<p>Several HIV-1 neutralizing antibodies (e.g. 2F5, 4E10) have been shown to react with self-antigens, suggesting that effective humoral responses to HIV-1 may be constrained by the tolerization of HIV-reactive B cells that also recognize self-antigens. I have tracked the development of 2F5-like HIV-1 gp41 membrane proximal external region (MPER)-reactive B cells throughout ontogeny using B-cell tetramer reagents. In BL/6 mice, MPER-binding populations are lost during normal B-cell development and immunization with HIV-1 MPER antigen does not elicit robust humoral responses. I have identified Kynureninase as a self-antigen that is recognized by 2F5 antibody and, therefore, is a molecule that could mediate the developmental loss of B cells reactive to an epitope shared by HIV gp41 and Kynureninase. To recover these MPER-reactive cells, I describe and characterize a stromal-cell independent culture system that efficiently supports pro-B cell to IgM+ B-cell development with near normal levels of IgH and Igkappa diversity. B-cell development in vitro closely follows the patterns of development in vivo with culture derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. Immature and transitional B-cell compartments are reduced, due to the induction of tolerance, in the bone marrow of 3H9 IgH knockin mice ; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of "forbidden", autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4-/- animals following the reconstitution of RAG-1-/- mice with IgM+ CD cells derived from BL/6 mice. I show that HIV-1 MPER-reactive B cells are recovered from both BL/6 and 2F5 IgH knockin bone marrow using this in vitro culture system. RAG-1-/- mice reconstituted with these culture-derived B and T cells generate strong germinal center and antibody responses to HIV-1 MPER antigens. These data demonstrate that the humoral immune response to this HIV-1 gp41 MPER antigen can be restored in mice when the constraints of B-cell tolerance have been relaxed.</p> / Dissertation

Resumo:As leishmanioses são protozoonoses transmitidas nas Américas por fêmeas de flebotomíneos do gênero Lutzomyia infectadas por Leishmania infantum chagasi durante a hematofagia. Embora os cães estejam bem estabelecidos como os principais reservatórios para a doença em áreas urbanas, vários relatos de gatos naturalmente infectados em todo o mundo podem indicar um papel importante desta espécie no ciclo da enfermidade. A detecção de anticorpos circulantes em gatos infectados é difícil, e estes animais são supostamente menos propensos a desenvolver sinais clínicos quando comparados aos cães. Este estudo teve como objetivo avaliar gatos residentes em área endêmica para leishmaniose visceral (LV) por métodos parasitológico e sorológico (ELISA e RIFI), PCR em tempo real (qPCR) e Intradermoreação de Montenegro (IDRM), a fim de verificar se este último pode ser uma ferramenta para identificar gatos infectados. Para tanto, foram utilizados 96 gatos adultos, independentemente do sexo, sintomáticos ou não, provenientes do município de Araçatuba, São Paulo. Considerando os resultados da qPCR e/ou do exame parasitológico, a frequência de infecção em felinos foi de 55,21% (53/96). Dos gatos infectados, 58,49% (31/53) eram assintomáticos, 62,26% (33/53) fêmeas e 41,51% (22/53) com idade entre 1 e 3 anos (p = 0,0002). A maioria dos felinos infectados apresentou baixos títulos de anticorpos (37/53, 69,81%) e não demonstrou alterações clínicas (24/37, 64,86%). Somente dois (2,08%) foram sororeativos na RIFI com títulos de anticorpos iguais a 1:40. Os 96 animais apresentaram IDRM negativa com leishmanina de L. infantum chagasi (4.107 parasitos/mL). Destes, 11 gatos foram selecionados e apenas um felino apresentou IDRM positiva com leishmanina de L. (L.) amazonensis (107 parasitos/mL). Os achados indicaram que a qPCR demonstrou eficácia e deve ser empregada...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract:Leishmaniasis are protozoan zoonotic diseases transmitted in the Americas by female sandflies of the genus Lutzomyia infected by Leishmania infantum chagasi during blood feeding. Although dogs are well established as the main reservoirs for the disease in urban areas, various reports of cats naturally infected worldwide may indicate an important role of this species in the disease cycle. Since the detection of circulating antibodies in infected cats is difficult, and infected cats in endemic areas are reportedly less likely to develop clinical signs when compared to dogs, this study aimed to evaluate cats living in endemic area by means of parasitological and serological (ELISA and IFAT), real time PCR and Montenegro skin test (MST), in order to use the later test to identify infected cats that did not develop antibody titers or clinical signs of the disease. For this purpose, 96 adult cats, regardless of sex, symptomatic or asymptomatic, from Araçatuba, São Paulo, were evaluated. Considering the results of qPCR and/or parasitological examination, the prevalence of infection in the evaluated population was 55.21% (53/96). Of the infected cats, 58.49% (31/53) were asymptomatic, 62.26% (33/53) females and 41.51% (22/53) aged between 1 and 3 years (p = 0.0002). Most of the infected cats had low antibody titers (37/53, 69.81%) and showed no clinical alterations (24/37, 64.86%). Only two (2.08%) were seroreactive by IFAT with titers of antibodies equal to 1:40. All 96 cats showed negative to MST with leishmanin of L. infantum chagasi (4.107 parasites/mL). Of these, 11 cats were selected and just one was positive to MST with leishmanin of L. (L.) amazonensis (107 parasites/mL). These findings indicate that qPCR demonstrated efficacy and should be used for visceral leishmaniasis diagnosis in cats and the MST, in the conditions in which it was tested, does not constitute a tool for identifying cats infected by L. infantum chagasi / Orientador:Mary Marcondes / Banca:Suely Regina Mogami Bomfim / Banca:Katia Denise Saraiva Bresciani / Banca:Márcia Dalastra Laurenti / Banca:Raimundo Souza Lopes / Doutor

Made available in DSpace on 2015-10-06T13:03:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-21. Added 1 bitstream(s) on 2015-10-06T13:18:20Z : No. of bitstreams: 1 000848977.pdf: 1255909 bytes, checksum: e72eb452aeeef227b5ad181b4d572e4f (MD5) / Leishmaniasis are protozoan zoonotic diseases transmitted in the Americas by female sandflies of the genus Lutzomyia infected by Leishmania infantum chagasi during blood feeding. Although dogs are well established as the main reservoirs for the disease in urban areas, various reports of cats naturally infected worldwide may indicate an important role of this species in the disease cycle. Since the detection of circulating antibodies in infected cats is difficult, and infected cats in endemic areas are reportedly less likely to develop clinical signs when compared to dogs, this study aimed to evaluate cats living in endemic area by means of parasitological and serological (ELISA and IFAT), real time PCR and Montenegro skin test (MST), in order to use the later test to identify infected cats that did not develop antibody titers or clinical signs of the disease. For this purpose, 96 adult cats, regardless of sex, symptomatic or asymptomatic, from Araçatuba, São Paulo, were evaluated. Considering the results of qPCR and/or parasitological examination, the prevalence of infection in the evaluated population was 55.21% (53/96). Of the infected cats, 58.49% (31/53) were asymptomatic, 62.26% (33/53) females and 41.51% (22/53) aged between 1 and 3 years (p = 0.0002). Most of the infected cats had low antibody titers (37/53, 69.81%) and showed no clinical alterations (24/37, 64.86%). Only two (2.08%) were seroreactive by IFAT with titers of antibodies equal to 1:40. All 96 cats showed negative to MST with leishmanin of L. infantum chagasi (4.107 parasites/mL). Of these, 11 cats were selected and just one was positive to MST with leishmanin of L. (L.) amazonensis (107 parasites/mL). These findings indicate that qPCR demonstrated efficacy and should be used for visceral leishmaniasis diagnosis in cats and the MST, in the conditions in which it was tested, does not constitute a tool for identifying cats infected by L. infantum chagasi

A tumorigênese cutânea é determinada pela combinação de diversos fatores genéticos e ambientais, que envolvem múltiplos eventos onde as células epiteliais podem progredir e se desenvolverem. Todo esse processo é associado com alterações da imunidade celular e humoral. Muitos fatores físicos e químicos podem predispor ao câncer de pele, como o carcinógeno DMBA, com ação iniciadora e promotora. Camundongos geneticamente selecionados para a alta (High) ou baixa (Low) produção de anticorpos constituem uma excelente ferramenta para o estudo da influência da imunidade humoral no desenvolvimento de tumores. Foram avaliados camundongos das linhagens High e Low submetidos ao tratamento com o DMBA na pele após 48 horas, 120 e 240 dias. Mostramos que a linhagem selecionada para a maior produção de anticorpos (High) é a mais sensível ao tratamento com formação de lesões que progrediram para o desenvolvimento de papilomas, apresentando maior incidência e multiplicidade tumoral que os animais Low. Os machos da linhagem High também desenvolveram tumores nos pulmões em decorrência do tratamento com o DMBA na pele. O perfil de citocinas avaliado mostrou que os animais Low tem maior expressão gênica de IFN-g e IL-6 do que os animais High, e estes maior expressão de IL-1b e Cxcl2 que os animais Low após 48 horas do tratamento. A secreção de IL-6 também foi maior nos animais Low com 48 horas, sendo que a produção de TGF-b foi maior nos animais High aos 120 dias. Estes resultados sugerem que na linhagem High o perfil de resposta celular seja do tipo Th2 com produção de IL-10 e TGF-b, o que favorece o surgimento de tumores, e na linhagem Low, a resposta celular seja do tipo Th1, pela presença de IFN-g e TNF-&alpha;, favorecendo o reparo tecidual. Como não foram encontradas diferenças na via de metabolização pelas enzimas do citocromo P450 e no polimorfismo do receptor Ahr, outros fatores podem estar relacionados aos fenótipos observados. Assim, estas linhagens geneticamente selecionadas que diferem quanto à capacidade de secreção de anticorpos, representam uma nova ferramenta para o estudo de fatores genéticos que influenciam o microambiente na predisposição ao câncer. / The skin tumorigenesis is determined by the combination of various genetic and environmental factors, involving multiple events, where epithelial cells can progress and develop. This entire process is associated with changes in cellular and humoral immunity. Many physical and chemical factors may predispose to skin cancer, such as DMBA carcinogen with initiating and promoting action. Mice genetically selected for high (High) or low (Low) antibody production are an excellent tool for studying the influence of humoral immunity in the development of tumors. High and Low mice were treated with DMBA on the skin and, after 48 hours, 120 and 240 days, they were evaluated. We showed that High mice are more sensitive to DMBA treatment, presenting lesions that progressed to the development of papillomas and showing higher incidence and tumor multiplicity than Low ones. Males of High strain have also developed lung tumors as a result of treatment with DMBA on the skin. The profile of cytokines evaluated of Low animals showed that gene expression of IFN-g and IL-6 is more elevated than the one observed in High mice; on the other hand, IL- 1b and CXCL2 are increased in High animals, 48 hours after treatment. The secretion of IL-6 was also greater in Low animals, and TGF-b was higher in High animals, after 120 days of treatment. These results suggest that the High mice response has a Th2 profile with secretion of IL- 10 and TGF-b, which favors the growth of tumors; on the other hand, Low mice have a Th1 response, due to the presence of IFNg and TNF&alpha;, favoring tissue repair. As no differences were found in the enzymes of cytochrome P450 and in the polymorphism of Ahr receptor, other factors may be related to the observed phenotypes. Thus, these genetically selected mice which differ in the ability to secrete antibodies represent a new tool for the study of genetic factors influencing the microenvironment in its predisposition to cancer.

Rodent-borne hantaviruses (family Bunyaviridae) cause two distinct human infections; hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is a common viral zoonosis, characterized by fever, renal dysfunction and hemostatic imbalance. Four HFRS-associated hantaviruses have been described: Hantaan virus and Seoul virus mainly found in Asia, Dobrava virus, encountered in the Balkan region and Puumala virus (PUU), causing mild HFRS (nephropathia epidemica; NE) in Europe. HPS, recently discovered in the Americas, involves adult respiratory distress syndrome with a high mortality rate and is caused by Sin Nombre virus. Hantaviruses are enveloped and carry a RNA genome which encodes a polymerase, two glycoproteins and a nucleocapsid protein. The latter elicits a strong humoral immune response in infected patients. The clinical diagnosis of hantavirus infections has until recently relied on serological confirmation by immunofluorescense assay (IFA) and enzyme-linked immunosorbent assay (ELISA) using cell culture derived viral antigens. Due to the hazardous nature of hantaviruses and variable virus yield in cell culture we aimed at using recombinant hantavirus proteins for serological purposes. We expressed PUU N in E. coli (PUU rN) and found that high levels of IgM to this protein could be detected at onset of NE. This indicated that it was useful as the sole antigen for serodiagnosis. Our finding was confirmed by comparing IFA and PUU rN ELISA using 618 sera collected at the regional diagnostic laboratory. Full-length PUU rN is difficult to purify due to aggregation to E. coli remnants. We therefore located the important domain for the humoral immune response by utilizing truncated PUU rN proteins to its amino-terminal region (amino acid 7-94). Amino acid 1-117 of N of the five major human hantavirus pathogens were produced in E. coli. Serological assays based on them could detect IgM and IgG serum responses in 380 HFRS and HPS patients from Sweden, Finland, Slovenia, China, Korea and the USA with high sensitivity. In an epidemiological investigation of hantavirus serum responses in European Russia we unexpectedly found antibody responses to the hantaviruses found in east Asia and the Balkan region in 1.5 %, speaking in favour for the presence of such virus in this region. The degree of cross reactivity within the hantavirus genus was adressed by following the serum responses in NE patients. We found an increase of cross reactivity during the maturation of the immune response from onset of disease up to three years by comparing the IgG reactivity towards the hantavirus aminoterminal rN proteins. The first human isolate of the causative agent of NE in Scandinavia was recovered in cell culture from phytohemagglutinin stimulated leukocytes. Serological analysis revealed that this virus belongs to the PUU hantavirus serotype, distinct from the rodent prototype PUU Sotkamo. The human PUU Umeå is unique but genetically similar to rodent isolates from northern Sweden. / digitalisering@umu.se

Oncolytic viruses (OVs) are novel biological agents that selectively infect and kill malignant cells. OVs can also generate anti-cancer immunity. Our lab exploited this phenomenon and developed an in vitro vaccine with infected leukemia cells with oncolytic virus vaccine – and named immunotherapy by leukemia-oncolytic virus (iLOV) – that provided in vivo protection in a murine model for acute lymphoblastic leukemia. This work further characterizes iLOV biology and the interaction of its immune responses. An in vitro immune response assay was optimized to detect and quantify the in vivo anti-leukemia immunity generated by iLOV. Anti-viral immunity is an obstacle for OV therapy. Although iLOV created anti-viral antibodies towards itself, these neutralizing antibodies did not hinder the vaccine’s ability to initiate complement or dendritic cell activation. We envision personalized versions of iLOV for leukemia patients in remission to prevent the possibility of relapse. This work highlights new advantages for infected cell vaccines and supports the progress of iLOV toward clinical testing.

Background: Throughout human history infectious agents such as viruses have been one of the biggest threats to public health. One example of infectious agents that can cause severe malignant conditions in humans is the Epstein-Barr virus (EBV). This virus has been researched for decades but still a lot of its potential malignant functions remain to be elucidated. Autoimmunity, and especially multiple sclerosis (MS), has been strongly associated to EBV infection for a long time but the exact mechanisms behind this relationship are still largely unknown. Aim: The main aim of this study was to investigate the evidence connecting an EBV-specific dysregulated immune response to MS. Methods: This paper is written as a systematic review examining the latest science within the studied field. PubMed was searched for articles published between 2010-2020. Results: In total 15 studies were reviewed. Five out of seven studies found an altered antibody response towards EBV in patients with MS, while one demonstrated somewhat mixed results and one could not support such a pattern. Seven out of eight studies found an altered T cell response towards EBV in MS patients, while one could only support such a trend. Conclusions: This review confirms that there is strong evidence for a dysregulated EBV-specific immune response in MS patients. Evidence for a causal relationship between the failure to control a reactivated EBV infection and the progression of disease is suggestive, but this needs to be confirmed by further studies.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Inflammation of the urethra—urethritis—is commonly diagnosed in men and women who have sexually transmitted infections (STI). Characteristic signs and symptoms of urethritis include urethral discharge and burning pain during urination (dysuria). However, these findings are non-specific and can be elicited by STI for which optimal treatment approaches differ. We wanted to investigate if immunoglobulins (antibodies) in the urine of men with acute urethritis could determine the etiologies of these cases. Previously, we conducted an observational case-control study of biological males to compare the urethral microbiota of participants with unambiguous, laboratory-confirmed urethritis (cases) and participants without urethral inflammation (controls). This revealed that nearly 2 in 5 men with nongonococcal urethritis tested negative for all common STI. We identified atypical urethral pathogens in approximately 1/3 of these STI-negative individuals using shotgun metagenomic sequencing. However, we did not detect microorganisms suspected to be urethral pathogens in the remaining 2/3 of STI-negative participants. We hypothesized that these men with “pathogen-negative” urethritis had persisting inflammation from a recent STI that already cleared spontaneously by the time of testing. We observed that urine IgA antibodies against Chlamydia trachomatis (Ctr) infectious particles were significantly more prevalent among men with pathogen-negative urethritis compared to controls. In contrast, we found that the prevalence of urine anti-Ctr IgA was similar between controls and urethritis cases with atypical infections. However, our efforts to detect antibodies against another common STI, Mycoplasma genitalium (Mgen), were complicated by low abundance in urine and the unexpected prevalence of Mgen-reactive antibodies among controls. Collectively, our results suggest that signs and symptoms of urethritis can continue after the causative STI(s) have been eliminated. Furthermore, male urine represents a practical, non-invasive source of pathogen-reactive antibodies that could be evaluated using point-of-care diagnostic tests to elucidate urethritis etiologies. Importantly, our results also suggest that sexual partners of men with pathogen-negative, nongonococcal urethritis are an unrecognized chlamydia reservoir. / 2024-05-22

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis, a disease of equids. During natural outbreaks of the disease, EAV can cause abortion in pregnant mares and persistent infection in stallions. Understanding how host cellular proteins interact with viral RNA and viral proteins, as well as their role in viral infection, will enable better characterization of the pathogenesis of EAV and establishment of persistent infection in stallions. Accordingly, we hypothesized that both viral factors and host genetically related factors could influence the outcome of EAV infection in horses. To test this hypothesis, we first combined contemporary molecular biology techniques with dual color flow cytometric analysis to characterize the interactions of viral structural proteins and the equine peripheral blood mononuclear cells in vitro. Results from this study demonstrated that interactions between GP2, GP3, GP4, GP5 and M envelope proteins of EAV play a major role in determining the CD14+ monocyte tropism while the tropism of CD3+ T lymphocytes is determined by GP2, GP4, GP5 and M envelope proteins but not the GP3 protein. Secondly, a genome wide association study using SNP genotyping identified a common haplotype associated with the in vitro CD3+ T lymphocyte/resistance to EAV infection among four breeds of horses. Subsequently, these studies were extended to establish a possible correlation between the in vitro susceptibility of CD3+ T lymphocytes to EAV and establishment of persistent infection in stallions. Interestingly, carrier stallions with susceptible CD3+ T lymphocyte phenotype to EAV may represent those at higher risk of becoming persistently infected. Finally, the precise effect of EAV on the immune system of horses, innate and humoral immunity, was studied. Horses were shown to mount a strong humoral antibody response to nonstructural proteins (nsps) 2, 4, 5 and 12 of EAV, whereas nsps 1, 2 and 11 suppressed the type I interferon production. The data presented in this dissertation suggest new directions for future EAV research using genomic and proteomic approaches to study host cell factors involved in EAV attachment and entry and establishment of persistent infection in the stallions.