<p>Today the immunosuppressive treatment is kept to a level were the incidence of acute rejection is below 20% within the first year after transplantation. As a consequence, a group of transplanted patients is over-immunosuppressed and at risk for infections. There is therefore an urgent need for tools which are able to determine the cellular immune response after organ transplantation. This knowledge would facilitate the task of prospectively opimize the immunosuppressive treatment and identify patients at risk of developing rejection episodes or infections.</p><p>To address this issue, a rat-kidney transplantation model for acute rejection was developed to study immune responses to allografts. Infiltrating lymphocytes were analysed using an in vitro culture system which allowed cells to propagate from the biopsies to culture medium. The numbers of outgrowing cells were correlated with morphological and immunohistochemical signs of rejection. When immunosuppressive treatment was administered for 2 and four days after acute rejection, histology did not reveal any improvement, however cellular propagation was reduced by 50 and 75%, respectively. Using the tissue culture technique in human transplanted kidney grafts, which was originally developed for the animal model, the number of propagated cells measured was profoundly higher in grafts with acute cellular rejection than from grafts in other groups. In some cases the number of propagated cells was better correlated with the clinical outcome than the diagnosis made by morphological evaluation. To determine immune responses to cytomegalovirus (CMV), we utilised Human Leukocyte Antigen (HLA) tetramer staining and stimulation of T cells with viral antigens. Both of these techniques independently detected CMV specific T cells in immunosuppressed and healthy individuals with latent or active infection. Although the frequency of CMV specific T cells did not differ between groups, there was a functional impairment in immunosuppressed patients as evidenced by reduced interferon-gamma production. In conclusion, these techniques can be used to determine the cellular immune response to allografts and cytomegalovirus and prove valuable for the optimization of immunosuppressive protocols and antiviral treatment. </p>

Today the immunosuppressive treatment is kept to a level were the incidence of acute rejection is below 20% within the first year after transplantation. As a consequence, a group of transplanted patients is over-immunosuppressed and at risk for infections. There is therefore an urgent need for tools which are able to determine the cellular immune response after organ transplantation. This knowledge would facilitate the task of prospectively opimize the immunosuppressive treatment and identify patients at risk of developing rejection episodes or infections. To address this issue, a rat-kidney transplantation model for acute rejection was developed to study immune responses to allografts. Infiltrating lymphocytes were analysed using an in vitro culture system which allowed cells to propagate from the biopsies to culture medium. The numbers of outgrowing cells were correlated with morphological and immunohistochemical signs of rejection. When immunosuppressive treatment was administered for 2 and four days after acute rejection, histology did not reveal any improvement, however cellular propagation was reduced by 50 and 75%, respectively. Using the tissue culture technique in human transplanted kidney grafts, which was originally developed for the animal model, the number of propagated cells measured was profoundly higher in grafts with acute cellular rejection than from grafts in other groups. In some cases the number of propagated cells was better correlated with the clinical outcome than the diagnosis made by morphological evaluation. To determine immune responses to cytomegalovirus (CMV), we utilised Human Leukocyte Antigen (HLA) tetramer staining and stimulation of T cells with viral antigens. Both of these techniques independently detected CMV specific T cells in immunosuppressed and healthy individuals with latent or active infection. Although the frequency of CMV specific T cells did not differ between groups, there was a functional impairment in immunosuppressed patients as evidenced by reduced interferon-gamma production. In conclusion, these techniques can be used to determine the cellular immune response to allografts and cytomegalovirus and prove valuable for the optimization of immunosuppressive protocols and antiviral treatment.

HIV infects over 33 million people worldwide with a new infection occurring every 9 seconds. Sex is the primary mode of transmission and the majority of new infections occur during unprotected sexual contact between an HIV-infected individual and an uninfected sexual partner(s) since HIV infected individuals tend to shed virus in their genital secretions. The infectiousness of an individual is closely tied to the amount of virus in blood, which is closely associated with HIV levels shed in semen or vaginal fluid or rectal secretions. Although, Highly Active Antiretroviral Therapy (HAART) is associated with complete suppression of HIV RNA in blood to undetectable levels, the impact of HAART on semen HIV RNA levels is less clear. I evaluated the correlation between systemic and mucosal HIV-specific CD8+ T cell immune responses and HIV RNA levels in blood and semen. Overall, there was a strong positive correlation between HIV RNA levels in blood and semen. Neither systemic nor mucosal (in semen) HIV-specific CD8+ responses were associated with HIV RNA levels in blood or semen, in fact CD8+ T cell immune responses in semen correlated with increased HIV RNA levels in semen. Furthermore, inflammatory cytokines (IL-6, and IL-8) CMV levels in semen were associated with increased semen HIV RNA shedding. HAART initiation was associated with complete suppression of HIV viremia, but a significant proportion of individuals on suppressive HAART continue to shed HIV RNA in semen even after 6 months, and this isolated virus was infectious and often present at high levels (> 5000 copies/mL). Nevertheless, long-term HAART was associated with complete immune reconstitution of CD4+ T cells in the sigmoid colon of HIV-infected individuals on long-term therapy. These findings demonstrate that neither systemic nor mucosal HIV-specific CD8+ responses, when assayed with IFN- production as an endpoint, were associated with reduced HIV RNA levels in blood or semen. Semen HIV RNA levels did correlate with local inflammatory cytokines and CMV reactivation. Furthermore, despite effective HAART a significant proportion of HIV-infected men continued to shed HIV RNA in semen. However, long-term completely suppressive HAART was associated with complete immune reconstitution of the sigmoid colon.

HIV infects over 33 million people worldwide with a new infection occurring every 9 seconds. Sex is the primary mode of transmission and the majority of new infections occur during unprotected sexual contact between an HIV-infected individual and an uninfected sexual partner(s) since HIV infected individuals tend to shed virus in their genital secretions. The infectiousness of an individual is closely tied to the amount of virus in blood, which is closely associated with HIV levels shed in semen or vaginal fluid or rectal secretions. Although, Highly Active Antiretroviral Therapy (HAART) is associated with complete suppression of HIV RNA in blood to undetectable levels, the impact of HAART on semen HIV RNA levels is less clear. I evaluated the correlation between systemic and mucosal HIV-specific CD8+ T cell immune responses and HIV RNA levels in blood and semen. Overall, there was a strong positive correlation between HIV RNA levels in blood and semen. Neither systemic nor mucosal (in semen) HIV-specific CD8+ responses were associated with HIV RNA levels in blood or semen, in fact CD8+ T cell immune responses in semen correlated with increased HIV RNA levels in semen. Furthermore, inflammatory cytokines (IL-6, and IL-8) CMV levels in semen were associated with increased semen HIV RNA shedding. HAART initiation was associated with complete suppression of HIV viremia, but a significant proportion of individuals on suppressive HAART continue to shed HIV RNA in semen even after 6 months, and this isolated virus was infectious and often present at high levels (> 5000 copies/mL). Nevertheless, long-term HAART was associated with complete immune reconstitution of CD4+ T cells in the sigmoid colon of HIV-infected individuals on long-term therapy. These findings demonstrate that neither systemic nor mucosal HIV-specific CD8+ responses, when assayed with IFN- production as an endpoint, were associated with reduced HIV RNA levels in blood or semen. Semen HIV RNA levels did correlate with local inflammatory cytokines and CMV reactivation. Furthermore, despite effective HAART a significant proportion of HIV-infected men continued to shed HIV RNA in semen. However, long-term completely suppressive HAART was associated with complete immune reconstitution of the sigmoid colon.

Recent studies suggest that some cases of prenatal depression may be associated with reactivation of latent infections of the herpesvirus family. The possible relationships among stress, prenatal depression, and herpes viral reactivation in pregnancy are understudied and the molecular pathways such as the neuroimmune biogenic amine pathway are unidentified. Chronic stress shifts the T helper-1 cell (Th1) cytokine profile to a Th2 profile, which favors virus induced pathogenesis and survival. Pregnancy is also associated with a similar Th2 dominance. In non-pregnant individuals, exposure to psychological or physical stress may be associated with latent herpes viral reactivation and could result in behavioral deficits and depression. Normally, type-1 cytokines such as Interferon-gamma (IFN -gamma) and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) induce indoleamine-2, 3-dioxygenase (IDO) activation which inhibits herpes virus replication and reactivation, decreases tryptophan production, and alters phenylalanine /tyrosine metabolism. Thus it is possible that prenatal depression may occur from tryptophan stealing through the IDO pathway which results in decreased serotonin as well as increased risk for latent herpes viral reactivation. The purpose of this study is to analyze the relationships among stress, herpes viral titers, depression, and metabolites of IDO activation, which involves tryptophan and guanosine-triphosphate-cyclohydrolase-1(GTP-CH1) pathways. This study builds on Influence of Lactation on Postpartum Stress and Immunity (Grant number: R01-NR05000) which investigated perinatal immune, endocrine, and inflammatory changes in pregnancy and the postpartum. A secondary data analysis was conducted on baseline data from women collected at 16 to 25 gestational weeks. This data set included some herpes viral titers, and additional ones were measured in stored plasma samples. The aim of this study is to examine relationships among stress, herpes viral reactivation, depression, and the IDO activation pathway. The results of this study provide information about the possible role of further relationships of prenatal stress, latent herpes viral reactivation, and depression mechanisms. The results will be important in health promotion and disease prevention during pregnancy.

INFECTIOUS COMPLICATIONS IN CHRONIC RENAL FAILURE Infections represent a serious problem in chronic kidney disease (cohort and they are) associated with signifficant morbidity and mortality. The thesis originated in the nephrology division of the Department of Internal Medicine I., Charles University Teaching Hospital and Medical Faculty in Pilsen, an institution with a long standing research activity in the field. In the theoretical part of this work, a general summary of infectious complications in chronic kidney disease population is presented. The other part of this work presents results of our research dealing with pharmacoeconomical aspects of cytomegalovirus infection and finally our results in the field of influenza vaccination. The Aim of the first presented work was to evaluate the cost impact of four different strategies for prevention of cytomegalovirus infection after renal transplantation. We provide post hoc analysis of 2 randomized studies performed in our department and calculating direct CMV-related expenses using valacyclovir prophylaxis, ganciclovir prophylaxis, preemptive vlaganciclovir treatment and deferred therapy. To simulate ihe impact of varying prices of prharmacotherapy or diagnostic procedures, a sensitivity analysis was performed. With respect to our results, valacyclovir...

A infecção por citomegalovírus (CMV) tem efeito duradouro na distribuição dos subtipos de células T, mas pouco se sabe sobre o seu impacto na função celular. Foi realizada uma análise de expressão global gênica em células CD4+ purificadas em 38 recipientes de transplante de células tronco hematopoéticas (HSCT) (mediana de idade 48 anos; variação 20-65 anos) estudados em média cinco anos pós transplante (mediana 4.75 anos; variação 1-20 years). A população estudada incluiu 18 pacientes com GVHD crônico ativo (cGVHD) e 20 pacientes considerados tolerantes. Tolerância foi definida como ausência de sinais e sintomas de cGVHD, assim como ausência de tratamento imunossupressor (IST) por pelo menos 2 meses com seguimento de 1 ano sem tratamento imunossupressor. A expressão gênica foi medida por Illumina bead arrays. O seroestato do CMV foi definido pela sorologia pré transplante (por ELISA). Não havia evidência documentada de reativação do CMV no momento de coleta das amostras estudadas. Doze de 54 genes candidatos associados à função imune foram associados de maneira significativa com CMV+ em pacientes com cGVHD ativo (p<0.05) (PDCD-1; GZMH, IFNG, PRF1, CST7, IL18RAP, ITGAM, CTSW, ITGAL, GBP1, CDKN1B, CXCR4), sendo que somente três genes (CD14; CD86; IER3) foram associados a CMV+ entre os pacientes tolerantes. A expressão de PD-1 significativamente maior em pacientes CMV+ e com cGVHD ativo foi confirmada em uma população independente através de estudos de imunofenotipagem. Os pacientes CMV+/cGVHD ativos tiveram um perfil compatível com ativação de células T efetoras, não sendo detectado com a mesma intensidade em pacientes tolerantes e pacientes CMV-/cGVHD ativos. Tendo como objetivo a latência da infecção, o citomegalovírus tentará evadir-se das tentativas do sistema imune de depuração viral, criando modificações que vão desde mudanças nos subtipos de linfócitos até a remodelação de cromatina por uma série de enzimas e microRNA. O ambiente caracteristicamente inflamatório do cGVHD, a produção aumentada de citocinas, a terapia imunossupressora e a reconstituição imune defeituosa das células T podem aumentar o risco de reativação do CMV, mesmo indetectável, seguido por supra-regulação de genes relacionados à ativação de células T e função efetora. O gene PD-1 pode estar supra-regulado em ativação de células T e função efetora, mas tem papel essencial na prevenção da expansão e função das células T efetoras, sendo um candidato alvo para a prevenção e tratamento do cGVHD. Entender o impacto do CMV na regulação de PD-1 em pacientes com cGVHD seria instrumental na implementação de novas terapias; portanto mais estudos com populações maiores são necessários para entendermos o impacto da imunomodulação secundária à infecção prévia por CMV no funcionamento das células T durante cGVHD. / Cytomegalovirus infection (CMV) is known to have a life-long effect on the distribution of T cell subsets, but little is known about the impact on cell function. We performed a gene expression analysis in purified CD4+ T cells from 38 hematopoietic cell transplant (HCT) recipients (median age 48; range 20-65) studied on average 5 years after HCT (median 4.75; range 1-20 years). The study population included 18 patients with active chronic GVHD (cGVHD) and 20 tolerant (TOL) patients. Tolerance was defined by absence of signs, symptoms of cGVHD and immunosuppressive therapy (IST) for at least 2 months and 1 year follow-up without immunosupressive therapy . Gene expression was measured on Illumina bead arrays. CMV status was defined by pre-transplant recipient CMV serology (by ELISA). There was no recorded evidence of CMV reactivation at the time of study. Twelve of 54 candidate genes associated with immune function and inflammation were found to be associated CMV positive serostatus in cGVHD patients at a significance threshold of p<0.05 (PDCD-1; GZMH, IFNG, PRF1, CST7, IL18RAP, ITGAM, CTSW, ITGAL, GBP1, CDKN1B, CXCR4), but only three genes (CD14; CD86; IER3) were associated with CMV serostatus in TOL patients. The significant higher PD-1 expression on CD4+ cells of CMV+/active cGVHD patients was confirmed by immunophenotype testing in an independent population. CMV+/active cGVHD patients had a profile consistent with T effector cell activation that was not present in TOL and CMV serostatus negative/active cGVHD patients. Pursuing latency, cytomegalovirus will try to evade the immune system attempts of viral clearance creating modifications varying from changes in lymphocyte subsets to chromatin remodeling by several enzymes and microRNA. The chronic GVHD characteristic inflammatory environment, increased cytokine production, immunosuppressive therapy, and impaired T cell immune reconstitution, may increase the risk of CMV reactivation, even if not detectable, followed by up-regulation of genes related to T cell activation and effector function. The PD-1 gene can be up-regulated in T cell activation and effector functions, but it also has an essential role in preventing the expansion and function of effector cells, being a candidate target for the prevention or treatment of chronic GVHD. Understanding the CMV impact on the PD-1 regulation in active cGVHD would be key on the implementation of new therapies.Thus, more studies in larger populations are needed in order to understand CMV previous infection immunomodulation impact in T cell function during chronic GVHD.

A infecção por citomegalovírus (CMV) tem efeito duradouro na distribuição dos subtipos de células T, mas pouco se sabe sobre o seu impacto na função celular. Foi realizada uma análise de expressão global gênica em células CD4+ purificadas em 38 recipientes de transplante de células tronco hematopoéticas (HSCT) (mediana de idade 48 anos; variação 20-65 anos) estudados em média cinco anos pós transplante (mediana 4.75 anos; variação 1-20 years). A população estudada incluiu 18 pacientes com GVHD crônico ativo (cGVHD) e 20 pacientes considerados tolerantes. Tolerância foi definida como ausência de sinais e sintomas de cGVHD, assim como ausência de tratamento imunossupressor (IST) por pelo menos 2 meses com seguimento de 1 ano sem tratamento imunossupressor. A expressão gênica foi medida por Illumina bead arrays. O seroestato do CMV foi definido pela sorologia pré transplante (por ELISA). Não havia evidência documentada de reativação do CMV no momento de coleta das amostras estudadas. Doze de 54 genes candidatos associados à função imune foram associados de maneira significativa com CMV+ em pacientes com cGVHD ativo (p<0.05) (PDCD-1; GZMH, IFNG, PRF1, CST7, IL18RAP, ITGAM, CTSW, ITGAL, GBP1, CDKN1B, CXCR4), sendo que somente três genes (CD14; CD86; IER3) foram associados a CMV+ entre os pacientes tolerantes. A expressão de PD-1 significativamente maior em pacientes CMV+ e com cGVHD ativo foi confirmada em uma população independente através de estudos de imunofenotipagem. Os pacientes CMV+/cGVHD ativos tiveram um perfil compatível com ativação de células T efetoras, não sendo detectado com a mesma intensidade em pacientes tolerantes e pacientes CMV-/cGVHD ativos. Tendo como objetivo a latência da infecção, o citomegalovírus tentará evadir-se das tentativas do sistema imune de depuração viral, criando modificações que vão desde mudanças nos subtipos de linfócitos até a remodelação de cromatina por uma série de enzimas e microRNA. O ambiente caracteristicamente inflamatório do cGVHD, a produção aumentada de citocinas, a terapia imunossupressora e a reconstituição imune defeituosa das células T podem aumentar o risco de reativação do CMV, mesmo indetectável, seguido por supra-regulação de genes relacionados à ativação de células T e função efetora. O gene PD-1 pode estar supra-regulado em ativação de células T e função efetora, mas tem papel essencial na prevenção da expansão e função das células T efetoras, sendo um candidato alvo para a prevenção e tratamento do cGVHD. Entender o impacto do CMV na regulação de PD-1 em pacientes com cGVHD seria instrumental na implementação de novas terapias; portanto mais estudos com populações maiores são necessários para entendermos o impacto da imunomodulação secundária à infecção prévia por CMV no funcionamento das células T durante cGVHD. / Cytomegalovirus infection (CMV) is known to have a life-long effect on the distribution of T cell subsets, but little is known about the impact on cell function. We performed a gene expression analysis in purified CD4+ T cells from 38 hematopoietic cell transplant (HCT) recipients (median age 48; range 20-65) studied on average 5 years after HCT (median 4.75; range 1-20 years). The study population included 18 patients with active chronic GVHD (cGVHD) and 20 tolerant (TOL) patients. Tolerance was defined by absence of signs, symptoms of cGVHD and immunosuppressive therapy (IST) for at least 2 months and 1 year follow-up without immunosupressive therapy . Gene expression was measured on Illumina bead arrays. CMV status was defined by pre-transplant recipient CMV serology (by ELISA). There was no recorded evidence of CMV reactivation at the time of study. Twelve of 54 candidate genes associated with immune function and inflammation were found to be associated CMV positive serostatus in cGVHD patients at a significance threshold of p<0.05 (PDCD-1; GZMH, IFNG, PRF1, CST7, IL18RAP, ITGAM, CTSW, ITGAL, GBP1, CDKN1B, CXCR4), but only three genes (CD14; CD86; IER3) were associated with CMV serostatus in TOL patients. The significant higher PD-1 expression on CD4+ cells of CMV+/active cGVHD patients was confirmed by immunophenotype testing in an independent population. CMV+/active cGVHD patients had a profile consistent with T effector cell activation that was not present in TOL and CMV serostatus negative/active cGVHD patients. Pursuing latency, cytomegalovirus will try to evade the immune system attempts of viral clearance creating modifications varying from changes in lymphocyte subsets to chromatin remodeling by several enzymes and microRNA. The chronic GVHD characteristic inflammatory environment, increased cytokine production, immunosuppressive therapy, and impaired T cell immune reconstitution, may increase the risk of CMV reactivation, even if not detectable, followed by up-regulation of genes related to T cell activation and effector function. The PD-1 gene can be up-regulated in T cell activation and effector functions, but it also has an essential role in preventing the expansion and function of effector cells, being a candidate target for the prevention or treatment of chronic GVHD. Understanding the CMV impact on the PD-1 regulation in active cGVHD would be key on the implementation of new therapies.Thus, more studies in larger populations are needed in order to understand CMV previous infection immunomodulation impact in T cell function during chronic GVHD.

A infecção por citomegalovírus (CMV) tem efeito duradouro na distribuição dos subtipos de células T, mas pouco se sabe sobre o seu impacto na função celular. Foi realizada uma análise de expressão global gênica em células CD4+ purificadas em 38 recipientes de transplante de células tronco hematopoéticas (HSCT) (mediana de idade 48 anos; variação 20-65 anos) estudados em média cinco anos pós transplante (mediana 4.75 anos; variação 1-20 years). A população estudada incluiu 18 pacientes com GVHD crônico ativo (cGVHD) e 20 pacientes considerados tolerantes. Tolerância foi definida como ausência de sinais e sintomas de cGVHD, assim como ausência de tratamento imunossupressor (IST) por pelo menos 2 meses com seguimento de 1 ano sem tratamento imunossupressor. A expressão gênica foi medida por Illumina bead arrays. O seroestato do CMV foi definido pela sorologia pré transplante (por ELISA). Não havia evidência documentada de reativação do CMV no momento de coleta das amostras estudadas. Doze de 54 genes candidatos associados à função imune foram associados de maneira significativa com CMV+ em pacientes com cGVHD ativo (p<0.05) (PDCD-1; GZMH, IFNG, PRF1, CST7, IL18RAP, ITGAM, CTSW, ITGAL, GBP1, CDKN1B, CXCR4), sendo que somente três genes (CD14; CD86; IER3) foram associados a CMV+ entre os pacientes tolerantes. A expressão de PD-1 significativamente maior em pacientes CMV+ e com cGVHD ativo foi confirmada em uma população independente através de estudos de imunofenotipagem. Os pacientes CMV+/cGVHD ativos tiveram um perfil compatível com ativação de células T efetoras, não sendo detectado com a mesma intensidade em pacientes tolerantes e pacientes CMV-/cGVHD ativos. Tendo como objetivo a latência da infecção, o citomegalovírus tentará evadir-se das tentativas do sistema imune de depuração viral, criando modificações que vão desde mudanças nos subtipos de linfócitos até a remodelação de cromatina por uma série de enzimas e microRNA. O ambiente caracteristicamente inflamatório do cGVHD, a produção aumentada de citocinas, a terapia imunossupressora e a reconstituição imune defeituosa das células T podem aumentar o risco de reativação do CMV, mesmo indetectável, seguido por supra-regulação de genes relacionados à ativação de células T e função efetora. O gene PD-1 pode estar supra-regulado em ativação de células T e função efetora, mas tem papel essencial na prevenção da expansão e função das células T efetoras, sendo um candidato alvo para a prevenção e tratamento do cGVHD. Entender o impacto do CMV na regulação de PD-1 em pacientes com cGVHD seria instrumental na implementação de novas terapias; portanto mais estudos com populações maiores são necessários para entendermos o impacto da imunomodulação secundária à infecção prévia por CMV no funcionamento das células T durante cGVHD. / Cytomegalovirus infection (CMV) is known to have a life-long effect on the distribution of T cell subsets, but little is known about the impact on cell function. We performed a gene expression analysis in purified CD4+ T cells from 38 hematopoietic cell transplant (HCT) recipients (median age 48; range 20-65) studied on average 5 years after HCT (median 4.75; range 1-20 years). The study population included 18 patients with active chronic GVHD (cGVHD) and 20 tolerant (TOL) patients. Tolerance was defined by absence of signs, symptoms of cGVHD and immunosuppressive therapy (IST) for at least 2 months and 1 year follow-up without immunosupressive therapy . Gene expression was measured on Illumina bead arrays. CMV status was defined by pre-transplant recipient CMV serology (by ELISA). There was no recorded evidence of CMV reactivation at the time of study. Twelve of 54 candidate genes associated with immune function and inflammation were found to be associated CMV positive serostatus in cGVHD patients at a significance threshold of p<0.05 (PDCD-1; GZMH, IFNG, PRF1, CST7, IL18RAP, ITGAM, CTSW, ITGAL, GBP1, CDKN1B, CXCR4), but only three genes (CD14; CD86; IER3) were associated with CMV serostatus in TOL patients. The significant higher PD-1 expression on CD4+ cells of CMV+/active cGVHD patients was confirmed by immunophenotype testing in an independent population. CMV+/active cGVHD patients had a profile consistent with T effector cell activation that was not present in TOL and CMV serostatus negative/active cGVHD patients. Pursuing latency, cytomegalovirus will try to evade the immune system attempts of viral clearance creating modifications varying from changes in lymphocyte subsets to chromatin remodeling by several enzymes and microRNA. The chronic GVHD characteristic inflammatory environment, increased cytokine production, immunosuppressive therapy, and impaired T cell immune reconstitution, may increase the risk of CMV reactivation, even if not detectable, followed by up-regulation of genes related to T cell activation and effector function. The PD-1 gene can be up-regulated in T cell activation and effector functions, but it also has an essential role in preventing the expansion and function of effector cells, being a candidate target for the prevention or treatment of chronic GVHD. Understanding the CMV impact on the PD-1 regulation in active cGVHD would be key on the implementation of new therapies.Thus, more studies in larger populations are needed in order to understand CMV previous infection immunomodulation impact in T cell function during chronic GVHD.

The aim of this research was to demonstrate the potential of the novel pre-concentration device, capillary microextraction of volatiles (CMV), for breath analysis. The CMV offers dynamic sampling of volatile organic compounds with its simple coupling to a GC inlet for GC/MS analysis, avoiding expensive thermal desorption instrumentation needed for sorbent tubes, as well as an increased surface area over a single SPME fiber. CMV collectively identified 119 compounds in the breath of 13 self-reported smokers and 7 nonsmokers. The presence and intensity of twelve compounds were used to classify all the nonsmokers 100% of the time using Principal Component Analysis to elucidate the groupings. In some cases, nicotine was not detected in smokers and they were confused with the nonsmokers. Nicotine was detected in the breath of 69% of smokers with an average mass of 143 ± 31 pg for cigarette smokers from the approximate 5 L sample of breath collected. The successful use of the CMV sampler and preconcentration of breath to distinguish between smokers and nonsmokers served as a proof of concept for future applications of the CMV for detection of marijuana smokers’ breath for impaired driver management.