Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Η λεπτίνη παράγεται από τα κύτταρα του λιπώδους ιστού, καθώς και από άλλους ιστούς συμπεριλαμβανομένων του πλακούντος και ρυθμίζει τη πρόσληψη τροφής, τη κατανάλωση ενέργειας, την αναπαραγωγική διαδικασία και τις ανοσολογικές λειτουργίες. Για τη διερεύνηση του ρόλου της λεπτίνης επί του νεογνικού ανοσολογικού συστήματος μετρήσαμε τα επίπεδα λεπτίνης και κυτταροκινών (INF-γ, TNF-α, IL-2, IL-4, IL-10 και IL-12) στον ορό αίματος ομφαλίου λώρου 481 φυσιολογικών νεογνών τα οποία γεννήθηκαν από υγιείς μητέρες. 317 ήταν νεογνά με ιδανικό βάρος γέννησης για την ηλικία κύησης (AGA) και 164 έφεραν αυξημένο βάρος για την ηλικία κύησης (LGA). Η ενδιάμεση συγκέντρωση λεπτίνης σε ολόκληρο το δείγμα ήταν 11.6 ng/ml. Στο 12% AGA νεογνών, τα επίπεδα λεπτίνης ήταν υψηλότερα του μέσου όρου (ενδιάμεση τιμή 34 ng/ml). Στο 33% των νεογνών αυτών (4% του ολικού δείγματος) τα επίπεδα λεπτίνης κυμαίνονταν μεταξύ 37.5-204 ng/ml, στην ομάδα αυτή βρέθηκαν επίσης υψηλά επίπεδα ιντερφερόνης-γ (μέση τιμή 27.11 pg/ml, διακύμανση 17.5-38.5 pg/ml). Κατόπιν μελετήσαμε εάν η λεπτίνη μπορεί να επηρεάσει τη γονιδιακή έκφραση κυτταροκινών στα Τ κύτταρα και μονοκύτταρα αίματος ομφαλίου λώρου. Η καλλιέργεια των κυττάρων ομφαλίου λώρου (Τ ή μονοκυττάρων) AGA νεογνών, τυχαία επιλεγμένων ή κυττάρων περιφερικού αίματος ενηλίκου, με λεπτίνη είχε σαν αποτέλεσμα τη γονιδιακή έκφραση IL-2, INF-γ και IL-4 από τα κύτταρα του ομφαλίου λώρου, καθώς και από τα Τ κύτταρα του ενηλίκου όπως και την έκφραση IL-10 κυρίως από τα μονοκύτταρα αίματος ομφαλίου λώρου. Σημαντικά υψηλότερη γονιδιακή έκφραση INF-γ παρατηρήθηκε σε Τ κύτταρα ομφαλίου λώρου θήλεος τα οποία καλλιεργήθηκαν με λεπτίνη συγκριτικά με τα Τ κύτταρα ομφαλίου λώρου άρρενος. Συμπερασματικά, η παρουσία υψηλών συγκεντρώσεων λεπτίνης και INF-γ στον ορό αίματος ομφαλίου λώρου φυσιολογικών AGA νεογνών, καθώς και το γεγονός ότι η λεπτίνη μπορεί άμεσα να διεγείρει τη γονιδιακή έκφραση κυτταροκινών σε κύτταρα ομφαλίου λώρου (Τ και μονοκύτταρα) δείχνει ότι τα υψηλά επίπεδα λεπτίνης μπορούν ανεξάρτητα να επηρεάσουν το ανοσολογικό σύστημα των φυσιολογικών νεογνών και να συμβάλλουν στη Th1 διαφοροποίηση της ανοσολογικής απόκρισης. / Leptin is a hormone synthesized by adipocytes and other tissues, including the placenta, and it regulates food intake and energy expenditure, reproductive and immune functions. To investigate the role of leptin in neonatal immunity, we measured serum leptin and cytokine (INF-γ, TNF-α, IL-2, IL-4, IL-10, IL-12) levels in cord blood (cb) of 481 healthy neonates, born to healthy mothers, of which 317 were appropriately grown for gestational age (AGA) and 164 were large for gestational age (LGA). The median serum leptin concentration in the whole sample was 11.6 ng/ml. In 12% AGA neonates, leptin levels were well above average, with a median of 34 ng/ml. In 33% of these neonates (4% of the total sample) leptin levels ranged between 37.5-204 ng/ml; in this group, significantly elevated levels of serum IFN- were also found (mean 27.11 pg/ml, range 17.5-38.5 pg/ml). We then investigated whether leptin can independently influence cytokine gene expression by cb T-cells and monocytes (Mc). Culture of cb T-cells or Mc, isolated from randomly selected AGA neonates or adult peripheral blood mononuclear cells (PBMC), with leptin, resulted in upregulation of IL-2, IFN- and IL-4 gene expression in cb and adult T-cells and IL-10 gene expression mainly in cb-Mc. Significantly higher expression of IFN- occurred in female cb-T-cells cultured with leptin, compared with male cb-T-cells. In conclusion, the concurrent presence of high concentrations in both leptin and INF- in cb of healthy AGA infants, together with the fact that leptin can directly upregulate cytokine gene expression in cb T and Mc cells, indicate that abnormally high leptin levels can independently influence the immune system of healthy newborns, and may mediate gender differences in the development of a Th1 polarized immune response.

Made available in DSpace on 2014-06-11T19:23:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-12Bitstream added on 2014-06-13T18:09:52Z : No. of bitstreams: 1 tanaka_mh_me_arafo.pdf: 647421 bytes, checksum: 7aca1036f8801f2b3143929ea57d6d5c (MD5) / A doença periodontal (DP) em indivíduos com Síndrome de Down (SD) se desenvolve com alta prevalência, precocemente, de modo rápido e generalizado em comparação com indivíduos não-sindrômicos. Foi demonstrado que portadores da SD apresentam resposta imune diminuída em relação aos cromossomicamente normais. O objetivo desta pesquisa foi investigar diferenças nos parâmetros clínicos periodontais e níveis de expressão dos genes Interferon-gama (IFNG), Interferon-gama receptor 1 (IFNGR1), Interferon-gama receptor 2 (IFNGR2), Interferon-alfa (IFNA), Interferon-alfa receptor 1 (IFNAR1), Interferon-alfa receptor 2 (IFNAR2), Janus-quinase 1 (JAK1), Transdutor de sinal e ativador da transcrição 1 (STAT1) e Fator de regulação de interferon 1 (IRF1) em indivíduos com SD que apresentam ou não DP e em indivíduos cromossomicamente normais. Fizeram parte deste estudo 80 indivíduos entre 7 e 57 anos de idade subdivididos em 4 grupos: SD com DP (A); indivíduos com SD sem DP (B); indivíduos não-sindrômicos (Controle) com DP (C) e indivíduos Controle sem DP (D). A expressão gênica foi investigada por meio de quantificação relativa utilizando a técnica da Reação em Cadeia da Polimerase (PCR) em Tempo Real. Para o índice sangramento à sondagem (SS) não houve diferença entre os grupos A e 21 C. A periodontite crônica localizada foi o tipo prevalente tanto entre indivíduos com SD como Controle. Considerando os parâmetros clínicos, não foram encontradas diferenças na periodontite crônica localizada entre os indivíduos com SD e Controle, assim como para a periodontite crônica generalizada. Com relação à análise genética, observou-se que indivíduos dos grupos com SD em relação aos grupos cromossomicamente normais (A+B-C+D) tiveram uma expressão de IFNG semelhante ao observado entre indivíduos do grupo... / Periodontal disease (PD) in individuals with Down Syndrome (DS) has an early, quickly and widespread onset and high prevalence when compared with individuals without the Syndrome. Only poor oral hygiene does not explain the severe periodontal destruction seen in DS patients. It has been shown that DS patients have a weaker immune response than people with normal number of chromosomes. The aim of this study was to investigate differences in periodontal clinical parameters and the expression levels of the genes Interferon-gamma (IFNG), Interferon-gamma receptor 1 (IFNGR1), Interferon-gamma receptor 2 (IFNGR2), interferon-alpha (IFNA), interferon-alpha receptor 1 (IFNAR1), Interferon-alpha receptor 2 (IFNAR2), Janus-kinase 1 (JAK1), Signal transducers and activators of transcription 1 (STAT1) and Interferon regulatory factor 1 (IRF1) in DS patients with and without periodontal disease in comparison with chromossomically normal individuals. A total of 80 individuals aged 7 to 57 years participated in this study and were divided into 4 groups: DS with PD (A); DS without PD (B); individuals without DS (control) with PD (C) and individuals without DS (control) and without PD (D). A quantitative RT-qPCR was used to investigate gene expression. There was no difference between groups A and C regarding the bleeding on probing 25 (BOP) index. The most prevalent type of periodontitis seen in this study was the localized chronic periodontitis, both in individuals with and without DS. Considering the clinical parameters, localized and generalized chronic periodontitis did not differ between individuals with and without DS. Regarding genetic analysis, individuals of the groups with DS in relation to the groups without DS (A+B-C+D) showed an IFNG expression similar to that seen among the individuals of groups control with PD (C-D). However, individuals... (Complete abstract click electronic access below)

The influence of interferon gamma and specific antibodies on the infection with E. cuniculi in vivo has been studied. Reconstruction of SCID mice with CD4+ T-lymphocytes from BALB/c mice and from mice with defect gene for interferon gamma was used. Effects of the treatment with mouse recombinant interferon gamma and anti-E. cuniculi sera on survival of E. cuniculi infected SCID mice were monitored. The influence of the immunization with E. cuniculi antigen on the survival of E. cuniculi infected mice with defect gene for interferon gamma was examined.

As pessoas vivendo com HIV tÃm probabilidade aumentada de desenvolver, apresentar formar graves, ter cepas multirresistentes e morrer por tuberculose. A profilaxia para infecÃÃo latente por Mycobacteium tuberculosis (ILTB) diminui a chance de ativaÃÃo de tuberculose (TB) numa mÃdia de 62% nessa populaÃÃo. Entretanto, o diagnÃstico da TB na sua forma latente à controverso. O teste tuberculÃnico (TT) à o Ãnico exame aprovado no Brasil para avaliaÃÃo dessa infecÃÃo, embora existam problemas tanto na sua realizaÃÃo quanto na sua interpretaÃÃo. Exames de liberaÃÃo de interferon-gama foram criados recentemente com o objetivo de aumentar a especificidade e a praticidade da investigaÃÃo da ILTB. Esse estudo se propÃs a avaliar como vem sendo feita a investigaÃÃo da ILTB e o desempenho do TT e do QuantiFERON-TB Gold In-Tube (QTF-GIT) em portadores de HIV. Foram selecionados ao todo 351 pacientes portadores de HIV e sem evidÃncia de TB ativa, admitidos em dois centros de referÃncia de Fortaleza-CE, no perÃodo de 2007-2010. Na admissÃo, 41,8% dos pacientes realizaram TT, 36,3% foram avaliados quanto a contato com TB e 28,4% tiveram radiografia de tÃrax. A profilaxia foi realizada para 73,3% dos pacientes com TT positivo. Houve diagnÃstico de ILTB em 25,3% dos pacientes de acordo com o TT e em 6,7% pelo QTF-GIT (p<0,001). A correlaÃÃo entre os resultados dos dois testes foi considerada fraca (k= -0,037). Resultado positivo do TT esteve associado com drogadiÃÃo (OR 7 CI: 1,53-32,11; p=0,01), contato com TB bacilÃfera (OR 13 CI: 2,7-62,83; p=0,001), profilaxia para ILTB prÃvia (OR 17,5 CI: 3,4-90,4; p<0,001), procedÃncia do interior do estado (OR 2,74 CI:1,04-7,22; p= 0,04). NÃo houve associaÃÃo entre QTF-GIT positivo e fatores de risco para TB. A mÃdia de contagem de linfÃcitos T CD4+ nos indivÃduos com TT positivo foi superior à mÃdia dos com TT negativo (535,8 vs. 373,4 cÃl/mm3; p=0,006), enquanto o inverso ocorreu em relaÃÃo ao QTF-GIT (277 vs. 438,3 cÃl/mm3; p= 0,055). A mÃdia do logaritmo da carga viral foi superior naqueles com QTF-GIT positivo (4,81 vs. 2,11 log10 cÃp/ml; p= 0,005). Mais da metade dos pacientes nÃo realizou TT, apesar da alta prevalÃncia de ILTB. O TT contou com maior nÃmero de testes positivos. O QTF-GIT mostrou-se superior para pacientes com elevada viremia e imunossupressÃo. Sugere-se o uso de ambos os testes de forma complementar para aumentar a chance de diagnÃstico de ILTB e diminuir os riscos de progressÃo da doenÃa. / People living with HIV have an enhanced chance to develop and to die of tuberculosis (TB). Many studies demonstrate that chemoprophylaxis for latent tuberculosis infection (LTBI) reduces the progression to active TB. Indeed, the diagnosis of LTBI is controversial. In Brazil, the only test approved for use is the tuberculin skin test (TST), however, this test is complicated by several problems due to application and interpretation of the exam. Recently developed interferon-gamma release assays (IGRA) using Mycobacterium tuberculosis-specific antigens have the advantage of decreased cross-reactivity and, therefore, increased specificity. The purpose of this study is to evaluate the adherence of LTBI diagnosis and to compare the results of the QuantiFERON-TB Gold In-Tube test (QTF-GIT) and TST in a population of HIV-positive individuals from a country with high prevalence of TB. A cross-sectional study was carried out with 351 HIV patients without active tuberculosis, attending outpatient in two reference centers, from November 2007- 2010. At admission, 41.8% had realized TST, 36.3% had been interrogated about TB exposure and 28.4% had performed a chest X-ray. Chemoprophylaxis was offered to 73.3% of TST positive patients. The TST and QTF-GIT results were positive in 25.3% and 6.7% (p<0.001) of the individuals, respectively. The agreement between the two tests was poor (k= -0.037). Drug use (OR 7, 95% CI 1.5-32.1; p=0.01), TB exposure (OR 13, 95% CI 2.7-62.83; p=0.001), previous LTBI prophylaxis (OR 17.5, 95% CI 3.4-90.4; p<0.001), and living outside the state capÃtal (OR 2.7, 95% CI 1-7.2; p= 0.04) were associated with a positive TST result. There is no association between QTF-GIT positive result and risk factors for TB. TST positive individuals had a higher mean CD4+ cell count than those with TST negative result (535.8 cell/mm3 vs. 373.4 cell/mm3; p=0.006), in contrast to QTF-GIT positive result (277 cell/mm3 vs. 438.3 cell/mm3; p= 0.055). Higher viral load was associated with QTF-GIT positive result (4.8 log10 cop/ml vs. 2.1 log10 cop/ml; p= 0.005). Despite of Brazil being a country with a high burden of TB, more than half the patients have not realized TST, which appears to be more sensitive than QTF-GIT for diagnosis of LTBI. Otherwise, QTF-GIT shows better results in patients with advanced immunosuppression and high viral load. We suggest the use of both tests to increase LTBI diagnosis and decrease the risk of disease progression.

Orientador: Márcio Zaffalon Casati / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T08:08:59Z (GMT). No. of bitstreams: 1 Monteiro_MabelledeFreitas_M.pdf: 1431989 bytes, checksum: 55bf42377e6ffeb3626cb5a8d6551c1c (MD5) Previous issue date: 2015 / Resumo: O Resumo poderá ser visualizado no texto completo da tese digital / Abstract: The Abstract is available with the full electronic digital document. / Mestrado / Periodontia / Mestra em Clínica Odontológica

>Magister Scientiae - MSc / Tuberculosis (TB) is a disease that results from infection by Mycobacterium tuberculosis, which is regarded the most common infecting organism. TB has killed countless numbers of people particularly in underdeveloped countries. TB bacteria can remain inactive or in dormant state for years without causing symptoms or spreading to other subjects, but as soon as the immune system of the host becomes weakened, the bacteria become active and infect mainly the lungs along with other parts of body. TB cases are further aggravated by other illnesses that affect the immune system, such as human immune virus (HIV), which is very prevalent in resource-poor countries. Interferon-gamma (IFN-γ) is a TB biomarker that has found to have all the qualities that are needed to help and cure Tuberculosis disease. Early diagnosis and treatment are essential measures for effectively controlling the disease. Traditional microbial culture-based tests are the most common methodologies currently used. Usually, these methods involve cell culture, cell counts, and cell enrichment, but this process is time-consuming and laborious, especially for the slow-growing bacteria like M. tuberculosis. Sputum smear is one of the methods currently used to detect acid fast bacilli (AFB) in clinical specimens or fluorescent staining. It is a cost-effective tool for diagnosing patients with TB and to monitor the progress of treatment especially in developing countries. The traditional method of inoculating solid medium such as Lowerstein-Jensen (L-J) or 7H10/7H11 media is also used currently it is slow and takes 6-8 weeks of incubation to diagnose the infection and further more time to determine the susceptibility patterns. The microscopic observation drug susceptibility (MODS) assay they are also used currently they rely on light microscopy to visualize the characteristic cording morphology of M. tuberculosis in liquid culture. MODS has shorter time to culture positivity (average 8 days) compared with LJ medium (average ~26 days), they are very expensive. The Gen-Probe assay specific for M. tuberculosis complex is a rapid detection that is also used, nucleic acid amplification (NAA) test results can be obtained as fast as in two hours (provided if a positive culture is present); it also has a high sensitivity of 99% and specificity of 99.2%. It holds the disadvantage of needing of positive culture that can take several days. Enzyme-linked immunosorbent assay (ELISA), is a test that uses antibodies and colour change to identify a substance. ELISA is an assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. It can be used to detection of Mycobacterium antibodies in tuberculosis. The Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) is used for the detection of M. tuberculosis it enables the amplification and detection of M. tuberculosis rRNA directly from respiratory specimens. The diagnostic methods employing genetechnology based on the amplification of DNA or RNA are expected to improve the speed, sensitivity, and specificity of Mycobacterium tuberculosis detection. TB rapid cultivation detection technique, such as MB/BacT system, BactecMGIT 960 system and flow cytometry. The BACTEC MGIT960 system (Becton Dickinson, Sparks, MD) performs incubation and reading of the tubes continuously inside the machine using a predefined algorithm to interpret the fluorescent signal and giving the results as positive or negative. When performing DST, the BACTEC MGIT960 interprets the results as susceptible or resistant to the antibiotic under study. Results are available within 8 days. A recent meta-analysis of the published studies found high accuracy and high predictive values associated with the use of BACTEC MGIT960. These methods are more sensitive and rapid than the traditional microbial culture-based methods. However, they cannot provide the detection results in real-time and most of these methods are centralized in large stationary laboratories because complex instrumentation and highly qualified technical staff are required. Recently, Food and Drug Administration (FDA) approved two new assays that were introduced. These two assays detect in vitro a specific immune response to M. tuberculosis. These tests are the QuantiFERON-TB Gold In-Tube (Cellestis/Qiagen, Carnegie, Australia) and the T-SPOT.TB assay (Oxford Immunotec, Abingdon, United Kingdom). Both assays use whole blood from the patient and measure the production of interferon gamma after the whole blood is exposed to specific antigens from M. tuberculosis. These tests are based on the knowledge that IFN-γ is a product of an active cell-mediated immune response induced by M. tuberculosis. However, TB detection remains a major obstacle due to several drawbacks of these methods. To date, the number of diagnosis approaches for TB has increased as the disease continues to be a major public health problem worldwide and most conventional detection technologies present difficulties in recognizing the presence of M. tuberculosis, since they are time consuming, do not provide clinically reliable results and significantly lack of sensitivity. This thesis focusedon developing two binary and one ternary-electrochemically quantum dots, all synthesised at room temperature in aqueous media for detecting (IFN-γ). Copper telluride (CuTe) and Zinc telluride (ZnTe) was prepared to check how does the two quantum dot behave individual and also to check on how they behave when they are combined and formed ternary quantum dots (CuZnTe). The electrochemical studies of the binary CuTe quantum dots, ZnTe quantum dots and the ternary CuZnTe core-shell quantum dots reveal that ternary quantum dots were stable and showed a significant enhancement in the conductivity of CuZnTe core-shell solution compared to that of CuTe and ZnTe, all studied in solution. The three different quantum dots were capped with three different capping reagents which are tetraethyl orthosilicate (TEOS), thioglycolic acid (TGA), (3-mercaptopropyl) trimethoxysilane (MPS). In the study, a label-free electrochemical immunosensor for the detection of interferon gamma (IFN-γ) was prepared for the first time using ternary quantum dots. The biosensor consists of water-soluble silica coated Copper Zinc telluride (CuZnTe core-shell) quantum dots conjugated to a gold electrode. The antibody-antigen were then conjugated on the CuZnTe core-shell QD modified gold electrode. Results from synthesis of two different binary quantum dots are also presented in the study and compared to the results of the CuZnTe core-shell QDs. The CuTe quantum dots had a small average size which was confirmed through HRTEM, SAXS and XRD analysis

IFNγ is an essential and pleiotropic activator of monocytes, but little is known about the effects IFNγ on cellular metabolism. Therefore, we sought to characterize and elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. First, we identified a critical role for IFNγ in the induction of immunoresponsive gene 1 (IRG1) and its product, itaconate. The immunometabolite, itaconate, has been reported to have antibacterial, anti-inflammatory and antioxidant activity. Irg1-/- mice, lacking itaconate, are highly susceptible and phenotypically similar to IFNγ knock out (GKO) mice upon infection with Mycobacterium tuberculosis. Therefore, we assessed the role of IRG1/itaconate in the context of non-tuberculous mycobacterial (NTM) infection, the most common type of infection in patients with immunodeficiencies caused by defects in IFNγ signaling. Our data suggest that impaired induction of itaconate in the context of mycobacterial infection may contribute to mycobacterial susceptibility and immune dysregulation in patients with defects in IFNγ signaling. Next, we evaluated the metabolic phenotype of IFNγ-stimulated human monocytes and found that IFNγ increased oxygen consumption rates (OCR), indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling of human macrophages revealed that this oxidative phenotype was dependent on IFNγ-induced, nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for complete induction of the respiratory burst in IFNγ stimulated human monocytes.

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