos, de acordo com o Índice de Atividade da Ileíte do RI (PDAI). Os pacientes não estavam em uso de medicações. O presente estudo foi aprovado pelo Comitê de Ética em Pesquisa Local e os participantes assinaram o termo de consentimento informado. Utilizou-se análise de variância (ANOVA), seguida por análise de significância (Teste de Tukey-Kramer) para a análise estatística. Nível de significância adotado foi p<0,05. Resultados: Houve maior expressão de TLR-4 em mucosa de RI de doentes operados por RCUI, quando comparada aos grupos Controle e PAF (p<0,05). Não houve diferença estatística das expressões de TLR-2 e p-JNK entre os diferentes grupos (p>0.05). Conclusão: Pacientes com RCUI apresentaram maior expressão de TLR4 na mucosa do RI, mesmo sem evidência clínica, endoscópica e histológica de inflamação no RI. Estes achados podem explicar a tendência de ativação de vias intracelulares deflagradas por antígenos bacterianos em pacientes com RCUI, o que pode contribuir com a produção de mediadores pró-inflamatórios, sendo coadjuvante do processo inflamatório inicial na mucosa do RI / Abstract: Introduction: Ileal pouch-anal anastomosis (IPAA) is the preferred surgical procedure for patients with refractory ulcerative colitis (UC) and familial adenomatous polyposis (FAP). However, pouchitis is the most common complication after ileal pouch-anal anastomosis (IPAA) in UC patients, being rare in FAP; and only occurs after ileostomy closure. Therefore, it is important to get more information about the role of the ileal pouch microbiota and mucosa susceptibility to inflammation. Therefore, we evaluated Toll-like receptors (TLRs) expression in normal endoscopic and histological mucosa of the ileal pouch in patients with UC and FAP, in order to find any abnormality in this pathway in asymptomatic patients, which may contribute to pouchitis. Patients and Method: Twelve patients (six with UC and six with FAP) who underwent to total rectocolectomy and "J" pouch reconstruction, were studied. Biopsies were obtained from the mucosa of the pouch by endoscopy. Normal ileum biopsies were obtained from six patients submitted to ileocolonoscopy with no abnormalities. The specimens were snap-frozen and the expressions of TLR2, TLR4 (bacterial recognition receptors) and JNK (nuclear signalization factor) were determined by immunoblot protein extract. The absence of pouchitis was assessed by clinical, histological and endoscopic parameters, according to the Pouchitis Disease Activity Index (PDAI). The patients were not under any medication. The committee approved the study and informed consent was signed by all participants. The ANOVA and Tukey-Kramer Tests were applied for statistical analysis. The level of significance was p<0.05. Results: Patients with UC had significantly higher protein levels of TLR4 than controls and FAP (p<0.05). The expressions of TLR2 and JNK were similar in the groups (p>0.05). Conclusion: Patients with UC had higher levels of TLR4, even in the absence of clinical, endoscopic and histological pouchitis. These findings may explain a tendency towards the up-regulation of intracellular pathways activated by bacterial antigens in UC patients, which could contribute to the production of proinflammatory mediators, being coadjuvant of the inflammatory process in the ileal pouch mucosa / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências

No description available.

Introduction: La pneumonie associée à la ventilation mécanique (VM) est fréquente chez les patients ventilés. L’étirement cyclique (EC) induit par la VM pourrait amorcer le poumon vers une réponse inflammatoire en cas d'exposition à des bactéries. Les Toll-like Receptors (TLR) reconnaissent les bactéries et déclenchent l'immunité. La VM pourrait moduler l'expression des TLR et leur réactivité aux agonistes. Le décubitus ventral (DV) réduit l’étirement du poumon. Méthodes: Les niveaux de TLR2 et la réponse à ses agonistes ont été mesures dans des cellules pulmonaires soumises à un EC, et dans un modèle de lapin ventilé. Une stimulation ex vivo du sang total prélevé sur lapins ventilés a été réalisée. Une pneumonie a été induite chez des lapins soumis à VM et maintenus en décubitus dorsal ou tournés en DV. Résultats: L’EC des cellules ainsi que des poumons de lapins augmente les niveaux de TLR2 et la réponse inflammatoire à ses agonistes. La VM et l’exposition du poumon à des agonistes TLR2 induisent synergiquement des lésions. Chez des lapins avec pneumonie sous VM la clairance bactérienne pulmonaire est réduite, la probabilité de bactériémie et le taux des cytokines circulantes augmentés. Le sang total provenant d'animaux sous VM libère de grandes quantités de cytokines après stimulation. Le DV est associe à des niveaux plus faibles de concentrations bactériennes et d'inflammation. Conclusions: La VM sensibilise le poumon aux ligands bactériens de TLR2, modifie la clairance bactérienne pulmonaire, favorise les lésions pulmonaires et de l'inflammation. La surexpression de TLR2 induite par l’EC pourrait expliquer ces différences. Le DV pourrait avoir un effet protecteur. / Introduction: Ventilator-associated pneumonia is common in patients subjected to mechanical ventilation (MV). Cyclic stretch subsequent to MV could prime the lung toward an inflammatory response if exposed to bacteria. Toll-like receptors (TLRs) recognize pathogens thus triggering immunity. MV could modulate TLRs expression and responsiveness to agonists. The prone position (PP) reduces lung stretch.Methods:TLR2 levels and response to the TLR2 ligands were measured in human pulmonary cells submitted to cyclic stretch, and either spontaneously breathing (SB) or MV rabbits. Ex vivo stimulation of whole blood taken from SB or MV rabbits was performed.Enterobacter aerogenes pneumonia was induced in rabbits subjected to MV and kept supine or turned to the PP. Results: Cyclic stretch of human cells as well as rabbitsÕ lung increased both TLR2 levels and inflammatory response to its agonist. MV and airways exposure to TLR2 ligands acted synergistically in causing lung injury.A decrease of lung bacterial clearance and a greater likelihood of bacteremia were observed in MV rabbits with S. aureus pneumonia. Circulating cytokines rose significantly only in these animals. MV induced TLR2 spleen overexpression. Whole blood obtained from MV animals released larger amounts of cytokines after stimulation. PP was associated with lower levels of bacterial concentrations and inflammation. Conclusions: MV sensitizes the lung to bacterial TLR2 ligands, alters lung bacterial clearance, promotes lung injury and inflammation. Both pulmonary and peripheral blood stretch-induced TLR2 overexpression could account at least in part for such differences. The PP could be protective.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. / Dissertation

A doença de Behçet tem sua fisiopatologia caracterizada por hiperatividade neutrofílica, particularmente em relação à quimiotaxia, e períodos de atividade da doença podem ser desencadeados por exposição a estreptococos. Uma vez que células do sistema imune inato são ativadas pelo ácido lipoteicoico (LTA) de bactérias gram-positivas via receptor toll-like (TLR) 2 e CD14, cujas expressões são reguladas pelos fatores estimulantes de colônias de granulócitos (G-CSF) e granulócitos-macrófagos (GM-CSF), o objetivo principal deste estudo foi determinar se há hiperexpressão de TLR2 em neutrófilos de DB ativa e se a quimiotaxia de polimorfonucleares (PMN) neutrófilos na DB poderia ser hiperestimulada pelo LTA. Além do TLR2, foram medidas as expressões de TLR4, CD14, CD114 (receptor de G-CSF) e CD116 (receptor de GM-CSF) nos neutrófilos e nos monócitos de pacientes com doença de Behçet (DB), as concentrações séricas de CD14 solúvel (CD14s) e as respostas quimiotáxicas dos PMNs de DB sob diferentes estímulos. A expressão dos receptores foi medida pela citometria de fluxo, as concentrações séricas por ELISA e as respostas quimiotáxicas foram avaliadas em câmara de Boyden. Nos PMNs, os receptores foram igualmente expressos nos dois grupos e, estimulados com LTA, suas respostas quimiotáxicas também foram similares. Somente à incubação com plasma os PMNs de DB desenvolveram hiperquimiotaxia em relação aos PMNs controles. A expressão do TLR2 foi maior em monócitos de DB em relação aos controles, e a concentração de CD14s sérica, de origem monocitária, foi maior nos pacientes com DB ativa. Em conjunto, os resultados demonstram que PMNs de DB, isoladamente, não reagem exacerbadamente ao LTA, e suas respostas migratórias são estritamente dependentes de fatores estimulantes solúveis. / Expressions of toll-like receptor (TLR) 2, TLR4, CD14, CD114 and CD116 were assessed on polymorphonuclear (PMN) neutrophils and monocytes of patients with Behçets disease (BD). PMN chemotactic responses under different stimulations were also measured. The objective was to determine if BD PMN chemotaxis may be overstimulated by lipoteichoic acid (LTA) from gram-positive bacteria. Receptor expressions were measured by flow cytometry and PMN chemotaxis was assessed in a Boyden chamber. Only TLR2 expression was higher on monocytes of the BD group than in control group. On PMNs, however, TLR2 expression was similar in both groups and, when stimulated with LTA, BD PMN cells showed chemotactic responses similar to the controls. These cells only exhibited increased chemotaxis when incubated with plasma. In conclusion, isolated BD PMN did not overreact to LTA, and its hyperchemotaxis is strictly dependent on soluble stimulating factors

A doença de Behçet tem sua fisiopatologia caracterizada por hiperatividade neutrofílica, particularmente em relação à quimiotaxia, e períodos de atividade da doença podem ser desencadeados por exposição a estreptococos. Uma vez que células do sistema imune inato são ativadas pelo ácido lipoteicoico (LTA) de bactérias gram-positivas via receptor toll-like (TLR) 2 e CD14, cujas expressões são reguladas pelos fatores estimulantes de colônias de granulócitos (G-CSF) e granulócitos-macrófagos (GM-CSF), o objetivo principal deste estudo foi determinar se há hiperexpressão de TLR2 em neutrófilos de DB ativa e se a quimiotaxia de polimorfonucleares (PMN) neutrófilos na DB poderia ser hiperestimulada pelo LTA. Além do TLR2, foram medidas as expressões de TLR4, CD14, CD114 (receptor de G-CSF) e CD116 (receptor de GM-CSF) nos neutrófilos e nos monócitos de pacientes com doença de Behçet (DB), as concentrações séricas de CD14 solúvel (CD14s) e as respostas quimiotáxicas dos PMNs de DB sob diferentes estímulos. A expressão dos receptores foi medida pela citometria de fluxo, as concentrações séricas por ELISA e as respostas quimiotáxicas foram avaliadas em câmara de Boyden. Nos PMNs, os receptores foram igualmente expressos nos dois grupos e, estimulados com LTA, suas respostas quimiotáxicas também foram similares. Somente à incubação com plasma os PMNs de DB desenvolveram hiperquimiotaxia em relação aos PMNs controles. A expressão do TLR2 foi maior em monócitos de DB em relação aos controles, e a concentração de CD14s sérica, de origem monocitária, foi maior nos pacientes com DB ativa. Em conjunto, os resultados demonstram que PMNs de DB, isoladamente, não reagem exacerbadamente ao LTA, e suas respostas migratórias são estritamente dependentes de fatores estimulantes solúveis. / Expressions of toll-like receptor (TLR) 2, TLR4, CD14, CD114 and CD116 were assessed on polymorphonuclear (PMN) neutrophils and monocytes of patients with Behçets disease (BD). PMN chemotactic responses under different stimulations were also measured. The objective was to determine if BD PMN chemotaxis may be overstimulated by lipoteichoic acid (LTA) from gram-positive bacteria. Receptor expressions were measured by flow cytometry and PMN chemotaxis was assessed in a Boyden chamber. Only TLR2 expression was higher on monocytes of the BD group than in control group. On PMNs, however, TLR2 expression was similar in both groups and, when stimulated with LTA, BD PMN cells showed chemotactic responses similar to the controls. These cells only exhibited increased chemotaxis when incubated with plasma. In conclusion, isolated BD PMN did not overreact to LTA, and its hyperchemotaxis is strictly dependent on soluble stimulating factors

Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

Orientador : Alexandre Leite Rodrigues de Oliveira / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:27:25Z (GMT). No. of bitstreams: 1 Freria_CamilaMarques_D.pdf: 9799936 bytes, checksum: 9a9057c6ab5ca87ea1bc6767c4f71490 (MD5) Previous issue date: 2013 / Resumo: Lesões nervosas periféricas e centrais levam à inflamação local e retrógrada, resultando em alterações axonais, perdas neuronais e sinápticas significativas. Juntamente a tais alterações, as células gliais tornam-se reativas, influenciando na remodelação do SNC após lesão. Os mecanismos que desencadeiam tais mudanças não são completamente compreendidos, mas é evidente que as moléculas classicamente relacionadas com o sistema imune estão envolvidas em tais eventos diretamente ou através da modulação da reatividade glial. Assim, nossa hipótese é que o controle da sinalização inflamatória após a lesão central ou periférica possa afetar indiretamente nos mecanismos endógenos de reparação no SNC, resultando em maior preservação das conexões neurais e melhor recuperação funcional. Para isso, realizamos lesões periféricas e centrais expondo os animais a diferentes microambientes de lesão a fim de investigar o papel das células gliais na sobrevivência, capacidade regenerativa axonal e estabilidade sináptica de motoneurônios medulares. Os resultados mostraram que, após lesão, a modulação da sinalização inflamatória através da administração de citocinas ou deleção de moléculas expressas na superfície das células gliais podem influenciar direta ou indiretamente na estabilidade dos circuitos neuronais, na regeneração axonal e sobrevivência neuronal. Desse modo, conclui-se que o controle da inflamação e da reatividade glial são, provavelmente, críticos para a plasticidade no Sistema Nervoso viabilizando, assim, novas estratégicas de tratamentos / Abstract: Central or peripheral lesions result in local and retrograde inflammation, leading to axonal degeneration, synaptic and/or neuronal loss. Additionally, after injury, reactive glial cells are recruited to the lesion site, influencing the plasticity of the nervous system. The mechanisms which trigger such changes are not completely understood, but evidences have shown that molecules classically related to the immune system are involved in such events directly or indirectly by glial modulation. Based on this, our hypotheses is that the control of inflammatory signaling after central or peripheral injury may indirectly affect the endogenous repair mechanisms, resulting in a greater synaptic preservation and better functional recovery. In this sense, animals were submitted to both central and peripheral lesions in order to investigate the effects of glial cells on neuron survival, axonal regeneration and synaptic plasticity. The results showed that, after lesion, the modulation of inflammatory signaling by cytokines or knocking down molecules on glial surface, directly or indirectly influence the stability of neural circuits, neuronal survival and axonal regeneration. Thus, we believe that this is important findings that may be critical to the development of new therapeutic strategies following nervous system injury / Doutorado / Clinica Medica / Doutora em Ciências

Respiratory syncytial virus (RSV) causes a common infection that is associated with a range of respiratory illnesses from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children < 1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected as contributing to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs including TLR2, TLR6, TLR3, TLR4, and TLR7 that can potentially interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting TNF-α, IL-6, CCL2 (MCP-1), and CCL5 (RANTES) production. As previously noted, TLR4 also contributed to cytokine activation (71, 90). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation and may play a role in modulating acquired immune responses through DCs. Despite the fact that RSV is the single most important cause of infant upper respiratory tract disease, there are no licensed vaccines available to prevent RSV disease. We have developed a virus-like particle (VLP) vaccine candidate for RSV. The VLP is composed of the NP and M proteins of Newcastle disease virus (NDV) and a chimera protein containing the cytoplasmic and transmembrane domains of the NDV HN protein and the ectodomain of the human RSV G protein (H/G). BALB/c mice immunized with 10 or 40 μg total VLP-H/G protein by intraperitoneal or intramuscular inoculation stimulated antibody responses to G protein as good as or better than comparable amounts of UV-inactivated RSV. Furthermore, VLP-H/G induced robust CTL responses in vaccinated animals. Immunization with two or even a single dose of these particles resulted in the complete protection of BALB/c mice from RSV replication in the lungs. Upon RSV challenge of VLP-H/G immunized mice, no enhanced pathology in the lungs was observed, although lungs of mice immunized in parallel with formalin-inactivated RSV (FI-RSV) showed the significant pathology that has been previously observed with FI-RSV vaccination. Thus, the VLP-H/G candidate vaccine was immunogenic in BALB/c mice and prevented replication of RSV in murine lungs with no evidence of immunopathology. These data support further development of virus-like particle vaccine candidates for RSV.

Of the eleven species of bacteria that comprise the genus Yersinia of the family Enterobacteriaceae, three species are pathogenic for humans. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause a mild, self-limiting mesenteric lymphadenitis or ileitis. Yersinia pestis causes a highly invasive often fatal disease known as plague. All three elaborate a type three secretion system that is essential for virulence and encoded on closely related plasmids. In Y. pestis, all the effectors, structural components and chaperones are encoded on the 70kb plasmid, pCD1. Of these, LcrV from Y. enterocolitica has been implicated in playing an immunosuppressive role through its interaction with host Toll-like receptor 2 (TLR2) and induction of IL-10. Through expression and purification of recombinant LcrV from Escherichia coliwe show that only high molecular weight species of rLcrV are able to stimulate TLR2. In a highly sensitive subcutaneous mouse infection model we demonstrate no difference in the time to death between TLR2-sufficient or deficient mice. Analysis of cytokine levels between these two genotypes also shows no significant difference between splenic IL-10 and IL-6 or levels of bacteria. We conclusively show that this interaction, if it does occur, plays no significant role in vivo. In a separate set of experiments, we also determined that the expression of F1, a peptide shown to be responsible for 37°C-dependent inhibition of invasion by Y. pestis in vitro, was significantly decreased under high oxygen conditions. This led us to re-examine the invasion phenotype both in vitro and in vivo. These results give new insights into virulence gene expression in Y. pestis by environmental cues other than temperature.