Analisaram-se as respostas Thl e Th2 desencadeadas por Mycobacterium tuberculosis virulento em do... more Analisaram-se as respostas Thl e Th2 desencadeadas por Mycobacterium tuberculosis virulento em doentes com tuberculose pulmonar (TP) e em dadores saudaveis vacinados pela BCG. Efectuaram-se compara~oes entre a capacidade que as celulas T apresentavam para proliferar e para produzir IFN-y e IL-5 em resposta aos derivados de proteins purificada (PPD), M. tuberculosis H37Rv (Mtb), e M. tuberculosis H37Rv inactivado pelo calor (hk Mtb).
The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have l... more The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed.
Graefes Archive for Clinical and Experimental Ophthalmology, Mar 21, 2020
Purpose Regulatory T cells (Tregs) have been intensively studied in a myriad of autoimmune diseas... more Purpose Regulatory T cells (Tregs) have been intensively studied in a myriad of autoimmune diseases. As for noninfectious uveitis (NIU), results have been contradictory, and studies have failed to demonstrate a consistent reduction in Treg cell frequency in patients with active disease. The present study aims to characterize T lymphocyte subsets, including naïve and memory Tregs as well as their respective CD39 expression, in the peripheral blood of NIU patients. Inflammatory as well as suppressive cytokine profiles were also evaluated. Methods T cell subpopulations were evaluated by multiparametric flow cytometry using anti-CD3, anti-CD4, anti-CD45, anti-CD45RA, anti-CD197, anti-CD25, anti-CD127, and anti-CD39. Treg cells were defined as CD3 + CD4 + CD25 hi CD127 low. A multiplex bead-based immunoassay was used to determine TNF-α, IFN-ɣ, IL-17A, IL-10, and TGF-β levels. Results Twenty-nine patients with active NIU were included as well as 15 sex-and age-matched controls. There were no significant differences in T lymphocyte subsets, including Tregs, between patients and controls. However, patients with a lower grade of anterior chamber or vitreous inflammatory cellular reaction showed higher memory Treg counts than controls, with no respective increase in CD39+ expression, and a tendency for higher IL-17A levels (p = 0.06). This IL-17A elevation was present in the total NIU group (p = 0.08) as well as a positive correlation between IL-17A levels and the absolute counts of memory Tregs (p = 0.013; R = 0.465). Patients with higher IL-17A levels also showed higher serum concentrations of memory (p = 0.001) and naïve (p = 0.003) Tregs as well as elevated TNF-α (p < 0.0001) and IFN-ɣ (p = 0.016) levels. Negative correlations were observed between IL-10 and TGF-β levels and the percentages of memory (p = 0.030; R = − 0.411) and total CD39+ Tregs (p = 0.051; R = − 0.373) in the peripheral blood of NIU patients. Conclusion Our results showed that total Treg levels were not reduced in patients with NIU. Further characterization of Treg subsets, including memory Tregs and respective CD39 expression, may provide additional insight on the role of Treg cells in NIU. Consistent high levels of circulating IL-17A in NIU patients are in accordance with previous studies and reinforce this cytokine's vital role in uveitis pathogenesis and its possible use as a therapeutic target.
DOAJ (DOAJ: Directory of Open Access Journals), Feb 1, 2005
O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobact... more O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobacterium tuberculosis em indivíduos portugueses saudáveis e em doentes com tuberculose pulmonar (TB), bem como efectuar uma correlação do estado imunitário e da gravidade clínica da doença tuberculosa. As células mononucleadas do sangue periférico (CMSP) foram cultivadas e estimuladas com M. tuberculosis H37Rv e com um derivado de proteína purificado (PPD) e a produção de IFN-γ e de IL-4/IL-5 foi quantificada através das técnicas de ELISA e de citometria de fluxo multi-parâmetros. As CMSP de 30 doentes com tuberculose demonstraram um decréscimo significativo na proliferação e produção de IFN-γ, quando estimuladas com M. tuberculosis vivo comparativamente com o grupo controlo. Dos 15 doentes com tuberculose estudados e após estimulação com M. tuberculosis, sete demonstraram um grande aumento na produção de IL-4 intracelular pelas células T CD8+ e γδ+. Os doentes com tuberculose demonstraram um aumento de IL-4 intracelular após as CMSP serem estimuladas com M. tuberculosis vivo, nomeadamente do fenótipo CD4+, mas mais notavelmente do subtipo CD8+ and γδ TCR+. O aumento da produção de IL-4 em doentes com tuberculose, verificou-se em indivíduos com um envolvimento avançado do parênquima pulmonar apresentando grandes cargas bacterianas nas expectorações. Estes resultados sugerem que pode ocorrer uma alteração no equilíbrio das citocinas tipo 1 e tipo 2, em doentes com tuberculose que se encontrem num estádio clínico avançado da doença.
Torque teno virus (TTV) was recently identified as a potential biomarker for the degree of immuno... more Torque teno virus (TTV) was recently identified as a potential biomarker for the degree of immunosuppression, and potentially as a predictor of rejection and infection in solid organ transplant patients. We evaluated TTV viral load in kidney transplant (KT) patients during the first year post-transplant to examine overall kinetics and their relationships with deleterious events, including episodes of infection and the formation of de novo donor-specific antibodies (DSAs). In a single-center, prospective observational cohort study, 81 KT patients were monitored at baseline, week 1, and month 1, 3, 6, 9 and 12, post-KT, and whenever required by clinical events. Kidney function, plasma TTV load, immunoglobulins and lymphocyte subpopulations were assessed at each time point. Twenty-six patients (32.1%) presented a total of 38 infection episodes post-KT. Induction immunosuppression with thymoglobulin, compared to basiliximab, was not associated with more infections (p = 0.8093). Patients...
O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobact... more O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobacterium tuberculosis em indivíduos portugueses saudáveis e em doentes com tuberculose pulmonar (TB), bem como efectuar uma correlação do estado imunitário e da gravidade clínica da doença tuberculosa. As células mononucleadas do sangue periférico (CMSP) foram cultivadas e estimuladas com M. tuberculosis H37Rv e com um derivado de proteína purificado (PPD) e a produção de IFN-γ e de IL-4/IL-5 foi quantificada através das técnicas de ELISA e de citometria de fluxo multi-parâmetros. As CMSP de 30 doentes com tuberculose demonstraram um decréscimo significativo na proliferação e produção de IFN-γ, quando estimuladas com M. tuberculosis vivo comparativamente com o grupo controlo. Dos 15 doentes com tuberculose estudados e após estimulação com M. tuberculosis, sete demonstraram um grande aumento na produção de IL-4 intracelular pelas células T CD8+ e γδ+. Os doentes com tuberculose demonstraram um aumento de IL-4 intracelular após as CMSP serem estimuladas com M. tuberculosis vivo, nomeadamente do fenótipo CD4+, mas mais notavelmente do subtipo CD8+ and γδ TCR+. O aumento da produção de IL-4 em doentes com tuberculose, verificou-se em indivíduos com um envolvimento avançado do parênquima pulmonar apresentando grandes cargas bacterianas nas expectorações. Estes resultados sugerem que pode ocorrer uma alteração no equilíbrio das citocinas tipo 1 e tipo 2, em doentes com tuberculose que se encontrem num estádio clínico avançado da doença.
Although alkaloids from the family Aizoaceae have anticancer activity, species of this family hav... more Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non‐toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi‐drug resistant cells susceptible to anticancer drugs. These non‐toxic concentrations also prime THP‐1 human monocyte‐derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP‐1 cells within 1 day of exposure to quantities n...
We evaluated immune responses to Mycobacterium tuberculosis in 10 health-care workers (HCWs) and ... more We evaluated immune responses to Mycobacterium tuberculosis in 10 health-care workers (HCWs) and 10 non-HCWs and correlated their immune status with the development of active tuberculosis (TB). Twenty individuals were randomly recruited, tested, and monitored longitudinally for TB presentation. Peripheral blood mononuclear cells (PBMCs) from donors were stimulated with M. tuberculosis and tested for cell proliferation and the production of interferon (IFN)-g, interleukin (IL)-5, and IL-4, by use of enzyme-linked immunosorbent or flow-cytometric assays. HCWs had higher levels of cell proliferation (24,258 cpm) and IFN-g (6373 pg/mL) to M. tuberculosis than did non-HCWs (cell proliferation, 11,462 cpm; IFN-g, 3228 pg/ mL). Six of 10 HCWs showed increased median percentages of CD8 + IL-4 + (4.7%) and gd + IL-4 + (2.3%) T cells and progressed to active TB. HCWs who remained healthy showed increased median percentages of CD8 + IFNg + (25.0%) and gd + IFN-g + (8.0%) and lower percentages of CD8 + IL-4 + (0.05%) and gd + IL-4 + (0.03%) T cells. Tuberculosis (TB) is responsible for more adult deaths annually than any other infectious agent [1, 2]. In developing countries, nosocomial transmission of TB can be a threat to health-care workers (HCWs), because there are increased numbers of cases of TB on open wards and minimal or absent TB infection controls [3-6]. In countries where TB is endemic, it is difficult to
International Journal of Antimicrobial Agents, 2002
The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood mon... more The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood monocyte derived macrophages (HPBMDM) and of human macrophage cell line THP-1 at extracellular concentrations below those achievable clinically. These macrophages have nominal killing activity against bacteria and therefore, would not influence any activity that the compounds may have against intracellular localised Staphylococcus aureus. The results indicated that whereas TZ has an in vitro minimum inhibitory concentration (MIC) against the strains of S. aureus of 18, 0.1 mg/l of TZ in the medium completely inhibits the growth of S. aureus that has been phagocytosed by macrophages. The latter concentration was non-toxic to macrophages, did not cause cellular expression of activation marker CD69 nor induction of CD3'/ T cell production of IFN-g, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-g, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus.
This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacteri... more This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-c production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased cd TCR+ cell cytotoxicity and decreased CD3+cd TCRÀ cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and cd TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased cd TCR+ cytotoxicity and reduced CD3+cd TCRÀ cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of cd TCR+ cells which did not prevent TB.
BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include moni... more BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.
Analisaram-se as respostas Thl e Th2 desencadeadas por Mycobacterium tuberculosis virulento em do... more Analisaram-se as respostas Thl e Th2 desencadeadas por Mycobacterium tuberculosis virulento em doentes com tuberculose pulmonar (TP) e em dadores saudaveis vacinados pela BCG. Efectuaram-se compara~oes entre a capacidade que as celulas T apresentavam para proliferar e para produzir IFN-y e IL-5 em resposta aos derivados de proteins purificada (PPD), M. tuberculosis H37Rv (Mtb), e M. tuberculosis H37Rv inactivado pelo calor (hk Mtb).
The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have l... more The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed.
Graefes Archive for Clinical and Experimental Ophthalmology, Mar 21, 2020
Purpose Regulatory T cells (Tregs) have been intensively studied in a myriad of autoimmune diseas... more Purpose Regulatory T cells (Tregs) have been intensively studied in a myriad of autoimmune diseases. As for noninfectious uveitis (NIU), results have been contradictory, and studies have failed to demonstrate a consistent reduction in Treg cell frequency in patients with active disease. The present study aims to characterize T lymphocyte subsets, including naïve and memory Tregs as well as their respective CD39 expression, in the peripheral blood of NIU patients. Inflammatory as well as suppressive cytokine profiles were also evaluated. Methods T cell subpopulations were evaluated by multiparametric flow cytometry using anti-CD3, anti-CD4, anti-CD45, anti-CD45RA, anti-CD197, anti-CD25, anti-CD127, and anti-CD39. Treg cells were defined as CD3 + CD4 + CD25 hi CD127 low. A multiplex bead-based immunoassay was used to determine TNF-α, IFN-ɣ, IL-17A, IL-10, and TGF-β levels. Results Twenty-nine patients with active NIU were included as well as 15 sex-and age-matched controls. There were no significant differences in T lymphocyte subsets, including Tregs, between patients and controls. However, patients with a lower grade of anterior chamber or vitreous inflammatory cellular reaction showed higher memory Treg counts than controls, with no respective increase in CD39+ expression, and a tendency for higher IL-17A levels (p = 0.06). This IL-17A elevation was present in the total NIU group (p = 0.08) as well as a positive correlation between IL-17A levels and the absolute counts of memory Tregs (p = 0.013; R = 0.465). Patients with higher IL-17A levels also showed higher serum concentrations of memory (p = 0.001) and naïve (p = 0.003) Tregs as well as elevated TNF-α (p < 0.0001) and IFN-ɣ (p = 0.016) levels. Negative correlations were observed between IL-10 and TGF-β levels and the percentages of memory (p = 0.030; R = − 0.411) and total CD39+ Tregs (p = 0.051; R = − 0.373) in the peripheral blood of NIU patients. Conclusion Our results showed that total Treg levels were not reduced in patients with NIU. Further characterization of Treg subsets, including memory Tregs and respective CD39 expression, may provide additional insight on the role of Treg cells in NIU. Consistent high levels of circulating IL-17A in NIU patients are in accordance with previous studies and reinforce this cytokine's vital role in uveitis pathogenesis and its possible use as a therapeutic target.
DOAJ (DOAJ: Directory of Open Access Journals), Feb 1, 2005
O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobact... more O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobacterium tuberculosis em indivíduos portugueses saudáveis e em doentes com tuberculose pulmonar (TB), bem como efectuar uma correlação do estado imunitário e da gravidade clínica da doença tuberculosa. As células mononucleadas do sangue periférico (CMSP) foram cultivadas e estimuladas com M. tuberculosis H37Rv e com um derivado de proteína purificado (PPD) e a produção de IFN-γ e de IL-4/IL-5 foi quantificada através das técnicas de ELISA e de citometria de fluxo multi-parâmetros. As CMSP de 30 doentes com tuberculose demonstraram um decréscimo significativo na proliferação e produção de IFN-γ, quando estimuladas com M. tuberculosis vivo comparativamente com o grupo controlo. Dos 15 doentes com tuberculose estudados e após estimulação com M. tuberculosis, sete demonstraram um grande aumento na produção de IL-4 intracelular pelas células T CD8+ e γδ+. Os doentes com tuberculose demonstraram um aumento de IL-4 intracelular após as CMSP serem estimuladas com M. tuberculosis vivo, nomeadamente do fenótipo CD4+, mas mais notavelmente do subtipo CD8+ and γδ TCR+. O aumento da produção de IL-4 em doentes com tuberculose, verificou-se em indivíduos com um envolvimento avançado do parênquima pulmonar apresentando grandes cargas bacterianas nas expectorações. Estes resultados sugerem que pode ocorrer uma alteração no equilíbrio das citocinas tipo 1 e tipo 2, em doentes com tuberculose que se encontrem num estádio clínico avançado da doença.
Torque teno virus (TTV) was recently identified as a potential biomarker for the degree of immuno... more Torque teno virus (TTV) was recently identified as a potential biomarker for the degree of immunosuppression, and potentially as a predictor of rejection and infection in solid organ transplant patients. We evaluated TTV viral load in kidney transplant (KT) patients during the first year post-transplant to examine overall kinetics and their relationships with deleterious events, including episodes of infection and the formation of de novo donor-specific antibodies (DSAs). In a single-center, prospective observational cohort study, 81 KT patients were monitored at baseline, week 1, and month 1, 3, 6, 9 and 12, post-KT, and whenever required by clinical events. Kidney function, plasma TTV load, immunoglobulins and lymphocyte subpopulations were assessed at each time point. Twenty-six patients (32.1%) presented a total of 38 infection episodes post-KT. Induction immunosuppression with thymoglobulin, compared to basiliximab, was not associated with more infections (p = 0.8093). Patients...
O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobact... more O objectivo deste estudo foi o de comparar as respostas imunes ao bacilo e antigénios do Mycobacterium tuberculosis em indivíduos portugueses saudáveis e em doentes com tuberculose pulmonar (TB), bem como efectuar uma correlação do estado imunitário e da gravidade clínica da doença tuberculosa. As células mononucleadas do sangue periférico (CMSP) foram cultivadas e estimuladas com M. tuberculosis H37Rv e com um derivado de proteína purificado (PPD) e a produção de IFN-γ e de IL-4/IL-5 foi quantificada através das técnicas de ELISA e de citometria de fluxo multi-parâmetros. As CMSP de 30 doentes com tuberculose demonstraram um decréscimo significativo na proliferação e produção de IFN-γ, quando estimuladas com M. tuberculosis vivo comparativamente com o grupo controlo. Dos 15 doentes com tuberculose estudados e após estimulação com M. tuberculosis, sete demonstraram um grande aumento na produção de IL-4 intracelular pelas células T CD8+ e γδ+. Os doentes com tuberculose demonstraram um aumento de IL-4 intracelular após as CMSP serem estimuladas com M. tuberculosis vivo, nomeadamente do fenótipo CD4+, mas mais notavelmente do subtipo CD8+ and γδ TCR+. O aumento da produção de IL-4 em doentes com tuberculose, verificou-se em indivíduos com um envolvimento avançado do parênquima pulmonar apresentando grandes cargas bacterianas nas expectorações. Estes resultados sugerem que pode ocorrer uma alteração no equilíbrio das citocinas tipo 1 e tipo 2, em doentes com tuberculose que se encontrem num estádio clínico avançado da doença.
Although alkaloids from the family Aizoaceae have anticancer activity, species of this family hav... more Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non‐toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi‐drug resistant cells susceptible to anticancer drugs. These non‐toxic concentrations also prime THP‐1 human monocyte‐derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP‐1 cells within 1 day of exposure to quantities n...
We evaluated immune responses to Mycobacterium tuberculosis in 10 health-care workers (HCWs) and ... more We evaluated immune responses to Mycobacterium tuberculosis in 10 health-care workers (HCWs) and 10 non-HCWs and correlated their immune status with the development of active tuberculosis (TB). Twenty individuals were randomly recruited, tested, and monitored longitudinally for TB presentation. Peripheral blood mononuclear cells (PBMCs) from donors were stimulated with M. tuberculosis and tested for cell proliferation and the production of interferon (IFN)-g, interleukin (IL)-5, and IL-4, by use of enzyme-linked immunosorbent or flow-cytometric assays. HCWs had higher levels of cell proliferation (24,258 cpm) and IFN-g (6373 pg/mL) to M. tuberculosis than did non-HCWs (cell proliferation, 11,462 cpm; IFN-g, 3228 pg/ mL). Six of 10 HCWs showed increased median percentages of CD8 + IL-4 + (4.7%) and gd + IL-4 + (2.3%) T cells and progressed to active TB. HCWs who remained healthy showed increased median percentages of CD8 + IFNg + (25.0%) and gd + IFN-g + (8.0%) and lower percentages of CD8 + IL-4 + (0.05%) and gd + IL-4 + (0.03%) T cells. Tuberculosis (TB) is responsible for more adult deaths annually than any other infectious agent [1, 2]. In developing countries, nosocomial transmission of TB can be a threat to health-care workers (HCWs), because there are increased numbers of cases of TB on open wards and minimal or absent TB infection controls [3-6]. In countries where TB is endemic, it is difficult to
International Journal of Antimicrobial Agents, 2002
The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood mon... more The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood monocyte derived macrophages (HPBMDM) and of human macrophage cell line THP-1 at extracellular concentrations below those achievable clinically. These macrophages have nominal killing activity against bacteria and therefore, would not influence any activity that the compounds may have against intracellular localised Staphylococcus aureus. The results indicated that whereas TZ has an in vitro minimum inhibitory concentration (MIC) against the strains of S. aureus of 18, 0.1 mg/l of TZ in the medium completely inhibits the growth of S. aureus that has been phagocytosed by macrophages. The latter concentration was non-toxic to macrophages, did not cause cellular expression of activation marker CD69 nor induction of CD3'/ T cell production of IFN-g, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-g, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus.
This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacteri... more This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-c production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased cd TCR+ cell cytotoxicity and decreased CD3+cd TCRÀ cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and cd TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased cd TCR+ cytotoxicity and reduced CD3+cd TCRÀ cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of cd TCR+ cells which did not prevent TB.
BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include moni... more BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.
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