Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and ... more Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier resulting in microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long-term in the gastro-intestinal lymphatic tract, where low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence, however it is unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host-cofactor for HIV replication in Th17 cells. We found that specific...
OBJECTIVES Untreated HIV infection was previously associated with IL-32 overexpression in gut epi... more OBJECTIVES Untreated HIV infection was previously associated with IL-32 overexpression in gut epithelial cells (IEC). Here, we explored IL-32 isoform expression in the colon of people living with HIV (PLWH) receiving antiretroviral therapy (ART) and IL-32 triggers/modulators in IEC. DESIGN Sigmoid colon biopsies (SCB) and blood were collected from ART-treated PLWH (HIV + ART; n = 17; mean age: 56 years; CD4 counts: 679 cells/μl; time on ART: 72 months) and age-matched HIV-uninfected controls (HIVneg; n = 5). The IEC line HT-29 was used for mechanistic studies. METHODS Cells from SCB and blood were isolated by enzymatic digestion and/or gradient centrifugation. HT-29 cells were exposed to TLR1-9 agonists, TNF-α, IL-17A, and HIV. IL-32α/β/γ/D/ε/θ and IL-17A mRNA levels were quantified by real-time RT-PCR. IL-32 protein levels were quantified by ELISA. RESULTS IL-32β/γ/ε isoform transcripts were detectable in the blood and SCB, with IL-32β mRNA levels being predominantly expressed in b...
The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4 + T cells is crit... more The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4 + T cells is critical for mucosal homeostasis, with HIV-1 causing significant alterations in people living with HIV (PLWH) despite antiretroviral therapy (ART). In a model of IEC and T cell co-cultures, we investigated the effects of IL-17A, the Th17 hallmark cytokine, on IEC ability to promote de novo HIV infection and viral reservoir reactivation. Our results demonstrate that IL-17A acts in synergy with TNF to boost IEC production of CCL20, a Th17-attractant chemokine, and promote HIV trans-infection of CD4 + T cells and viral outgrowth from reservoir cells of ART-treated PLWH. Importantly, the Illumina RNA-sequencing revealed an IL-17A-mediated pro-inflammatory and pro-viral molecular signature, including a decreased expression of type I interferon (IFN-I)-induced HIV restriction factors. These findings point to the deleterious features of IL-17A and raise awareness for caution when designing therapies aimed at restoring the paucity of mucosal Th17 cells in ART-treated PLWH.
The frequency and functions of Th17-polarized CCR6+RORyt+CD4+ T cells are rapidly compromised upo... more The frequency and functions of Th17-polarized CCR6+RORyt+CD4+ T cells are rapidly compromised upon HIV infection and are not restored with long-term viral suppressive antiretroviral therapy (ART). In line with this, Th17 cells represent selective HIV-1 infection targets mainly at mucosal sites, with long-lived Th17 subsets carrying replication-competent HIV-DNA during ART. Therefore, novel Th17-specific therapeutic interventions are needed as a supplement of ART to reach the goal of HIV remission/cure. Th17 cells express high levels of peroxisome proliferator-activated receptor gamma (PPARy), a transcriptional factor that represses the transcription of the HIV provirus and the rorc gene, which encodes for the Th17-specific master regulator RORyt/RORC2. Thus, we hypothesized that the pharmacological inhibition of PPARy will facilitate HIV reservoir reactivation while enhancing Th17 effector functions. Consistent with this prediction, the PPARy antagonist T0070907 significantly increa...
Background Increased intestinal barrier permeability and subsequent gut microbial translocation a... more Background Increased intestinal barrier permeability and subsequent gut microbial translocation are significant contributors to inflammatory non-AIDS comorbidities in people living with HIV (PLWH). Evidence in animal models have shown that markers of intestinal permeability and microbial translocation vary over the course of the day and are affected by food intake and circadian rhythms. However, daily variations of these markers are not characterized yet in PLWH. Herein, we assessed the variation of these markers over 24 h in PLWH receiving antiretroviral therapy (ART) in a well-controlled environment. Methods As in Canada, PLWH are predominantly men and the majority of them are now over 50 years old, we selected 11 men over 50 receiving ART with undetectable viremia for more than 3 years in this pilot study. Blood samples were collected every 4 h over 24 h before snacks/meals from 8:00 in the morning to 8:00 the next day. All participants consumed similar meals at set times, and ha...
Revista Peruana de Medicina Experimental y Salud Pública, Apr 5, 2018
Objetivos. Evaluar el efecto antiinflamatorio de la fracción flavonoide de Lepechinia meyenii (Wa... more Objetivos. Evaluar el efecto antiinflamatorio de la fracción flavonoide de Lepechinia meyenii (Walp.) Epling sobre leucocitos de pacientes con artritis reumatoide (AR). Materiales y métodos. Se recolectaron plantas de la especie Lepechinia meyenii (Walp.) Epling extrayendo diferentes fracciones flavonoides por cromatografía de columna y de capa fina. Se evaluó la producción de anión superóxido mediante la técnica de ensayo reducción nitroblue tetrazolium, en neutrófilos obtenidos de sangre de pacientes con AR, separados en tres grupos: control negativo, que consistió de neutrófilos (5x10 5 células), control positivo, formado por neutrófilos activados con PMA (phorbol myristate acetate) (150 ng/mL) y los tratamientos, formados por neutrófilos activados y tratados con diferentes concentraciones de la fracción flavonoide LM8 (60, 120 y 180 ug/mL). La expresión de genes proinflamatorios se estudió por RTqPCR, en leucocitos mononucleares obtenidos de pacientes con AR separados en tres grupos: control negativo, que consistió de leucocitos mononucleares (5x10 5 células), control positivo formado por leucocitos mononucleares activados con fitohemaglutinina (PHA) (150 ug/mL) y el tratamiento formado por leucocitos mononucleares activados y tratados con la fracción flavonoide LM8 (120 ug/mL). Resultados. Se purificaron varias fracciones flavonoides, resultando la fracción LM8 con el mejor efecto inmunomodulador. Dicha fracción disminuyó la producción de anión superóxido en una manera dependiente de la concentración. Por otro lado, disminuyó la expresión de TNFα, IL8 e IL17 en leucocitos mononucleares Conclusiones. Estos resultados son alentadores respecto al efecto inmunomodulador de esta planta medicinal peruana y justifican continuar su estudio para una posible aplicación clínica.
La tuberculosis es una enfermedad infecciosa de suma importancia en salud pública y de extensión ... more La tuberculosis es una enfermedad infecciosa de suma importancia en salud pública y de extensión mundial: cada año hay un estimado de 8,7 millones de nuevos casos en el mundo, causando 1,4 millones de muertes. El presente estudio busca evaluar y comparar la expresión génica de los linfocitos T CD4+ de individuos con tuberculosis latente y activa de la ciudad de Arequipa. Fueros aislados por selección negativa linfocitos T CD4+ utilizando beads magnéticos de 10 pacientes con tuberculosis latente y 20 con tuberculosis activa. Se evaluó la expresión génica de las citoquinas pro-inflamatorias IFN-gamma, IL-17, IL-8 e IL-6 y de la citoquina anti inflamatoria IL-4 y el factor de transcripción FOXP3 por RT PCR. El presente estudio encontró que una mayor expresión génica de IFN-gamma, IL-6, IL-8 e IL-17 están asociados a tuberculosis latente mientras que una mayor expresión de IL-4 y FOXP3 están asociados a tuberculosis activa. Entendiendo los mecanismos inmunológicos asociados a tuberculos...
OBJECTIVE The aim of this study was to explore the contribution of blood and colon myeloid cells ... more OBJECTIVE The aim of this study was to explore the contribution of blood and colon myeloid cells to HIV persistence during antiretroviral therapy (ART). DESIGN Leukapheresis was collected from HIV-infected individuals with undetectable plasma viral load during ART (HIV + ART; n = 15) and viremics untreated (HIV+; n = 6). Rectal sigmoid biopsies were collected from n = 8 HIV+ART. METHODS Myeloid cells (total monocytes (Mo), CD16/CD16 Mo, CD1c dendritic cells) and CD4 T cells were isolated by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) from peripheral blood. Matched myeloid and CCR6CD4 T cells were isolated from blood and rectal biopsies by FACS. Levels of early (RU5 primers), late (Gag primers) and/or integrated HIV-DNA (Alu/HIV primers) were quantified by nested real-time PCR. Replication-competent HIV was amplified by co-culturing cells from HIV-positive individuals with CD3/CD28-activated CD4 T cells from uninfected donors. RESULTS Early/late but not integrated HIV reverse transcripts were detected in blood myeloid subsets of four out of 10 HIV+ART; in contrast, integrated HIV-DNA was exclusively detected in CD4 T cells. In rectal biopsies, late HIV reverse transcripts were detected in myeloid cells and CCR6CD4 T cells from one out of eight and seven out of eight HIV+ART individuals, respectively. Replication-competent HIV was outgrown from CD4 T cells but not from myeloid of untreated/ART-treated HIV-positive individuals. CONCLUSION In contrast to CD4 T cells, blood and colon myeloid cells carry detectable HIV only in a small fraction of HIV+ART individuals. This is consistent with the documented resistance of Mo to HIV infection and the rapid turnover of Mo-derived macrophages in the colon. Future assessment of multiple lymphoid and nonlymphoid tissues is required to include/exclude myeloid cells as relevant HIV reservoirs during ART.
Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tiss... more Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tissue localization. Their susceptibility to HIV-1 is subject to variations from permissiveness to resistance, owing in part to regulatory microRNAs. Here, we used RNA sequencing (RNA-seq) to examine the expression of >400 microRNAs in productively infected and bystander cells of HIV-1-exposed macrophage cultures. Two microRNAs upregulated in bystander macrophages, miR-221 and miR-222, were identified as negative regulators of CD4 expression and CD4-mediated HIV-1 entry. Both microRNAs were enhanced by tumor necrosis factor alpha (TNF-α), an inhibitor of CD4 expression. MiR-221/miR-222 inhibitors recovered HIV-1 entry in TNF-α-treated macrophages by enhancing CD4 expression and increased HIV-1 replication and spread in macrophages by countering TNF-α-enhanced miR-221/miR-222 expression in bystander cells. In line with these findings, HIV-1-resistant intestinal myeloid cells express higher...
Objectives: The objective of this article is to investigate the contribution of colon and blood C... more Objectives: The objective of this article is to investigate the contribution of colon and blood CD4 þ T-cell subsets expressing the chemokine receptor CCR6 to HIV persistence during antiretroviral therapy. Design: Matched sigmoid biopsies and blood samples (n ¼ 13) as well as leukapheresis (n ¼ 20) were collected from chronically HIV-infected individuals receiving antiretroviral therapy. Subsets of CD4 þ T cells with distinct differentiation/polarization profiles were identified using surface markers as follows: memory (T M , CD45RA À), central memory (T CM ; CD45RA À CCR7 þ), effector (T EM/TM ; CD45RA À CCR7 À), Th17 (CCR6 þ CCR4 þ), Th1Th17 (CCR6 þ CXCR3 þ), Th1 (CCR6 À CXCR3 þ), and Th2 (CCR6 À CCR4 þ). Methods: We used polychromatic flow cytometry for cell sorting, nested real-time PCR for HIV DNA quantification, ELISA and flow cytometry for HIV p24 quantification. HIV reactivation was induced by TCR triggering in the presence/absence of all-trans retinoic acid. Results: Compared with blood, the frequency of CCR6 þ T M was higher in the colon. In both colon and blood compartments, CCR6 þ T M were significantly enriched in HIV DNA when compared with their CCR6 À counterparts (n ¼ 13). In blood, integrated HIV DNA levels were significantly enriched in CCR6 þ versus CCR6 À T CM of four of five individuals and CCR6 þ versus CCR6 À T EM of three of five individuals. Among blood T CM , Th17 and Th1Th17 contributed the most to the pool of cells harboring integrated HIV DNA despite their reduced frequency compared with Th2, which were infected the least. HIV reactivation was induced by TCR triggering and/or retinoic acid exposure at higher levels in CCR6 þ versus CCR6 À T M , T CM , and T EM. Conclusion: CCR6 is a marker for colon and blood CD4 þ T cells enriched for replication-competent HIV DNA. Novel eradication strategies should target HIV persistence in CCR6 þ CD4 þ T cells from various anatomic sites.
Traditionell wird die Anforderungserhebung vor allem als eine reine Erfassung existierender Wünsc... more Traditionell wird die Anforderungserhebung vor allem als eine reine Erfassung existierender Wünsche gesehen. In letzter Zeit setzt sich jedoch die Ansicht durch, dass Anforderungen für neue Produkte meist erst entwickelt werden müssen; Anforderungserhebung also mit Innovation verbunden ist. Damit ist auch das Interesse an der Unterstützung von Kreativität in der Anforderungserhebung stark gewachsen. Kreativitätsunterstützung wird meist im Zusammenhang mit moderierten Workshops gesehen. Hier stellen wir jedoch einen Ansatz dar, bei dem ein Assistenzsystem basierend auf modelliertem Wissen versucht zusätzliche Anhaltspunkte für die kreative Entwicklung von Anforderungen zu geben.
Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and ... more Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier resulting in microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long-term in the gastro-intestinal lymphatic tract, where low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence, however it is unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host-cofactor for HIV replication in Th17 cells. We found that specific...
OBJECTIVES Untreated HIV infection was previously associated with IL-32 overexpression in gut epi... more OBJECTIVES Untreated HIV infection was previously associated with IL-32 overexpression in gut epithelial cells (IEC). Here, we explored IL-32 isoform expression in the colon of people living with HIV (PLWH) receiving antiretroviral therapy (ART) and IL-32 triggers/modulators in IEC. DESIGN Sigmoid colon biopsies (SCB) and blood were collected from ART-treated PLWH (HIV + ART; n = 17; mean age: 56 years; CD4 counts: 679 cells/μl; time on ART: 72 months) and age-matched HIV-uninfected controls (HIVneg; n = 5). The IEC line HT-29 was used for mechanistic studies. METHODS Cells from SCB and blood were isolated by enzymatic digestion and/or gradient centrifugation. HT-29 cells were exposed to TLR1-9 agonists, TNF-α, IL-17A, and HIV. IL-32α/β/γ/D/ε/θ and IL-17A mRNA levels were quantified by real-time RT-PCR. IL-32 protein levels were quantified by ELISA. RESULTS IL-32β/γ/ε isoform transcripts were detectable in the blood and SCB, with IL-32β mRNA levels being predominantly expressed in b...
The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4 + T cells is crit... more The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4 + T cells is critical for mucosal homeostasis, with HIV-1 causing significant alterations in people living with HIV (PLWH) despite antiretroviral therapy (ART). In a model of IEC and T cell co-cultures, we investigated the effects of IL-17A, the Th17 hallmark cytokine, on IEC ability to promote de novo HIV infection and viral reservoir reactivation. Our results demonstrate that IL-17A acts in synergy with TNF to boost IEC production of CCL20, a Th17-attractant chemokine, and promote HIV trans-infection of CD4 + T cells and viral outgrowth from reservoir cells of ART-treated PLWH. Importantly, the Illumina RNA-sequencing revealed an IL-17A-mediated pro-inflammatory and pro-viral molecular signature, including a decreased expression of type I interferon (IFN-I)-induced HIV restriction factors. These findings point to the deleterious features of IL-17A and raise awareness for caution when designing therapies aimed at restoring the paucity of mucosal Th17 cells in ART-treated PLWH.
The frequency and functions of Th17-polarized CCR6+RORyt+CD4+ T cells are rapidly compromised upo... more The frequency and functions of Th17-polarized CCR6+RORyt+CD4+ T cells are rapidly compromised upon HIV infection and are not restored with long-term viral suppressive antiretroviral therapy (ART). In line with this, Th17 cells represent selective HIV-1 infection targets mainly at mucosal sites, with long-lived Th17 subsets carrying replication-competent HIV-DNA during ART. Therefore, novel Th17-specific therapeutic interventions are needed as a supplement of ART to reach the goal of HIV remission/cure. Th17 cells express high levels of peroxisome proliferator-activated receptor gamma (PPARy), a transcriptional factor that represses the transcription of the HIV provirus and the rorc gene, which encodes for the Th17-specific master regulator RORyt/RORC2. Thus, we hypothesized that the pharmacological inhibition of PPARy will facilitate HIV reservoir reactivation while enhancing Th17 effector functions. Consistent with this prediction, the PPARy antagonist T0070907 significantly increa...
Background Increased intestinal barrier permeability and subsequent gut microbial translocation a... more Background Increased intestinal barrier permeability and subsequent gut microbial translocation are significant contributors to inflammatory non-AIDS comorbidities in people living with HIV (PLWH). Evidence in animal models have shown that markers of intestinal permeability and microbial translocation vary over the course of the day and are affected by food intake and circadian rhythms. However, daily variations of these markers are not characterized yet in PLWH. Herein, we assessed the variation of these markers over 24 h in PLWH receiving antiretroviral therapy (ART) in a well-controlled environment. Methods As in Canada, PLWH are predominantly men and the majority of them are now over 50 years old, we selected 11 men over 50 receiving ART with undetectable viremia for more than 3 years in this pilot study. Blood samples were collected every 4 h over 24 h before snacks/meals from 8:00 in the morning to 8:00 the next day. All participants consumed similar meals at set times, and ha...
Revista Peruana de Medicina Experimental y Salud Pública, Apr 5, 2018
Objetivos. Evaluar el efecto antiinflamatorio de la fracción flavonoide de Lepechinia meyenii (Wa... more Objetivos. Evaluar el efecto antiinflamatorio de la fracción flavonoide de Lepechinia meyenii (Walp.) Epling sobre leucocitos de pacientes con artritis reumatoide (AR). Materiales y métodos. Se recolectaron plantas de la especie Lepechinia meyenii (Walp.) Epling extrayendo diferentes fracciones flavonoides por cromatografía de columna y de capa fina. Se evaluó la producción de anión superóxido mediante la técnica de ensayo reducción nitroblue tetrazolium, en neutrófilos obtenidos de sangre de pacientes con AR, separados en tres grupos: control negativo, que consistió de neutrófilos (5x10 5 células), control positivo, formado por neutrófilos activados con PMA (phorbol myristate acetate) (150 ng/mL) y los tratamientos, formados por neutrófilos activados y tratados con diferentes concentraciones de la fracción flavonoide LM8 (60, 120 y 180 ug/mL). La expresión de genes proinflamatorios se estudió por RTqPCR, en leucocitos mononucleares obtenidos de pacientes con AR separados en tres grupos: control negativo, que consistió de leucocitos mononucleares (5x10 5 células), control positivo formado por leucocitos mononucleares activados con fitohemaglutinina (PHA) (150 ug/mL) y el tratamiento formado por leucocitos mononucleares activados y tratados con la fracción flavonoide LM8 (120 ug/mL). Resultados. Se purificaron varias fracciones flavonoides, resultando la fracción LM8 con el mejor efecto inmunomodulador. Dicha fracción disminuyó la producción de anión superóxido en una manera dependiente de la concentración. Por otro lado, disminuyó la expresión de TNFα, IL8 e IL17 en leucocitos mononucleares Conclusiones. Estos resultados son alentadores respecto al efecto inmunomodulador de esta planta medicinal peruana y justifican continuar su estudio para una posible aplicación clínica.
La tuberculosis es una enfermedad infecciosa de suma importancia en salud pública y de extensión ... more La tuberculosis es una enfermedad infecciosa de suma importancia en salud pública y de extensión mundial: cada año hay un estimado de 8,7 millones de nuevos casos en el mundo, causando 1,4 millones de muertes. El presente estudio busca evaluar y comparar la expresión génica de los linfocitos T CD4+ de individuos con tuberculosis latente y activa de la ciudad de Arequipa. Fueros aislados por selección negativa linfocitos T CD4+ utilizando beads magnéticos de 10 pacientes con tuberculosis latente y 20 con tuberculosis activa. Se evaluó la expresión génica de las citoquinas pro-inflamatorias IFN-gamma, IL-17, IL-8 e IL-6 y de la citoquina anti inflamatoria IL-4 y el factor de transcripción FOXP3 por RT PCR. El presente estudio encontró que una mayor expresión génica de IFN-gamma, IL-6, IL-8 e IL-17 están asociados a tuberculosis latente mientras que una mayor expresión de IL-4 y FOXP3 están asociados a tuberculosis activa. Entendiendo los mecanismos inmunológicos asociados a tuberculos...
OBJECTIVE The aim of this study was to explore the contribution of blood and colon myeloid cells ... more OBJECTIVE The aim of this study was to explore the contribution of blood and colon myeloid cells to HIV persistence during antiretroviral therapy (ART). DESIGN Leukapheresis was collected from HIV-infected individuals with undetectable plasma viral load during ART (HIV + ART; n = 15) and viremics untreated (HIV+; n = 6). Rectal sigmoid biopsies were collected from n = 8 HIV+ART. METHODS Myeloid cells (total monocytes (Mo), CD16/CD16 Mo, CD1c dendritic cells) and CD4 T cells were isolated by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) from peripheral blood. Matched myeloid and CCR6CD4 T cells were isolated from blood and rectal biopsies by FACS. Levels of early (RU5 primers), late (Gag primers) and/or integrated HIV-DNA (Alu/HIV primers) were quantified by nested real-time PCR. Replication-competent HIV was amplified by co-culturing cells from HIV-positive individuals with CD3/CD28-activated CD4 T cells from uninfected donors. RESULTS Early/late but not integrated HIV reverse transcripts were detected in blood myeloid subsets of four out of 10 HIV+ART; in contrast, integrated HIV-DNA was exclusively detected in CD4 T cells. In rectal biopsies, late HIV reverse transcripts were detected in myeloid cells and CCR6CD4 T cells from one out of eight and seven out of eight HIV+ART individuals, respectively. Replication-competent HIV was outgrown from CD4 T cells but not from myeloid of untreated/ART-treated HIV-positive individuals. CONCLUSION In contrast to CD4 T cells, blood and colon myeloid cells carry detectable HIV only in a small fraction of HIV+ART individuals. This is consistent with the documented resistance of Mo to HIV infection and the rapid turnover of Mo-derived macrophages in the colon. Future assessment of multiple lymphoid and nonlymphoid tissues is required to include/exclude myeloid cells as relevant HIV reservoirs during ART.
Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tiss... more Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tissue localization. Their susceptibility to HIV-1 is subject to variations from permissiveness to resistance, owing in part to regulatory microRNAs. Here, we used RNA sequencing (RNA-seq) to examine the expression of >400 microRNAs in productively infected and bystander cells of HIV-1-exposed macrophage cultures. Two microRNAs upregulated in bystander macrophages, miR-221 and miR-222, were identified as negative regulators of CD4 expression and CD4-mediated HIV-1 entry. Both microRNAs were enhanced by tumor necrosis factor alpha (TNF-α), an inhibitor of CD4 expression. MiR-221/miR-222 inhibitors recovered HIV-1 entry in TNF-α-treated macrophages by enhancing CD4 expression and increased HIV-1 replication and spread in macrophages by countering TNF-α-enhanced miR-221/miR-222 expression in bystander cells. In line with these findings, HIV-1-resistant intestinal myeloid cells express higher...
Objectives: The objective of this article is to investigate the contribution of colon and blood C... more Objectives: The objective of this article is to investigate the contribution of colon and blood CD4 þ T-cell subsets expressing the chemokine receptor CCR6 to HIV persistence during antiretroviral therapy. Design: Matched sigmoid biopsies and blood samples (n ¼ 13) as well as leukapheresis (n ¼ 20) were collected from chronically HIV-infected individuals receiving antiretroviral therapy. Subsets of CD4 þ T cells with distinct differentiation/polarization profiles were identified using surface markers as follows: memory (T M , CD45RA À), central memory (T CM ; CD45RA À CCR7 þ), effector (T EM/TM ; CD45RA À CCR7 À), Th17 (CCR6 þ CCR4 þ), Th1Th17 (CCR6 þ CXCR3 þ), Th1 (CCR6 À CXCR3 þ), and Th2 (CCR6 À CCR4 þ). Methods: We used polychromatic flow cytometry for cell sorting, nested real-time PCR for HIV DNA quantification, ELISA and flow cytometry for HIV p24 quantification. HIV reactivation was induced by TCR triggering in the presence/absence of all-trans retinoic acid. Results: Compared with blood, the frequency of CCR6 þ T M was higher in the colon. In both colon and blood compartments, CCR6 þ T M were significantly enriched in HIV DNA when compared with their CCR6 À counterparts (n ¼ 13). In blood, integrated HIV DNA levels were significantly enriched in CCR6 þ versus CCR6 À T CM of four of five individuals and CCR6 þ versus CCR6 À T EM of three of five individuals. Among blood T CM , Th17 and Th1Th17 contributed the most to the pool of cells harboring integrated HIV DNA despite their reduced frequency compared with Th2, which were infected the least. HIV reactivation was induced by TCR triggering and/or retinoic acid exposure at higher levels in CCR6 þ versus CCR6 À T M , T CM , and T EM. Conclusion: CCR6 is a marker for colon and blood CD4 þ T cells enriched for replication-competent HIV DNA. Novel eradication strategies should target HIV persistence in CCR6 þ CD4 þ T cells from various anatomic sites.
Traditionell wird die Anforderungserhebung vor allem als eine reine Erfassung existierender Wünsc... more Traditionell wird die Anforderungserhebung vor allem als eine reine Erfassung existierender Wünsche gesehen. In letzter Zeit setzt sich jedoch die Ansicht durch, dass Anforderungen für neue Produkte meist erst entwickelt werden müssen; Anforderungserhebung also mit Innovation verbunden ist. Damit ist auch das Interesse an der Unterstützung von Kreativität in der Anforderungserhebung stark gewachsen. Kreativitätsunterstützung wird meist im Zusammenhang mit moderierten Workshops gesehen. Hier stellen wir jedoch einen Ansatz dar, bei dem ein Assistenzsystem basierend auf modelliertem Wissen versucht zusätzliche Anhaltspunkte für die kreative Entwicklung von Anforderungen zu geben.
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