Mechanism of interleukin-8 induction by human cytomegalovirus UL76 protein

Dissertation presented to obtain the Ph.D degree in Biology

Transplante renal : desigualdades no acesso

RESUMO - Em Portugal estima-se que existam cerca de 14 mil insuficientes renais crónicos e estima-se que em 2025 sejam 24 mil. As alternativas de tratamento são: hemodiálise, diálise peritoneal ou o transplante renal. Das alternativas de tratamento, o transplante renal é considerado a melhor alternativa terapêutica proporcionando melhor qualidade de vida, aumentando a sobrevida dos doentes, caracterizando-se por ser menos oneroso e por apresentar melhor custo- efectivo, quando comparado com hemodiálise ou diálise peritoneal. Portugal situa-se entre os primeiros da Europa, relativo ao número de transplantes renais efectuados (56,1 por milhão de habitante), em 2010 efectuaram-se 573 transplantes renais. Apesar disso, muitos são os doentes que continuam em lista de espera a aguardar transplante, em média os doentes esperam cerca de dois a três anos por um transplante renal, quando o tempo ideal seria três a seis meses. Por outro lado, estudos internacionais demonstram que existem desigualdades no acesso ao transplante renal, assim à semelhança de outros países torna-se pertinente estudar a realidade portuguesa em relação à temática da desigualdade no acesso, dado o objectivo primordial do Serviço Nacional de Saúde de garantir a equidade nos cuidados de saúde. Este trabalho tem como objectivo principal avaliar se factores como o sexo, idade a localização geográfica influenciam o acesso ao transplante renal, contribuindo para desigualdades no acesso. Este trabalho baseou-se na base de dados dos doentes inscritos em lista de espera para transplante renal, respeitante à área de abrangência do Centro de Histocompatibilidade do Sul. Caracterizou-se a população quanto ao sexo, idade, concelho, região de saúde e unidade de transplantação. Determinou-se ainda, os tempos médios de espera para inscrição em lista activa e para transplante por sexo, idade, região de saúde e unidade de transplantação.Dos resultados obtidos salienta-se que as desigualdades encontradas no acesso ao transplante renal verificam-se entre o início do tratamento até à inscrição em lista activa para transplante. Depois dos doentes em lista activa, o tempo de espera médio não é influenciado significativamente pelo sexo, idade ou localização geográfica.

Restriction enzyme analysis of the human cytomegalovirus genome in specimens collected from immunodeficient patients in Belém, State of Pará, Brazil

INTRODUCTION: Human cytomegalovirus is an opportunistic betaherpesvirus that causes persistent and serious infections in immunodeficient patients. Recurrent infections occur due to the presence of the virus in a latent state in some cell types. It is possible to examine the virus using molecular methods to aid in the immunological diagnosis and to generate a molecular viral profile in immunodeficient patients. The objective of this study was to characterize cytomegalovirus genotypes and to generate the epidemiological and molecular viral profile in immunodeficient patients. METHODS: A total of 105 samples were collected from immunodeficient patients from the City of Belém, including newborns, hemodialysis patients, transplant recipients and HIV+ patients. An IgG and IgM antibody study was completed using ELISA, and enzymatic analysis by restriction fragment length polymorphism (RFLP) was performed to characterize viral genotypes. RESULTS: It was observed that 100% of the patients had IgG antibodies, 87% of which were IgG+/IgM-, consistent with a prior infection profile, 13% were IgG+/IgM+, suggestive of recent infection. The newborn group had the highest frequency (27%) of the IgG+/IgM+ profile. By RFLP analysis, only one genotype was observed, gB2, which corresponded to the standard AD169 strain. CONCLUSIONS: The presence of IgM antibodies in new borns indicates that HCMV continues to be an important cause of congenital infection. The low observed genotypic diversity could be attributed to the small sample size because newborns were excluded from the RFLP analysis. This study will be continued including samples from newborns to extend the knowledge of the general and molecular epidemiology of HCMV in immunodeficient patients.

Avaliação económica em transplantação renal : abordagem estratégica do processo de doação e análise de custo-utilidade do programa de transplantação renal versus hemodiálise

SUMÁRIO - O desafio atual da Saúde Pública é assegurar a sustentabilidade financeira do sistema de saúde. Em ambiente de recursos escassos, as análises económicas aplicadas à prestação dos cuidados de saúde são um contributo para a tomada de decisão que visa a maximização do bem-estar social sujeita a restrição orçamental. Portugal é um país com 10,6 milhões de habitantes (2011) com uma incidência e prevalência elevadas de doença renal crónica estadio 5 (DRC5), respetivamente, 234 doentes por milhão de habitantes (pmh) e 1.600 doentes/pmh. O crescimento de doenças associadas às causas de DRC, nomeadamente, diabetes Mellitus e hipertensão arterial, antecipam uma tendência para o aumento do número de doentes. Em 2011, dos 17.553 doentes em tratamento substitutivo renal, 59% encontrava-se em programa de hemodiálise (Hd) em centros de diálise extra-hospitalares, 37% viviam com um enxerto renal funcionante e 4% estavam em diálise peritoneal (SPN, 2011). A lista ativa para transplante (Tx) renal registava 2.500 doentes (SPN 2009). O Tx renal é a melhor modalidade terapêutica pela melhoria da sobrevida, qualidade de vida e relação custo-efetividade, mas a elegibilidade para Tx e a oferta de órgãos condicionam esta opção. Esta investigação desenvolveu-se em duas vertentes: i) determinar o rácio custo-utilidade incremental do Tx renal comparado com a Hd; ii) avaliar a capacidade máxima de dadores de cadáver em Portugal, as características e as causas de morte dos dadores potenciais a nível nacional, por hospital e por Gabinete Coordenador de Colheita e Transplantação (GCCT), e analisar o desempenho da rede de colheita de órgãos para Tx. Realizou-se um estudo observacional/não interventivo, prospetivo e analítico que incidiu sobre uma coorte de doentes em Hd que foi submetida a Tx renal. O tempo de seguimento mínimo foi de um ano e máximo de três anos. No início do estudo, colheram-se dados sociodemográficos e clínicos em 386 doentes em Hd, elegíveis para Tx renal. A qualidade de vida relacionada com a saúde (QVRS) foi avaliada nos doentes em Hd (tempo 0) e nos transplantados, aos três, seis, 12 meses, e depois, anualmente. Incluíram-se os doentes que por falência do enxerto renal transitaram para Hd. Na sua medição, utilizou-se um instrumento baseado em preferências da população, o EuroQol-5D, que permite o posterior cálculo dos QALY. Num grupo de 82 doentes, a QVRS em Hd foi avaliada em dois tempos de resposta o que permitiu a análise da sua evolução. Realizou-se uma análise custo-utilidade do Tx renal comparado com a Hd na perspetiva da sociedade. Identificaram-se os custos diretos, médicos e não médicos, e as alterações de produtividade em Hd e Tx renal. Incluíram-se os custos da colheita de órgãos, seleção dos candidatos a Tx renal e follow-up dos dadores vivos. Cada doente transplantado foi utilizado como controle de si próprio em diálise. Avaliou-se o custo médio anual em programa de Hd crónica relativo ao ano anterior à Tx renal. Os custos do Tx foram avaliados prospetivamente. Considerou-se como horizonte temporal o ciclo de vida nas duas modalidades. Usaram-se taxas de atualização de 0%, 3% e 5% na atualização dos custos e QALY e efetuaram-se análises de sensibilidade one way. Entre 2008 e 2010, 65 doentes foram submetidos a Tx renal. Registaram-se, prospetivamente, os resultados em saúde incluíndo os internamentos e os efeitos adversos da imunossupressão, e o consumo dos recursos em saúde. Utilizaram-se modelos de medidas repetidas na avaliação da evolução da QVRS e modelos de regressão múltipla na análise da associação da QVRS e dos custos do transplante com as características basais dos doentes e os eventos clínicos. Comparativamente à Hd, observou-se melhoria da utilidade ao 3º mês de Tx e a qualidade de vida aferida pela escala EQ-VAS melhorou em todos os tempos de observação após o Tx renal. O custo médio da Hd foi de 32.567,57€, considerado uniforme ao longo do tempo. O custo médio do Tx renal foi de 60.210,09€ no 1º ano e 12.956,77€ nos anos seguintes. O rácio custo-utilidade do Tx renal vs Hd crónica foi de 2.004,75€/QALY. A partir de uma sobrevivência do enxerto de dois anos e cinco meses, o Tx associou-se a poupança dos custos. Utilizaram-se os dados nacionais dos Grupos de Diagnóstico Homogéneos e realizou-se um estudo retrospectivo que abrangeu as mortes ocorridas em 34 hospitais com colheita de órgãos, em 2006. Considerou-se como dador potencial o indivíduo com idade entre 1-70 anos cuja morte ocorrera a nível hospitalar, e que apresentasse critérios de adequação à doação de rim. Analisou-se a associação dos dadores potenciais com características populacionais e hospitalares. O desempenho das organizações de colheita de órgãos foi avaliado pela taxa de conversão (rácio entre os dadores potenciais e efetivos) e pelo número de dadores potenciais por milhão de habitantes a nível nacional, regional e por Gabinete Coordenador de Colheita e Transplantação (GCCT). Identificaram-se 3.838 dadores potenciais dos quais 608 apresentaram códigos da Classificação Internacional de Doenças, 9.ª Revisão, Modificações Clínicas (CID- 9-MC) que, com maior frequência, evoluem para a morte cerebral. O modelo logit para dados agrupados identificou a idade, o rácio da lotação em Unidades de Cuidados Intensivos e lotação de agudos, existência de GCCT e de Unidade de Transplantação, e mortalidade por acidente de trabalho como fatores preditivos da conversão dum dador potencial em efetivo e através das estimativas do modelo logit quantificou-se a probabilidade dessa conversão. A doação de órgãos deve ser assumida como uma prioridade e as autoridades em saúde devem assegurar o financiamento dos hospitais com programas de doação, evitando o desperdício de órgãos para transplantação, enquanto um bem público e escasso. A colheita de órgãos deve ser considerada uma opção estratégica da atividade hospitalar orientada para a organização e planeamento de serviços que maximizem a conversão de dadores potenciais em efetivos incluindo esse critério como medida de qualidade e efetividade do desempenho hospitalar. Os resultados deste estudo demonstram que: 1) o Tx renal proporciona ganhos em saúde, aumento da sobrevida e qualidade de vida, e poupança de custos; 2) em Portugal, a taxa máxima de eficácia da conversão dos dadores cadavéricos em dadores potenciais está longe de ser atingida. O investimento na rede de colheita de órgãos para Tx é essencial para assegurar a sustentabilidade financeira e promover a qualidade, eficiência e equidade dos cuidados em saúde prestados na DRC5.

Use of tacrolimus in rescue therapy of acute and chronic rejection in liver transplantation

PURPOSE: To study the indications and results of tacrolimus as rescue therapy for acute cellular or chronic rejection in liver transplantation. PATIENTS AND METHODS: Eighteen liver transplant recipients who underwent rescue therapy with tacrolimus between March 1995 and August 1999 were retrospectively studied. The treatment indication, patients, and graft situation were recorded as of October 31st, 1999. The response to tacrolimus was defined as patient survival with a functional graft and histological reversal of acute cellular, or for chronic rejection, bilirubin serum levels decreasing to up to twice the upper normal limit. RESULTS: Fourteen cases (77.8%) presented a good response. The response rate for the different indications was: (1) acute cellular + sepsis - 0/1 case; (2) recurrent acute cellular - 1/1 case; (3) OKT3-resistant acute cellular - 2/2 cases; (4) steroid-resistant acute cellular + active viral infection - 3/3 cases; (5) chronic rejection - 8/11 cases (72.7% response rate). The 4 patients who did not respond died. CONCLUSION: Tacrolimus rescue therapy was successful in most cases of acute cellular and chronic rejection in liver transplantation.

Permanent and temporary pacemaker implantation after orthotopic heart transplantation

PURPOSE:To determine the indication for and incidence and evolution of temporary and permanent pacemaker implantation in cardiac transplant recipients. METHODS: A retrospective review of 114 patients who underwent orthotopic heart transplantation InCor (Heart Institute USP BR) between March 1985 and May 1993. We studied the incidence of and indication for temporary pacing, the relationship between pacing and rejection, the need for pemanent pacing and the clinical follow-up. RESULTS: Fourteen of 114 (12%)heart transplant recipients required temporary pacing and 4 of 114 (3.5%) patients required permanent pacing. The indication for temporary pacing was sinus node dysfunction in 11 patients (78.5%) and atrioventricular (AV) block in 3 patients (21.4%). The indication for permanent pacemaker implantation was sinus node dysfunction in 3 patients (75%) and atrioventricular (AV) block in 1 patient (25%). We observed rejection in 3 patients (21.4%) who required temporary pacing and in 2 patients (50%) who required permanent pacing. The previous use of amiodarone was observed in 10 patients (71.4%) with temporary pacing. Seven of the 14 patients (50%) died during follow-up. CONCLUSION: Sinus node dysfunction was the principal indication for temporary and permanent pacemaker implantation in cardiac transplant recipients. The need for pacing was related to worse prognosis after cardiac transplantation.

Treatment of persistent rejection with methotrexate in stable patients submitted to heart transplantation

OBJECTIVE:To evaluate the use of methotrexate for the treatment of recurrent rejection in heart transplant recipients. METHODS: We studied 6 patients submitted to heart transplantation that showed rejection grade > or = 3A (ISHLT) in two consecutives endomyocardial biopsy specimens. The dose was 11.26±3.75mg/week. The evaluated data were: ventricular function, endomyocardial biopsy, white cell count and number of rejection episodes before and after methotrexate administration. RESULTS: There was a reduction in the number of rejection episodes (5.17±1.47 before methotrexate; 2.33±1.75 after 6 months and 3.17±2.99 after 12 months of treatment, p=0.0193). The ventricular function was normal with ejection fraction of 76.5±4.80 before and 75.6±4.59 after methotrexate (p=0.4859). One patient did not finish the treatment because he showed signs of rejection associated with severe pericardial effusion. Five patients had a reduction in the white cell count (8,108±23.72 before and 5650±1350 after methotrexate, p=0.0961). One pulmonary infection with complete resolution after antibiotic treatment was observed. CONCLUSION: Methotrexate in low doses is an effective adjunct therapy in the treatment of recurrent rejection after heart transplantation.

HCV infection after liver transplantation is associated with lower levels of alloreactive CD4+CD25+CD45RO+IL-7R+high+ T cells

Aim: Expression of IL-7R discriminates alloreactive CD4 T cells (Foxp3 negative), from IL-7Rlow regulatory CD4 T cells (Foxp3 positive). Chronic hepatitis C virus infection (HCV) reduces expression of IL-7R on T cells thus promoting persistence of infection. The aim of this study was to analyze the effect of HCV infection on the expression of IL-7R of activated CD4+ T cells in liver transplant patients. Patients and methods: We analyzed PBMC from liver transplant recipients for the expression of CD4, CD25, FoxP3, IL-7R (24 HCV negative and 29 HCV-chronically infected). We compared these data with non-transplanted individuals (52 HCV-chronically infected patients and 38 healthy donors). Results: In HCV-infected liver transplant recipients, levels of CD4+CD25+CD45RO+IL-7R+ T cells were significantly reduced (10.5+/-0.9%) when compared to non-HCV-infected liver transplant recipients (17.6+/-1.4%) (P<0.001), while both groups (HCV-infected and negative transplant recipients) had significantly higher levels than healthy individuals (6.6+/-0.9%) (P<0.0001). After successful antiviral therapy (sustained antiviral response), 6 HCV-infected transplant recipients showed an increase of CD4+CD25+CD45RO+IL-7R+ T cells, reaching levels similar to that of non-HCVinfected recipients (10.73+/-2.63% prior therapy versus 21.7+/-6.3% after clearance of HCV). (P<0.05) In 4 non-responders (i.e. HCVRNA remaining present in serum), levels of CD4+CD25+CD45RO+IL-7R+ T cells remained unmodified during and after antiviral treatment (11.8+/- 3.3% versus 11.3+/-3.3% respectively). Conclusions: Overall, these data indicate that CD4+CD25+CD45RO+IL-7R+ T cells appear to be modulated by chronic HCV infection after liver transplantation. Whether lower levels of alloreactive T cells in HCV-infected liver transplant recipients are associated with a tolerogenic profile remains to be studied.

Prise en charge par le praticien des infections après transplantation d'organes solides [Management of infection after organ transplantation by the primary care physician]

The primary care physician is frequently consulted in first line for infectious complications in organ transplant recipients. Many infections without signs of severity can nowadays be managed on an outpatient basis. However, a number of clinical situations specific to transplant recipients may require special attention and knowledge. In particular, the general practitioner must be aware of the potential interactions between immunosuppressive and antimicrobial therapies, the risk of renal dysfunction as a consequence of diarrhea or urinary tract infection, and the diagnostic of CMV disease as a cause of fever without obvious source occurring several months after transplantation. Collaboration with the transplantation specialists is recommended in order to assure an optimal management of these patients.

Methicillin-resistant Staphylococcus aureus infection after lung transplantation: 5-year review of clinical and molecular epidemiology.

BACKGROUND: Data on the epidemiology of MRSA infection in lung transplantation is limited. METHODS: We performed a 5-year retrospective study to assess the incidence and microbiologic and clinical characteristics of methicillin-resistant Staphylococcus aureus (MRSA) infection in a cohort of 163 lung transplant recipients. RESULTS: Seventeen patients with MRSA colonization and/or infection were identified, for a calculated incidence rate of 76.1 cases per 1,000 transplanted-years. Pulsed-field gel electrophoresis identified 3 different distinct MRSA profiles, all of them consistent with hospital-associated MRSA infection. CONCLUSION: Despite negative polymerase chain reaction (PCR) for the virulence factor Panton-Valentine leukocidin, MRSA infections resulted in significant disease and morbidity.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

Polyfunctional HCV-specific T-cell responses are associated with effective control of HCV replication.

HCV infection has a severe course of disease in HIV/HCV co-infection and in liver transplant recipients. However, the mechanisms involved remain unclear. Here, we evaluated functional profiles of HCV-specific T-cell responses in 86 HCV mono-infected patients, 48 HIV/HCV co-infected patients and 42 liver transplant recipients. IFN-gamma and IL-2 production and ability of CD4 and CD8 T cells to proliferate were assessed after stimulation with HCV-derived peptides. We observed that HCV-specific T-cell responses were polyfunctional in HCV mono-infected patients, with presence of proliferating single IL-2-, dual IL-2/IFN-gamma and single IFN-gamma-producing CD4+ and dual IL-2/IFN-gamma and single IFN-gamma-producing CD8+ cells. In contrast, HCV-specific T-cell responses had an effector profile in HIV/HCV co-infected individuals and liver transplant recipients with absence of single IL-2-producing HCV-specific CD4+ and dual IL-2/IFN-gamma-producing CD8+ T cells. In addition, HCV-specific proliferation of CD4+ and CD8+ T cells was severely impaired in HIV/HCV co-infected patients and liver transplant recipients. Importantly, "only effector" T-cell responses were associated with significantly higher HCV viral load and more severe liver fibrosis scores. Therefore, the present results suggest that immune-based mechanisms may contribute to explain the accelerated course of HCV infection in conditions of HIV-1 co-infection and liver transplantation.

QuantiFERON®-CMV assay for the assessment of cytomegalovirus cell-mediated immunity.

Cytomegalovirus (CMV) infection has historically been a major complication among immunocompromised patients, such as solid-organ and stem-cell transplant recipients and patients with advanced HIV infection. While the introduction of antiretroviral therapy has almost eradicated CMV infection in HIV-infected patients, CMV disease remains a significant problem in transplant recipients once antiviral prophylaxis is discontinued. QuantiFERON(®)-CMV allows the assessment of cellular immunity against CMV by detecting the production of IFN-γ following in vitro stimulation with CMV antigens. Preliminary studies have shown a correlation between a lack of detectable cell-mediated immunity measured by the QuantiFERON-CMV assay and a higher incidence of CMV infection and disease in immunocompromised patients. Measurement of cell-mediated immunity against CMV appears to be a promising strategy to identify patients at highest risk for the development of CMV disease and, therefore, to individualize preventive strategies for CMV in transplant recipients.

New nonculture-based methods for the diagnosis of invasive candidiasis.

PURPOSE OF REVIEW: Invasive candidiasis is a severe infectious complication occurring mostly in onco-hematologic and surgical patients. Its conventional diagnosis is insensitive and often late, leading to a delayed treatment and a high mortality. The purpose of this article is to review recent contributions in the nonconventional diagnostic approaches of invasive candidiasis, both for the detection of the epidose and the characterization of the etiologic agent. RECENT FINDINGS: Antigen-based tests to detect invasive candidiasis comprise a specific test, mannan, as well as a nonspecific test, beta-D-glucan. Both have a moderate sensitivity and a high specificity, and cannot be recommended alone as a negative screening tool or a positive syndrome driven diagnostic tool. Molecular-based tests still have not reached the stage of rapid, easy to use, standardized tests ideally complementing blood culture at the time of blood sampling. New tests (fluorescence in-situ hybridization or mass spectrometry) significantly reduce the delay of identification of Candida at the species level in positive blood cultures, and should have a positive impact on earlier appropriate antifungal therapy and possibly on outcome. SUMMARY: Both antigen-based and molecular tests appear as promising new tools to complement and accelerate the conventional diagnosis of invasive candidiasis with an expected significant impact on earlier and more focused treatment and on prognosis.

Organ donation: Public attitudes and stakeholder engagement in Northern Ireland 2013

In June 2013, a representative sample of the public (n=1,012) responded to a survey about their attitudes towards organ donation. At the same time, a process of stakeholder engagement began, which involved 16 discussion groups with key stakeholders as requested by the Health Minister (including organ donation charities, those on the transplant waiting list, transplant recipients, donor families, and Health and Social Care staff). Discussion groups took place between June and August 2013 and proformas were also completed. The central purpose of this public and stakeholder engagement process was to inform the direction of a public information campaign that will be developed by the Public Health Agency (PHA).The�report highlights the findings from this programme of work.