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BACKGROUND: Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression-free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter-laboratory variation in MM MRD reporting. METHODS: Seventeen participants from 13 countries performed blinded analysis of the same eight de-identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re-analyzed for MRD and other bone marrow cells (Stage 2). Inter-laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3). RESULTS: In Stage 1, participants agreed on MRD+/MRD− status 89% and 68% of the time respectively. Inter-observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD− status improved to 74%. Better consistency enumerating all parameters among operators resulted in near-unanimous agreement on sample adequacy. CONCLUSION: Uniform flow cytometry data analysis substantially reduced inter-laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

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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 o ...

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 naive 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. 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+CD25hiCD127low. A multiplex bead-based immunoassay was used to determine TNF-α, IFN-ɣ, IL-17A, IL-10, and TGF-β levels. 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 naive (p = 0.003) Tregs as well as elevated TNF-α (p

Background Minimal/measurable residual disease (MRD) testing by flow cytometry (FC) has been proposed as a potential surrogate clinical endpoint in plasma cell myeloma (PCM) clinical trials. As a result, effort has gone into standardizing this approach on PCM patients. Aims To assess inter-laboratory variation in FC MRD testing of PCM patients in an independent inter-laboratory study. Methods A dilution series of five stabilized bone marrow samples manufactured to contain 0%, 0.1%, 0.01%, 0.001%, and 0.0001% neoplastic plasma cells (PCs) were tested blind, using standardized FC PCM MRD assays by 10 international laboratories. Results Laboratories' assays broadly adhered to the consensus guidelines; however, some deviations were identified in panel design, fluorochrome conjugates, and lysis reagents. Despite this, all laboratories that returned results detected neoplastic PCs down to 0.001% of leucocytes. 6/8 laboratories detected neoplastic PCs at a level of 0.0001%. Quantitative data returned by laboratories showed good consensus and linearity with increasing variation at lower levels of MRD. However, examples of analytical and post analytical error were identified. Summary/conclusion Broadly standardized PCM MRD FC assays can attain the lower limit of detection (LOD) required by current and future clinical trials, an important consideration in establishing PCM MRD testing as a surrogate clinical marker in PCM clinical trials. Laboratories' assays showed good linearity, encouraging the prediction of survival based on log reduction in neoplastic PC populations in future clinical trials. However, the deviations from consensus guidelines identified in this study would suggest that if PCM MRD assays are further standardized interlaboratory variation could be reduced. © 2018 International Clinical Cytometry Society.

BACKGROUND Malignant epithelial cells may be detected in different specimens, by immunophenotyping using flow cytometry (FCM). CD326 (epithelial-specific antigen, clone Ber-Ep4) was used to identify epithelial cells, CD45 to discriminate between leucocytes (positive for this antigen) and non-hematological cells (negative for this antigen), and CD33 to identify monocytes/macrophages. This combination is particularly useful in effusions to characterize large cells and distinguish between monocyte/macrophages (CD45+ CD33+ CD326-), mesothelial cells (CD45 ± (dim) CD33 - CD326-) and epithelial cells (CD45 - CD33 - CD326 +). We evaluated the efficiency of flow cytometry to detect malignant epithelial cells in 238 fresh samples, including effusions, lymph node biopsies, fine needle aspirates, bone marrow aspirates, cerebrospinal fluid, among others. These are specimens expected to lack epithelial cells. FCM results were then compared to the results of smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks, when available. Final diagnosis was the gold standard and a very good sensitivity (96.7%) and specificity (99.3%) were obtained. We concluded that the detection of CD326 positive cells using FCM is strongly indicative of the presence of carcinoma cells. © 2015 International Clinical Cytometry Society.

Background Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing. Methods A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM. Results/Conclusion Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 106 and 5 × 106 bone marrow cells to be measured, respectively. © 2015 International Clinical Cytometry Society

Early-stage chronic lymphocytic leukemia (CLL) with neurologic involvement is a rare condition and should require a careful follow-up. Although no standard protocol exists for this condition, intrathecal chemotherapy, combined with systemic chemoimmunotherapy, has been used previously. This case describes the treatment of a patient with CLL and symptomatic compromise of the central nervous system. Our results suggest that a combination of chemotherapy, radiotherapy, and ibrutinib, administered sequentially over a 2-year period, led to a near-complete resolution of the cerebral spinal fluid neoplastic infiltration. Importantly, this response has been maintained with ibrutinib monotherapy for more than 12 months.

Terra rossa is a residual soil that is derived from the chemical dissolution of carbonate rocks. This type of soil occurs in the region of the so-called Estremoz anticlinal, being the result of the weathering of extensive marble occurrences which are exploited for ornamental stone. The research work performed and disclosed in the present paper shows that terra rossa possesses appropriate compositional and technological properties for its use on ceramic applications, particularly for the local traditional pottery. In potential marble quarry sites several thick soil horizons can be found, and whenever the soil is removed marble is shown up. As a rule, the removed soil is deposited around the quarry, and so far no use has been given to it. Pottery has a long tradition in the Alentejo region, and Vila de Redondo and Aldeia de S. Pedro do Corval are known as emblematic centers of this activity. However, time has shown that the scarcity of high quality clay deposits has become an important issue. Therefore, potters are buying clay from other regions of Portugal, in particular in the area of Caldas da Rainha, located in the central west of the country and far from Alentejo, or even importing it from Spain. The cooperative research work carried out in the Laboratories of DGUE, the Department of Geosciences at the University of Aveiro (DGUA) and CENCAL, and at the pottery owned by the artisan Mestre Xico Tarefa, in Vila de Redondo, has shown that terra rossa, due to its physicochemical, mineralogical and technological properties, can be used as a raw material in ceramic manufacturing, particularly in the traditional pottery produced both in Redondo and S. Pedro de Corval.

Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia.

BACKGROUND: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. METHODS: Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. RESULTS: Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. CONCLUSIONS: The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.

BACKGROUND: The WHO and iwCLL diagnostic criteria for CLL rely on morphology and immunophenotype based on the co-expression of CD19/CD5/CD23 on B-cells with weak CD20 and monoclonal sIg expression. These diagnostic criteria are likely to persist in the near future because there is no specific diagnostic molecular abnormality for CLL. The current criteria have some limitations affecting reproducibility, particularly flexibility in marker expression with many centres using a scoring system that permits absence of CD5 or CD23. Potentially informative new markers have been identified but there is no consensus yet on which should be routinely assessed. AIM: To identify reproducible criteria and to achieve a consensus on markers recommended for the diagnosis of CLL METHODS: ERIC/ESCCA members were invited to classify 35 flow-cytometry markers as being required or recommended for the diagnosis of CLL. Consensus was considered to be achieved if >75% of participants agreed on the marker classification. A diagnostic panel was identified by the steering committee and characteristics of component markers that could be reproducibly validated within an individual laboratory were identified. The proposed panel was assessed in 13 different centres. RESULTS: Responses were received from 154 members (100 laboratory staff, 14 clinicians and 36 from both laboratory and clinic) with a diagnostic workload >20 cases per week in 23/154 (15%), 5-20 in 82/154 (53%) and CONCLUSIONS: We present flow-cytometry criteria for the diagnosis of CLL largely consistent with current practice. In addition, reproducible definitions of the required expression pattern and performance characteristics of reagents are provided. Prospective evaluation of the proposed criteria as well as a parallel project to facilitate definitive diagnosis of CD5+ B-LPD cases that do not meet the proposed criteria are underway. Figure 1. required and recommended markers for use in the diagnosis of CLL with reagent specification based on expression patterns in normal peripheral blood. Figure 1. required and recommended markers for use in the diagnosis of CLL with reagent specification based on expression patterns in normal peripheral blood. ‡ Weak expression = median fluorescence intensity at least 20% lower than median for normal peripheral blood B-cells, reference range determined within each laboratory, based on ICSH/ISLH/CLIA guidelines for reproducibility *consensus, not specifically validated Disclosures Rawstron: Abbvie: Honoraria; Pharmacyclics: Research Funding; Celgene: Honoraria; Roche: Honoraria; BD Biosciences: Patents & Royalties; Gilead: Honoraria, Research Funding. Cuneo:Roche: Speakers Bureau; Gilead: Speakers Bureau; Jannsen: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Mulligan:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding. Hillmen:Celgene: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Hallek:Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:AbbVie: Consultancy; Janssen: Consultancy; Roche: Consultancy, Research Funding; Adaptive: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; GSK: Research Funding; Acerta Pharma BV: Research Funding; Pharmacyclics: Consultancy.

RESUMO: Foram comparadas em dadores com tuberculose pulmonar e indivíduos saudáveis as respostas blastogénicas e a produção de citocinas induzidas por dois filtrados de cultura de Mycobacterium tuberculosis, colectados em diferentes tempos. Tanto no grupo de dadores saudáveis, como no grupo de doentes com tuberculose pulmonar, as proteínas de curto prazo filtradas da cultura de M. tuberculosis (short term culture filtrate proteins-ST-CFP) induziram uma produção mais marcada de INF-γ do que as proteínas de médio prazo (mid term culture filtrate proteins-MT-CFP), enquanto que as MT-CFP induziram uma linfoproliferação mais elevada. Verificou-se uma heterogeneidade considerável nos controlos e os resultados indicaram que o contacto crónico e regular com doentes com tuberculose apresentaram respostas elevadas às ST-CFP. Em relação à estimulação com os perfis antigénicos, tanto a linfoproliferação como a produção de INF-γ se encontravam reduzidas em dadores com tuberculose pulmonar, quando comparados com os controlos. A presença dos antigénios ESAT-6, 10 kDa, 19 kDa e 30 kDa do M. tuberculosis e do lipoarabinomanano (LAM), foi comparada em ambas as preparações utilizando o Western blotting. A MT-CFP contém ligeiramente mais lipoarabinomanano do que a ST-CFP e quantidades mais elevadas de ESAT-6 estão presentes na ST-CFP. A análise ao ARNm para o INF-γ, IL-4 e IL-10 confirmou que a produção de INF-γ estava reduzida em doentes com tuberculose pulmonar, sem qualquer aumento concomitante na produção de IL-4. Estes resultados mostram que variações ao longo do tempo de cultura do M. tuberculosis podem desencadear in vitro diferentes respostas imunitárias mediadas por células.REV PORT PNEUMOL 2002; VIII (6): 629-643 ABSTRACT: Blastogenic responses and cytokine production induced by two culture filtrates of Mycobacterium tuberculosis collected at different time points were compared in donors with pulmonary tuberculosis and healthy Mantoux-positive BCG vaccinated controls. The short term culture filtrate proteins (ST-CFP) induced stronger IFN-γ production than the mid term culture filtrate proteins (MT-CFP), in both healthy BCG-vaccinated controls and patients with pulmonary tuberculosis, while the MT-CFP induced higher lymphocyte proliferation. Considerable heterogeneity was found within the healthy subjects, results indicated that chronic tuberculosis contacts elicited higher responses toward ST-CFP. Pulmonary tuberculosis donors demonstrated reduced proliferation and IFN-γ production towards the antigen profiles when compared to the healthy subjects. The presence of ESAT-6, 10 kDa, 19 kDa, 30 kDa antigens of M. tuberculosis and lipoarabinomannan (LAM) were compared in both preparations using Western blotting. Although the MT-CFP contained slightly more lipoarabinomannan than the ST-CFP and higher quantities of the ESAT-6 was present in ST-CFP, there were no other obvious differences in the composition of the two culture filtrates. Analysis of mRNA for IFN-γ, IL-4 and IL-10 confirmed that IFN-γ production was reduced in patients with pulmonary tuberculosis without any concomitant increase in IL-4. These results show that variations in time of culture of M. tuberculosis, can elicit different cell-mediated immune responses in vitro.REV PORT PNEUMOL 2002; VIII (6): 629-643 Palavras-chave: Tuberculose (TB), proteínas filtradas de cultura, Proliferação linfocitária, Interferão-γ, Key-words: Tuberculosis (TB), culture filtrate proteins, Lymphocyte proliferation, Interferon-γ

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-gamma, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-gamma, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus.

Malignant epithelial cells may be detected in different specimens, by immunophenotyping using flow cytometry (FCM). CD326 (epithelial-specific antigen, clone Ber-Ep4) was used to identify epithelial cells, CD45 to discriminate between leucocytes (positive for this antigen) and non-hematological cells (negative for this antigen), and CD33 to identify monocytes/macrophages. This combination is particularly useful in effusions to characterize large cells and distinguish between monocyte/macrophages (CD45+ CD33+ CD326-), mesothelial cells (CD45 ± (dim) CD33 - CD326-) and epithelial cells (CD45 - CD33 - CD326 +). We evaluated the efficiency of flow cytometry to detect malignant epithelial cells in 238 fresh samples, including effusions, lymph node biopsies, fine needle aspirates, bone marrow aspirates, cerebrospinal fluid, among others. These are specimens expected to lack epithelial cells. FCM results were then compared to the results of smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks, when available. Final diagnosis was the gold standard and a very good sensitivity (96.7%) and specificity (99.3%) were obtained. We concluded that the detection of CD326 positive cells using FCM is strongly indicative of the presence of carcinoma cells. © 2015 International Clinical Cytometry Society.

Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing.A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM.Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 10(6) and 5 × 10(6) bone marrow cells to be measured, respectively.

RESUMO: Avaliaram-se in vitro as respostas das citocinas Tipo 1 e Tipo 2 de dadores portugueses saudáveis vacinados à nascença com BCG, com Mantoux positivo (induraçãoâ¥5 mm) e de doentes com tuberculose pulmonar (TP). Foi efectuado o estudo por ELISA da produção de IFN-γ e IL-5 por Células Mononucleares do Sangue Periférico (CMSP) dos dadores após estimulação com M. tuberculosis e antigénio solúvel PPD. Foi confirmada a presença intracelular de IFN-γ e de IL-4 em subpopulações de células T por análise multiparamétrica, em citometria de fluxo. As CMSP dos doentes com tuberculose estimuladas com PPD e M. tuberculosis demonstraram uma diminuição na produção de IFN-γ sem aumento da produção de IL-5 em resposta ao painel de antigénios. Nos doentes com tuberculose observou-se uma frequência diminuída de IFN-γ intracelular nas células T CD4+ e CD8+, em comparação com os grupos controlos. Após estimulação das CMSP com M. tuberculosis, os doentes demonstraram um aumento médio de IL-4 intracelular no fenótipo T CD4+, mais evidente na subpopulação T CD8+. O aumento da secreção de IL-4 nos doentes com tuberculose verificou-se sobretudo nos indivíduos em estado avançado da doença. Os resultados obtidos por Citometria de Fluxo contradizem de alguma forma os obtidos por ELISA. A secreção de IL-4 intracelular representa uma medida mais sensível da produção de citocinas tipo 2 do que a quantificação de IL-5 por técnicas de ELISA. Estes novos resultados sugerem que pode ocorrer uma mudança de T 1 para T 2 em doentes com tuberculose em estado avançado.REV PORT PNEUMOL 2001; VII (2): ABSTRACT: In vitro Type 1 and Type 2 cytokine responses were assessed in healthy Portuguese donors who were BGC vaccinated at birth and Mantoux positive (induration 5â¥mm) and pulmonary tuberculosis patients (TB). We evaluated the production of IFN-γ and IL-5, after PBMC from donors were stimulated with live M. tuberculosis H37Rv and the soluble antigen PPD, by standard ELISA techniques. These studies were extended to confirm intracellular presence of IFN-γ and IL-4 in specific T cell subsets by multi-parameter flow cytometry. PBMC from tuberculosis patients demonstrated significantly reduced amounts of IFN-γ when stimulated with PPD and M. tuberculosis, with no increase in IL-5 production towards all the antigens, when compared to the control group. Intracellular staining for IFN-γ in tuberculosis patients showed reduced frequencies of CD4+ and CD8+ T cell intracellular IFN-γ in comparison to healthy subjects. Tuberculosis patients demonstrated a mean increase of intracellular IL-4 after PBMC were stimulated with M. tuberculosis in the CD4+ phenotype, but more notably in the CD8+ subset. The increased secretion of IL-4 in tuberculosis patients was primarily in individuals with an advanced clinical form of the disease. Interestingly the findings using flow cytometry techniques somewhat contradicts the results obtained by ELISA. Intracellular IL-4 secretion is therefore a more sensitive measure of Type 2 cytokine production than quantitation of IL-5 by ELISA. These results suggest that a type 1 switch to a type 2 can occur, in patients with tuberculosis in an advanced stage.REV PORT PNEUMOL 2001; VII (2): Palavras-chave: Imunidade Celular, Tuberculose Pulmonar, Respostas de citocinas Tipo 1 e Tipo 2, Key-words: Cellular Immunity, Pulmonary tuberculosis, Type 1 and Type 2 Cytokine responses

RESUMO: Analisaram-se as respostas Th1 e Th2 desencadeadas por Mycobacterium tuberculosis virulento em doentes com tuberculose pulmonar (TP) e em dadores saudáveis vacinados pela BCG. Efectuaramse comparações entre a capacidade que as células T apresentavam para proliferar e para produzir IFN-γ e IL-5 em resposta aos derivados de proteíns purificada (PPD), M. tuberculosis H37Rv (Mtb), e M. tuberculosis H37Rv inactivado pelo calor (hk Mtb).Este estudo demoostrou que os individuos saudá-vels vacínados com BCG evidenciaram um máximo de proliferação e produção de IFN-γ em resposta ao painel de antigénios, e que Mtb vívo destencadeou uma resposta significativamente mais forte que a obtida pelo Mtb inactivado pelo calor. Embora os doentes com tuberculose pulmonary mostrassem respostas medias mais baxies de proliferação e produção de IFN-γ cm relação aos eontrolos saúdaveis, a resposta proliferativa ao PPD não foi significativamente reduzida, enquanto que a resposta ao Mtb e hk Mtb foram, significativas estatisticamitante. Em conclusão na tuberculose pulmonar a produção de IFN-γ pode estar reduzida sem um concomitante aumento de IL-5, confirmandose assim não existir uma mudança de resposta Th1 para Th2 na tuberculose pulmonar.REV PORT PNEUMOL 1998; IV (4):393-402 ABSTRACT: The contribution of Th1 and Th2 responses elicited by virulent Mycobacterium tuberculosis was investigated, in healthy BCG vaccinated individuals and pulmonary tuberculosis patients. Comparisons were made between the T cell capacity to proliferate, produce IFN-γ and IL-5 in response to the soluable antigen purified protein derivative (PPD), live M. tuberculosis H37Rv (Mtb) and heat killed M tuberculosis H37Rv (hk Mtb). These studies demonstrated that control individuals showed the strongest mean proliferative and IFN-γ responses towards the antigen antigen panel and Mth elicited a significantly stronger response than hk Mtb. Although, pulmonary tuberculosis patients showed lower mean proliferation and IFN-γ towards all the antigen when compared to that control group, proliferative responses to PPD were found not to be significantly reduced, while the reduction in the response to Mtb and hk Mtb preparations were statistically significant. In conclusion, in pulmonary tuberculose infection mean prodution of IFN-γ may be reduced but no concomitant increase in IL-5 production occurred. These data confirm that there is no switch from a Th1 response to a Th2 response in tuberculosis infection.REV PORT PNEUMOL 1998; IV (4): 393-402 Key-words: Cellular Immunity, Cytokines, Pulmonary tuberculoses, Th1 and Th2 responses, Palavaras-chave: Imunidade Celular, Citocinas, Tuberculose Pulmonar, respostas Th1 e Th2

Several risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. OBJECTIVES: This study was designed to assess and compare the immunological status during the first 2 years in steroid-naive young children with >or= three episodes of physician-confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age-matched healthy controls (n=30). METHODS: Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) T cells and their cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN-gamma, TGF-beta and IL-10), CTLA-4 and Foxp3 mRNA expression were evaluated (real-time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12-myristate 13-acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). RESULTS: Flow cytometry results showed a significant reduction in the absolute number of CD4(+)CD25(high) and the absolute and percentage numbers of CD4(+)CD25(+)CTLA-4(+) in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4(+)CD25(+) (P=0.01) and CD4(+)CD25(high) (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA-4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA-4 (P=0.03) and IFN-gamma (P=0.04) expression was diminished in wheezy children compared with healthy children. High-risk children had lower expression of IFN-gamma (P=0.03) compared with low-risk and healthy children and lower expression of CTLA-4 (P=0.01) compared with healthy children. CONCLUSIONS: Although our findings suggest that some immunological parameters are impaired in children with recurrent wheeze, particularly with a high risk for asthma, further studies are needed in order to assess their potential as surrogate predictor factors for asthma in early life.

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.

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 normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections.

Chlorpromazine (CPZ) has in vitro antimicrobial activity against Staphylococcus aureus at concentrations that greatly exceed those achieved clinically. It is concentrated by tissues that are rich in macrophages and it is active against phagocytosed mycobacteria when the concentration in the medium is compatible with that achieved clinically. In this report we show that nontoxic concentrations of CPZ below clinical levels have killing activity against S. aureus phagocytosed by human monocyte-derived macrophages that have nominal killing activity against these bacteria. Little or no resistance to the antimicrobial activity of this compound is anticipated to result because of its large number of cellular targets. Therefore, CPZ may have a role in the management of intracellular staphylococcal infections that normally require the use of antibiotics whose potential toxicity exceeds that associated with short-term management with CPZ.