Yes / Alopecia areata (AA) is thought to be an autoimmune process. In other autoimmune diseases, the innate immune system and Toll‐like receptors (TLRs) can play a significant role. Expression of TLR7, TLR9 and associated inducible genes was evaluated by quantitative PCR in peripheral blood mononuclear cells (PBMCs) from 10 healthy individuals and 19 AA patients, categorized according to disease duration, activity and hair loss extent. Microdissected scalp biopsies from five patients and four controls were also assessed by quantitative PCR and immunohistology. TLR9 was significantly upregulated 2.37 fold in AA PBMCs. Notably, TLR9 was most significantly upregulated in patients with active AA, as shown by a positive hair pull test, compared to stable AA patients. In hair follicle bulbs from AA patients, IFNG and TLR7 exhibited statistically significant 3.85 and 2.70 fold increases in mRNA, respectively. Immunohistology revealed TLR7 present in lesional follicles, while TLR9 positive cells were primarily observed peri‐bulbar to AA affected hair follicles. The increased expression of TLR7 and TLR9 suggest components of the innate immune system may be active in AA pathogenesis. / National Alopecia Areata Foundation; Canadian Dermatology Foundation; Michael Smith Foundation for Health Research, Grant/Award Number: CI‐SCH‐00480(06‐1); Canadian Institutes of Health Research, Grant/Award Number: MOP‐167368 and MSH‐192593‐140450

No description available.

BACKGROUND: The type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-β. In this study the impact of poliovirus infection on the type I interferon response has been examined. METHODS: The type I IFN response was assessed by measuring IFN-β mRNA levels using qRT-PCR and normalizing to levels of β-actin mRNA. The status of host factors involved in activation of the type I IFN response was examined by immunoblot, immunofluorescence microcopy and qRT-PCR. RESULTS: The results show that poliovirus infection results in induction of very low levels of IFN-β mRNA despite clear activation of NF-κB and ATF-2. In contrast, analysis of IRF-3 revealed no transcriptional induction of an IRF-3-responsive promoter or homodimerization of IRF-3 indicating it is not activated in poliovirus-infected cells. Exposure of poliovirus-infected cells to poly(I:C) results in lower levels of IFN-β mRNA synthesis and IRF-3 activation compared to mock-infected cells. Analysis of MDA-5 and IPS-1 revealed that these components of the RLR pathway were largely intact at times when the type I IFN response was suppressed. CONCLUSIONS: Collectively, these results demonstrate that poliovirus infection actively suppresses the host type I interferon response by blocking activation of IRF-3 and suggests that this is not mediated by cleavage of MDA-5 or IPS-1.

The emergence of viruses with zoonotic potential, i.e. with the potential ability to cross species barriers to infect unnatural hosts, poses a huge threat to humans. It is therefore essential to develop new methodologies to rapidly and efficiently generate attenuated virus vaccine candidates to attempt to control the threat. Viruses need to be able to at least partially inhibit the host’s innate defence mechanism, known as the interferon (IFN) system, to replicate efficiently in vivo and establish a productive infection. It has been previously reported that viruses that have lost their ability to circumvent the host’s IFN response, or IFN-sensitive viruses, are promising candidates for live attenuated virus vaccines. Here we report on the development of a cell-based method to attempt to rapidly select IFN-sensitive viruses that can not block IFN signalling, from wild-type virus populations. Lentivirus vectors containing selection markers (HSV-tk – Herpes Simplex virus thymidine kinase gene and pac – puromycin resistance gene) under the control of a tight IFN-inducible promoter (the murine Mx1 promoter) were generated and used to specifically engineer HEp2 cell lines, termed Mx GIPSE and Mx TIPSE, for this purpose. The developed methodology relies on the engineered cell lines and a selection procedure using exogenous IFN-α and puromycin: if a cell is infected with IFN-resistant virus, it will die in the presence of IFN-α and puromycin because IFN signalling will be blocked, thereby blocking the activation of the Mx1 promoter and consequent expression of pac; if a cell is infected with an IFN-sensitive virus, it will survive in the presence of IFN-α and puromycin because the Mx1 promoter will become activated through the IFN signalling pathway, leading to the expression of pac. IFN-sensitive viruses can then be rescued from the surviving cells, and amplified using IFN-permissive cell lines expressing viral IFN antagonist proteins (proteins that block the host’s IFN response). When tested on PIV5 strains CPI- (an IFN-sensitive virus) and CPI+ (an IFN-resistant virus), the developed method allowed the survival and amplification of cells infected with CPI-, whilst cell death was observed for cells infected with CPI+. Whilst the developed methodology seems promising, further developments of the system are required. The possibilities of using the developed methodology in combination with other techniques, such as FACS sorting and immune selection, to rapidly select IFN-sensitive mutant viruses from wild-type and mutagenised virus populations are discussed. The potential to use Mx TIPSE cells to select IFN-resistant revertant viruses from IFN-sensitive virus populations is also discussed. In addition, a high throughput screening assay has been developed using the engineered Mx GIPSE and Mx TIPSE cell lines to search for compounds that block IFN signalling or that block the action of viral IFN antagonist proteins. Compounds that block IFN signalling would potentially be useful as anti-inflammatory drugs whilst compounds that block the action of viral IFN antagonist proteins would be valuable as antiviral drugs.

Aim of this thesis was to study the contribution of the hosts immune system during tumor regression. A wild-type rejection model was studied in which tumor regression is mediated through an adaptive, T cell host response (Research article 1). Additionally, the relationship between VACV infection and cancer rejection was assessed by applying organism-specific microarray platforms to infected and non-infected xenografts. It could be shown that tumor rejection in this nude mouse model was orchestrated solely by the hosts innate immune system without help of the adaptive immunity. In a third study the inflammatory baseline status of 75 human cancer cell lines was tested in vitro which was correlated with the susceptibility to VACV and Adenovirus 5 (Ad5) replication of the respective cell line (Manuscript for Research article 3). Although xenografts by themselves lack the ability to signal danger and do not provide sufficient proinflammatory signals to induce acute inflammation, the presence of viral replication in the oncolytic xenograft model provides the "tissue-specific trigger" that activates the immune response and in concordance with the hypothesis, the ICR is activated when chronic inflammation is switched into an acute one. Thus, in conditions in which a switch from a chronic to an acute inflammatory process can be induced by other factors like the immune-stimulation induced by the presence of a virus in the target tissue, adaptive immune responses may not be necessary and immune-mediated rejection can occur without the assistance of T or B cells. However, in the regression study using neu expressing MMC in absence of a stimulus such as a virus and infected cancer cells thereafter, adaptive immunity is needed to provoke the switch into an acute inflammation and initiate tissue rejection. Taken together, this work is supportive of the hypothesis that the mechanisms prompting TSD differ among immune pathologies but the effect phase converges and central molecules can be detected over and over every time TSD occurs. It could be shown that in presence of a trigger such as infection with VACV and functional danger signaling pathways of the infected tumor cells, innate immunity is sufficient to orchestrate rejection of manifested tumors. / Ziel dieser Arbeit war, die Beteiligung des Wirts-eigenen Immunsystems bei der Tumoregression zu analysieren. Mittels eines Wildtyp-Regressionsmodells, wurde der Anteil des adaptiven Immunsystems studiert (Research-Artikel 1). Mit Hilfe von Organismus-spezifischen Mikroarrays und Genexpressionsanalysen konnte in einem Nacktmausmodell gezeigt werden, dass erfolgreiche, durch onkolytische VACV-vermittelte Tumortherapie auch ohne Beteiligung des adaptiven Immunsystems möglich ist (Research Artikel 2). In einer dritten Studie wurden 75 humane Tumorzelllinien auf ihren intrinsischen Entzündungsstatus hin getestet und bezüglich eines Zusammenhanges von diesem mit der Replikationsfähigkeit von VACV und Adenovirus 5 (Ad5) analysiert (Manuskript für den Research-Artikel 3). Obwohl Xenografts allein kein ausreichendes „Gefahrsignal“ geben und durch das Fehlen einer pro-inflammatorischen Stimulierung keine akute Entzündung verursachen können, ist die Infektion mit onkolytischem VACV ausreichend, um den Gewebe-spezifischen „Trigger“ darzustellen. In diesem Fall wird die Immunantwort aktiviert und nach der Hypothese des „Immunologic Constant of Rejection“ (ICR) geschieht dies, wenn eine chronische in eine akute Inflammation verändert wird. In dem beschriebenen onkolytischen Regressionsmodell ist die Präsenz des Virus ausreichend, um das Immunsystem zu aktivieren, d.h. die chronische Entzündung im Tumor in eine akute umzuwandeln. Dabei ist die adaptive Immunität mit T- und B-Zell-Aktivierung nicht notwendig für die Rückbildung des Tumors. In Abwesenheit eines solchen Stimulus, wie in der ersten Studie mit neu-exprimierenden MMCs, wird die Spezifität der adaptiven Immunantwort benötigt, um die akute Inflammation anzustoßen und die Tumorregression voranzutreiben. Zusammengefasst unterstützt diese Arbeit die Hypothese, dass die Mechanismen, die zu „tissue specific destruction“ (TSD) führen, in verschiedenen immunologischen Erkrankungen zwar divergieren, der Effektor-Mechanismus aber stets der Gleiche ist. Es zeigte sich, dass in Anwesenheit eines „triggers“, wie z.B. der VACV-Infektion und intakten „danger signaling pathways“ der Tumorzellen, die angeborene Immunität allein ausreicht, um die Tumorrückbildung zu vermitteln.

Um dos objetivos deste trabalho foi avaliar e comparar a taxa de prenhez de receptoras bovinas nulíparas, após transferência de embriões fecundados in vitro ou in vivo, associados ou não a um embrião partenogenético. Um total de 239 novilhas foram utilizadas, e as médias das taxas de prenhez aos 30 (TP30d) e 60 dias (TP 60d), foram comparadas em arranjo fatorial 8x2 entre G1 e G2 (fecundados in vitro), e em análise fatorial 3x2, entre G3 e G4 (fecundados in vivo). O delineamento utilizado foi: G1) transferência de 1 embrião fecundado in vitro (n=86); G2) transferência de 1 embrião partenogenético associado a 1 embrião fecundado in vitro (n=81); G3) transferência de 1 embrião fecundado in vivo (n=36) e G4) transferência de 1 embrião partenogenético associado a 1 embrião fecundado in vivo (n=36). A taxa de prenhez aos 30 dias para o G1 foi de 31,3% (27/86) e do G2 de 37,0% (30/81); P= 0,28. Para o G3, de 55,5% (20/36) e G4, de 75,0% (27/36); P=0,09. Não houve diferença (P=0,92) para taxa de prenhez aos 60 dias para o G1 em relação ao G2, com taxas de 24,6% (22/86) e 28,2% (23/81); respectivamente. Também não houve diferença (P=0,08) aos 60 dias para o grupo G3 em relação ao G4, com taxas de 44,4 % (16/36) e 61,1% (22/36), respectivamente. As perdas embrionárias no período foram de 14,81% para o G1, 33,34% para o G2, 25,00% para o G3 e 18,52% para o G4. As médias de peso ao nascimento dos animais pertencentes ao G1 e G2, foram de 26,3 Kg (±1,2; n=6) e 28,8 Kg (±2,1; n=7) respectivamente. As médias dos dias de gestação das receptoras dos grupos G1e G2 foram de 290 dias (±2,1) e 292,8 dias (±1,1) respectivamente. Para avaliar o embrião partenogenético em relação aos embriões fecundados in vitro após 9 dias de cultivo embrionário, foi determinado os resultados para as seguintes variáveis dependentes: taxa de clivagem, taxa de blastocisto, taxa de blastocisto eclodido, número de núcleos e freqüência do RNAm do IFN-&tau; em 6 sessões de produção embrionária. Os resultados foram analisados em fatorial 6x2. Foi observada diferença (P<0.05) nas taxas de clivagem, blastocisto e eclosão entre os tratamentos, houve diferença entre número de núcleos, 231 para os fecundados e 156 para os partenogenéticos (P=0.001), e para a expressão do gene do IFN-&tau;, os embriões partenogenéticos expressaram mais o gene do IFN em relação aos fecundados (P=0.001). Finalmente, Western blots foram realizados para identificar a proteína do IFN-&tau; bovino em embriões fecundados e partenogenéticos após 12 dias de cultivo. Bandas correspondente ao IFN-&tau; foram encontradas nos meios de cultivo onde permaneceram os embriões fecundados e partenogenéticos com peso molecular médio de 24 kDa. Não se detectou uma banda específica ao IFN-&tau; nos extratos embrionários. Tendo em vista o apresentado, conclui-se que apesar dos embriões partenogenéticos expressarem o gene e secretarem a proteína do IFN-&tau;, a transferência de um embrião partenogenético, associado a embriões fecundados in vitro ou in vivo, não favoreceu (P<0.05) ao aumento da taxa de prenhez, mas não prejudicou o desenvolvimento da gestação a termo em bovinos. / The aim of this work was to evaluate the bovine pregnancies rate of in vitro and in vivo produced embryos associated or not to a parthenogenetic embryo in heifers. Two hundred thirty nine recipients were used. The average of pregnancies rate were compared between G1 and G2 (in vitro) and between G3 and G4. (in vivo). The groups were: G1) 1 in vitro fertilized embryo (n=86); G2) 1 parthenogenetic embryo associated with 1 in vitro fertilized embryo (n=81); G3) 1 in vivo fertilized embryo (n=36) and G4) 1 parthenogenetic embryo associated with 1 in vivo fertilized embryo (n=36). Pregnancy rate at day 30 for G1 was 31.3% (27/86) similar to G2 37.0% (30/81); P=0.28. Group 3 rate was 55.5% (20/36) and also similar to G4, 75.0% (27/36); P=0.09. There was no difference (P=0.92) to pregnancy rate at day 60 for G1 compared with G2. The G1 and G2 rates were 26.74% (23/86) and G2 24.69% (20/81) respectively. Also, there were no difference (P=0.08) between G3 and G4. The G3 and G4 rates were 44.4 % (16/36) and 61.1% (22/36) respectively. The embryonic loss in this period was 14.81% for G1, 33.34% for G2, 25.00% for G3 and 18.52% for G4. The calf average weight of these animals from G1 and G2 were respectively 26.3Kg (±1.2; n=6) and 28.8 Kg (±2.1; n=7). The average pregnancy length of the recipients from G1 and G2 were respectively 290 days (±2.1) and 292.8 days (±1.1). To evaluate the parthenogenetic embryo quality, the cleavage rate, blastocyts rate, hatching rate, the number of nuclei, immunocitochemistry for IFN-, and the mRNA frequency of IFN-&tau; was determined in 9 days harvesting parthenogenetic and in vitro fertilized embryos. Difference (P<0.05) was observed in cleaveage, blastocysts and hatching rates, nuclei counting between fertilized embryo (n=231) compared to the parthenogenetic embryo (n=156) and the mRNA relative quantification of the IFN-&tau; gene showed that fertilized embryos have lower expression compared to parthenogenetic embryo (P<0.01). Finally, the western blots analysis for IFN-&tau; protein was performed in 12 days harvested embryos. Bands for IFN-&tau; were found in culture medium from fertilized and parthenogenetic embryos with average molecular weight of 24 kDa. The IFN-&tau; specific protein was not found in the embryonic extracts. In view of the presented results we conclude that despite the production and secretion of IFN-&tau; the association of the parthenogenetic embryo with the fertilized embryo do not favored the pregnancy establishment, however do not interfered in the development of bovine pregnancy to term.

Vinte e seis pacientes portadores de Hepatite C crônica e co-infectados pelo HIV, sem outras causas para hepatopatia e virgens para a terapia do VHC, foram tratados com Interferon Peguilado Alfa 2a de 40 KDa associado a Ribavirina por 48 semanas. Coletamos 20 amostras - horas 0, 4, 8, 12, 18, 24, 30, 36, 42, 48, dias 3, 4,7, 8, 15, 22, 29, 43, 57 e semana 12 - para quantificação do HCV (TaqMan®) e do HIV após o início da terapia. Os dados basais evidenciavam que o Genótipo 1 correspondia a 15 pacientes, enquanto o Genótipo 3 a 11. A mediana do VHC foi de 1.285.000 UI/ml, CD4 médio 573/mm3 e a média da carga viral do HIV 1109 cp/ml. Em uma análise por intenção de tratamento, o percentual obtido de resposta virológica sustentada (RVS) foi 26,9%. A única correlação estatisticamente significativa entre as variáveis analisadas e parâmetros cinéticos calculados, e a obtenção de RVS ocorreu entre RVS e Genótipo 3 (p < 0,04). Houve ajuste para os parâmetros cinéticos calculados para 18 pacientes. Pacientes com RVS apresentaram maiores taxas de eficiência do Interferon, eliminação de células infectadas e eliminação de vírions livres, assim como as Fases 1 e 2 foram mais rápidas entre eles. A ausência de redução do VHC de um log10 em 24 ou 48 horas, obteve um VPN de 100% para RVS. Ausência de queda de dois log10 nos dias oito e 29 obteve VPN de 92,31% e 100%, havendo associação entre a presença dessa resposta virológica precoce (RVP) e a obtenção de RVS (p=0,003 e p=0,01). A cinética viral mostra-se uma ferramenta útil no manejo clínico e predição muito precoce da ausência de RVS, o que é muito importante, particularmente para os co-infectados com o HIV, ante o complexo manuseio clínico dessa situação. / Twenty six co-infected patients were treated with PegIFN A2A and Ribavirin for 48 weeks. Twenty samples were drew at hours 0, 4, 8, 12, 18, 24, 30, 36, 42, 48, days 3, 4, 7, 8, 22, 29, 43, 57 and week 12 to HIV and HCV (TaqMan®) quantification after began medication. Basal data showed Genotype 1 in 15 patients, 3 in 11. Median HCV were 1.285.000 UI/ml, mean CD4 573/mm3 and mean HIV 1109 cp/ml. SVR was associated only with Genotype 3 (p<0,04). Overal SVR (ITT) were 26,9%. We obtained fitted viral kinetics parameters for 18 patients. Patients with SVR showed higher Interferon efficacy, infected cells clearance, free virion clearance and faster phase 1 and 2. Reduction on HCV load of less than one log10 at 24 or 48 hs had a NPV of 100% for SVR. Less than two log decay at days 8 and 29 had 92,31 and 100% of NPV and the presence of those reduction were associated with SVR p=0,003 and p=0,01). Viral kinetics are an important tool regarding clinic management and very early prediction of absence of SVR, wich is very important for the HIV co-infected patients for whom the clicical management is very complex.

Perdas embrionárias e alterações gestacionais são frequentemente observadas em prenhezes de embriões bovinos manipulados in vitro. Sabe-se que tais anormalidades são resultantes de alterações epigenéticas ocasionadas pela manipulação dos gametas e/ou do embrião durante as técnicas de reprodução assistida (ARTs), com destaque para as técnicas de fecundação in vitro (FIV) e da clonagem por transferência nuclear de células somáticas (SCNT). Tais alterações resultam em distúrbios no desenvolvimento do concepto em algum momento crítico entre a fertilização e o parto, fornecendo bons modelos de estudos sobre a fisiopatologia de perdas embrionárias e dos distúrbios de desenvolvimento. Caracterizam-se como momentos críticos após a transferência do embrião (TE) o desenvolvimento embrionário no útero, o reconhecimento materno da gestação, a placentação e o desenvolvimento da placenta e do feto, os quais tem de ser transpassados sem nenhuma falha, permitindo um desenvolvimento normal do concepto até o termo. Sendo assim, o presente trabalho abordou três fases distintas do amplo período gestacional em prenhezes por ARTs. O Estudo 1 foi realizado durante o período peri-reconhecimento materno da gestação e objetivou descrever a abundância de expressão de genes estimulados pelo interferon tau (ISGs) de células mononucleares do sangue periférico (PBMCs) maternas em gestações oriundas de ARTs no primeiro mês de gestação; o Estudo 2 compreendeu os primeiros 35 dias de gestação e objetivou descrever as mudanças morfológicas e vasculares do complexo útero-concepto-ovário e o estímulo à expressão de ISGs em PBMCs em gestações de conceptos clonados por SCNT com diferentes fenótipos de desenvolvimento, sendo esses denominados gestação anembrionada e CL persistente; e o Estudo 3 foi conduzido durante o período pré-parto e objetivou descrever as alterações na produção de esteroides sexuais e corticosteroides em gestações produzidas por ARTs. Três hipóteses foram testadas: (1) Gestações de conceptos clonados por SCNT apresentam uma baixa e mais tardia estimulação de ISGs em PBMCs maternas quando comparadas com gestações de conceptos produzidos por FIV e IA; (2) O concepto clonado por SCNT apresenta um menor estímulo sobre mudanças morfológicas e vasculares do complexo útero-ovário e ISGs em PBMCs maternas durante os primeiros 35 dias de gestação, quando comparado com conceptos oriundos de IA; e (3) Gestações de embriões oriundos de ARTs apresentam alterações na dinâmica esteroidogênica no pré-parto quando comparados com gestações de IA. No estudo 1 foram coletadas amostras de sangue de gestações produzidas por inseminação artificial (IA), FIV e clonagem por SCNT, nos dias 15, 18, 20, 22, 24, 26, 28 e 31 pós-ovulação e foi realizada mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 2, gestações produzidas por IA e clonagem por SCNT, foram submetidas a escaneamentos ultrassonográficos dos ovários, útero e concepto a cada 3 dias do dia 14 ao 35 (dia 0 = ovulação) e amostras de sangue foram coletadas nos dias 15, 18, 20, 22, 24, 26, 28 e 31 para mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 3, foram coletadas amostras de sangue no último mês em gestações naturais, oriundas de FIV e de clonagem por SCNT para análise hormonal de 10 esteroides utilizando o método de espectrometria de massas multi-hormonal de alta resolução LC-MS/MS. O primeiro estudo mostrou semelhanças na expressão de genes estimulados pelo IFNT em gestações oriundas de ARTs e produzidas por IA. Entretanto, a estimulação nas gestações oriundas de ARTs aparentou ser quatro dias mais prolongada, sugerindo uma maior funcionalidade do trofectoderma em conceptos oriundos de ARTs. O segundo estudo demonstrou um aumento na expressão de ISGs em PBMCs maternas tanto em gestações de conceptos normais quanto em anormais, justificando a manutenção da função luteal mesmo na ausência de detecção do concepto por ultrasonografia. No terceiro estudo, demonstrou-se alterações na esteroidogênese nas gestações de embriões FIV e clonados no último mês de gestação, sendo essas compatíveis com a hiperativação da enzima aromatase durante todo o último mês de gestações oriundas de FIV e hiperativação das enzimas P450C11 e P450C21 trinta dias antes do parto em gestações oriundas de clonagem por SCNT. O presente estudo concluiu que conceptos oriundos de FIV e clonagem por SCNT apresentam um prolongamento no estímulo de ISGs pelo IFNT, conceptos clonados anormalos apresentam estímulo de ISGs, o que justifica a manutenção da função luteal, e, por fim, a cascata esteroidonênica que culmina com o parto apresenta-se alterada em gestações oriundas de FIV e clonagem por SCNT. / Pregnancy losses and gestational abnormalities are frequently observed in pregnancies from in vitro produced embryos in bovines. It is known that these abnormalities are due to epigenetic changes from the manipulation of gametes and/or embryo during the use of assisted reproduction techniques (ARTs), especially for the in vitro fertilization (IFV) and cloning by somatic cells nuclear transfer (SCNT). These changes results in disturbances of conceptus development in any critical stage between the fertilization and parturition, which provides good models for the study of physiopathology of embryo losses and disturbances of development. Critical stages after the embryo transfer (ET) to the uterus are characterized as the maternal recognition of pregnancy, placentation, and fetal-placental development, which needs to be surpassed without failures, in order to develop a normal conceptus until term. Therefore, the present work approached three distinct phases of the wide gestational period in pregnancies from ARTs. The Study 1 was conducted during the maternal peri-recongnition of pregnancy period and aimed to describe the expression of interferon stimulated genes (ISGs) in maternal peripheral blood mononuclear cells (PBMCs) in pregnancies derived of ARTs; the Study 2 comprise the first 35 days of pregnancy and aimed to describe morphological and vascular changes of the complex uterus-ovaries-conceptus, as well as the expression of ISGs in maternal PBMCs in pregnancies of conceptus cloned by SCNT with different phenotypes of development, denominated as anembryonic gestation and persistent CL; the Study 3 was conducted during the pre-partum period and aimed to describe changes in the production of sexual steroids and corticosteroids during the last month of pregnancies derived of ARTs. Three hypothesis were tested: (1) Pregnancies of conceptus cloned by SCNT presented a decrease and delay in the stimulation of ISGs in maternal PBMCs when compared with conceptuses produced by IFV and AI; (2) Stimulus from the conceptus for changes in the morphology and vasculature of the the uterus-ovarian complex, detected by ultrasonography in B and Doppler modes, and the stimulation of ISGs in maternal PBMCs during the first 35 days of pregnancy of conceptus cloned by SCNT are less intense when compared with conceptus derived from AI; and (3) Pregnancies derived of ARTs present changes in the steroidogenic dynamics in the pre-partum, when compared with pregnancies derived from AI. In Study 1 blood samples were collected from pregnancies produced by AI, IVF, and cloning by SCNT, at days 15, 18, 20, 22, 24, 26, 28, and 31 post-ovulation for the measurement of abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 2, pregnancies derived of AI and cloning by SCNT, were submitted to ultrasonographic scans for the evaluation and description of morphological and vascular changes in ovaries, uterus, and conceptus every 3 days from day 14 to 35 (day 0 = ovulation) and blood samples were collected on days 15, 18, 20, 22, 24, 26, 28 e 31 for the measurement of the abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 3, blood samples were collected during the last month of pregnancies naturally conceived, derived of IVF, and cloned by SCNT for the analysis of 10 steroids using the method of mass spectrometry high resolution LC-MS/MS. The first study showed similarities in the ISGs expression stimulation in pregnancies derived of ARTs and AI. However, the stimulation in the ART derived pregnancies was apparently 4 days longer, suggesting a greater placental function in conceptus derived of ARTs. The second study showed an increase in ISG expression in both normal and abnormal conceptus development, which justifies the maintenance of CL in the absence of a conceptus structure detected by ultrasonography. In the third study, was detected changes in the steroidogenesis of pregnancies derived of IFV and cloning by SCNT during the last month of pregnancy, which are compatible with the hyperactivation of the aromatase enzyme during the last month of IFV derived pregnancies, and hyperactivation of the enzymes P450C11 and P450C21 thirty days before parturition in pregnancies derived of cloning by SCNT. The present study concludes that conceptus derived of IFV and cloning by SCNT present a prolonged stimulus of ISGs, cloned conceptus with anomalous development presents a stimulus of ISGs, which justifies the CL function maintenance, and, ultimately, the steroidogenic cascade that culminates with the term is altered in pregnancies derived from IFV and cloning by SCNT.

A imunoterapia baseada em MoDC constitui uma estratégia para tratamento de indivíduos HIV+. Protocolos para obtenção de MoDC em geral utilizam IL-4 e GM-CSF (IL4-DC). Alguns estudos utilizam as IFN-DC (IFN-&alpha; + GM-CSF), que exibem um fenótipo combinado de DC mielóide, DC plasmocitóide (pDC) e célula NK. Esse perfil misto pode aperfeiçoar a imunoterapia para pacientes HIV+. Para tanto, monócitos de pacientes HIV+ foram cultivados com GM-CSF e IL-4 ou IFN-&alpha; por 5 dias e estimuladas por 48 horas com pulso de HIV inativado por AT-2 e/ou coquetel de citocinas pró-inflamatórias. Avaliamos a expressão de moléculas de superfície de IFN-DC e ativação de linfócitos T por citometria de fluxo; produção de citocinas IL-12 e IL-10 por ELISA. IFN-DC apresentaram morfologia e fenótipo basais ativados e características de pDC e célula NK, diferente das IL4-DC. As IFN-DC foram capazes de produzir IL-12, estimular a proliferação e produção de IFN-&gamma; de linfócitos TCD4 e CD8, porém similares às IL4-DCs. IFN-DC são capazes de estimular resposta de linfócitos T tanto quanto IL4-DC. / Immunotherapy based on MDDCs is a strategy for treating HIV-infected patients. Alternatively to the conventional protocol for DC differentiation based on IL-4 and GM-CSF (IL4-DC) some studies suggest the use of IFN-DC (IFN-&alpha; + GM-CSF). These cells exhibit a combined phenotype of myeloid DC, plasmacytoid DC (pDC) and NK. Considering the mixed profile of IFN-DCs alternative protocols can bring novel elements for immunotherapy. Monocytes isolated from HIV-infected patients were cultured in the presence of GM-CSF and IL-4 or IFN-&alpha;. On day 5 DCs were pulsed with AT-2-inactivated HIV and stimulated for 48 hours with a cocktail of proinflammatory cytokines. We assessed IFN-DC surface markers expression and T cell activation by flow cytometry; IL-10 and IL-12 production by ELISA. IFN-DC showed activated morphological and phenotypic features during basal state of maturity and exhibited features of pDC and NK different from IL4-DC. The IFN-DC like IL4-DC were able to produce IL-12 and stimulated T cells. So, the IFN-DC were able to stimulate the T cells as well as IL4-DCs.

Sia Sin Fun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 105-121). / Abstracts in English and Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vi / ABBREVIATIONS --- p.xi / LIST OF FIGURES --- p.xv / LIST OF TABLES --- p.xvi / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- The interferon / Chapter 1.1.1 --- Classification of interferons --- p.1 / Chapter 1.1.1.1 --- Type IIFN --- p.2 / Chapter 1.1.1.2 --- Type II IFN --- p.3 / Chapter 1.1.2 --- Biosynthesis of IFN --- p.3 / Chapter 1.1.3 --- IFN-α/β receptor and signal transduction --- p.8 / Chapter 1.1.4 --- Functions induced by IFN / Chapter 1.1.4.1 --- Antiviral activity of IFN-α/β --- p.11 / Chapter 1.1.4.1.1 --- PKR (double-stranded RNA-dependent protein kinase) --- p.11 / Chapter 1.1.4.1.2 --- The 2-5A synthetase/RNase L system (The 2-5A system) --- p.16 / Chapter 1.1.4.1.3 --- Mx proteins --- p.17 / Chapter 1.1.4.2 --- Immunomodulatory function of IFN-α/β --- p.18 / Chapter 1.1.4.3 --- Inhibition of cell growth --- p.18 / Chapter 1.1.4.4 --- Control of apoptosis --- p.19 / Chapter 1.1.5 --- The significance of IFN system --- p.20 / Chapter 1.1.6 --- Subtype of murine IFN-α --- p.21 / Chapter 1.1.7 --- Production of IFN in response to infection --- p.23 / Chapter 1.1.8 --- Existing methods to determine the IFN-α subtypes productionin response to stimulus --- p.24 / Chapter 1.2 --- Influenza virus --- p.27 / Chapter 1.2.1 --- Classification --- p.27 / Chapter 1.2.2 --- The structure of influenza virus --- p.29 / Chapter 1.2.3 --- The viral genome and proteins --- p.29 / Chapter 1.2.4 --- Replicative cycle of influenza virus / Chapter 1.2.4.1 --- "Virus adsorption, entry and uncoating" --- p.31 / Chapter 1.2.4.2 --- Transcription and replication of vRNA --- p.31 / Chapter 1.2.4.3 --- Synthesis of viral proteins --- p.32 / Chapter 1.2.4.4 --- Packaging and budding of progeny virus --- p.33 / Chapter 1.2.5 --- Viral inhibition of the IFN response --- p.33 / Chapter 1.3 --- Aim of study --- p.35 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Overall procedures --- p.37 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- "Cell line, bacterial strain and vector" --- p.40 / Chapter 2.2.2 --- Chemicals --- p.40 / Chapter 2.2.3 --- "Culture media, buffer and other solutions" --- p.41 / Chapter 2.2.4 --- Reagents and nucleic acids --- p.41 / Chapter 2.2.5 --- Reaction kits --- p.42 / Chapter 2.2.6 --- Solutions --- p.42 / Chapter 2.2.7 --- Solutions of reaction kits --- p.43 / Chapter 2.2.8 --- Major equipments --- p.44 / Chapter 2.2.9 --- Primers --- p.44 / Chapter 2.2.10 --- Cell lysate --- p.45 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Design of IFN-α whole coding region and subtype specific primers using OLIGO´ёØ ver 50 --- p.46 / Chapter 2.3.2 --- Construction of plasmid DNA with the whole coding region of IFN-α gene / Chapter 2.3.2.1 --- Isolation of genomic DNA from L929 cells --- p.46 / Chapter 2.3.2.2 --- Amplification of whole coding region of IFN-α subtypes --- p.47 / Chapter 2.3.2.3 --- Preparation of plasmid DNA --- p.48 / Chapter 2.3.2.4 --- Restriction digestion of the vector pBluescript SKII (-) with SmaI --- p.48 / Chapter 2.3.2.5 --- Purification of DNA fragments from agarose gel --- p.49 / Chapter 2.3.2.6 --- Blunt end ligation --- p.49 / Chapter 2.3.2.7 --- Transformation --- p.49 / Chapter 2.3.2.8 --- Screening by polymerase chain reaction --- p.50 / Chapter 2.3.2.9 --- DNA sequencing --- p.50 / Chapter 2.3.3 --- Test on IFN-α subtype specific primers / Chapter 2.3.3.1 --- Determination of the specificity of IFN-α subtype specific primers by PCR --- p.51 / Chapter 2.3.3.2 --- Determination of the amplification of a particular subtype in the presence of excess of other templates --- p.51 / Chapter 2.3.4 --- Induction of expression of IFN in fibroblast L929 cells / Chapter 2.3.4.1 --- By chemical agents Poly(I) Poly(C) and DEAE --- p.52 / Chapter 2.3.4.2 --- By infection with influenza virus (A/NWS/33 and B/Lee/40) --- p.52 / Chapter 2.3.5 --- Detection of IFN-α subtypes expression / Chapter 2.3.5.1 --- Isolation of total RNA by guandidium thiocyanate - cesium chloride ultracentrifugation --- p.53 / Chapter 2.3.5.2 --- Synthesis of first strand cDNA --- p.54 / Chapter 2.3.5.3 --- Normalization of RNA samples --- p.54 / Chapter 2.3.5.4 --- RT-PCR amplification of the IFN-α subtypes --- p.54 / Chapter 2.3.5.5 --- "RT-PCR amplification of IFN-γ, IFN-α receptor 1，IFN-α receptor 2 (transmembrane and soluble form) and IFN-(3" --- p.55 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Designing of primers for IFN-α genes --- p.56 / Chapter 3.1.1 --- Design of IFN-α primers for amplification of whole coding region --- p.56 / Chapter 3.1.2 --- Design of IFN-α subtype specific primers --- p.58 / Chapter 3.2 --- Cloning of the IFN-α subtype genes / Chapter 3.2.1 --- Purification of genomic DNA --- p.59 / Chapter 3.2.2 --- PCR conditions used for amplification of whole coding region of mIFN-α subtypes --- p.61 / Chapter 3.2.3 --- Subcloning the whole coding region of IFN-α genes from amplified fragments --- p.63 / Chapter 3.3 --- Optimization of specific amplification condition for subtype specific primers by cross-PCR --- p.64 / Chapter 3.4 --- Determination of the amplification of a particular subtype in the excess of other templates --- p.67 / Chapter 3.5 --- Determination of the gene expression in poly (I) poly (C) treated L929 cells / Chapter 3.5.1 --- Spectrophotometric analysis of total RNA --- p.70 / Chapter 3.5.2 --- Agarose gel electrophoresis of RNA --- p.72 / Chapter 3.5.3 --- Normalization of RNA sample --- p.73 / Chapter 3.5.4 --- Detection of IFN-α subtypes mRNA in L929 cell induced with poly(I) poly(C)-DEAE dextran --- p.74 / Chapter 3.5.5 --- "Detection of IFN-γ, IFN-α/β receptor and IFN-β expressionin Poly(I) Poly(C)-DEAE dextran or DEAE dextran treated L929 cells" --- p.70 / Chapter 3.6 --- Measurement of gene expression in L929 cells infected with influenza virus / Chapter 3.6.1 --- Spectrophotometric analysis of total RNA --- p.83 / Chapter 3.6.2 --- Agarose gel electrophoresis of RNA samples --- p.84 / Chapter 3.6.3 --- Normalization of RNA samples --- p.86 / Chapter 3.6.4 --- Detection of IFN-α subtypes mRNA in L929 cell infected with influenza A/NWS/33 virus --- p.87 / Chapter 3.6.5 --- Detection of IFN-α subtypes mRNA in L929 cells infected with influenza B/Lee/40 virus --- p.90 / Chapter 3.6.6 --- "Detection of IFN-γ, IFN-α/β receptor and IFN-β expression in L929 cells infected with A/NWS/33 and B/Lee/40" --- p.93 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.97 / REFERENCES --- p.105 / APPENDIX --- p.122