In this study, we examined the usefulness of various markers on blood cell populations in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). We also evaluated bone marrow specimens, which generally are considered less suitable than blood owing to variable expression of glycosyl phosphatidylinositol (GPI)-linked antigens during hematopoietic cell differentiation. All 15 patients in our cohort had subpopulations of CD16/CD55-deficient granulocytes and CD14/CD55-deficient monocytes ("PNH clones"). The PNH clone size of granulocytes and monocytes was greater than that of erythrocytes or lymphocytes in the majority of the cases. It is interesting that CD59 showed limited usefulness for detecting PNH+ monocytes. Normal monocytes exhibited significantly dimmer CD59 expression than normal granulocytes. PNH-deficient monocytes expressed only marginally lower CD59, making it a less robust marker for highlighting PNH+ monocytes compared with CD14 or CD55. Finally, our study demonstrated the definitive usefulness of a limited combination of markers (CD16/CD55/CD45/CD14) in detecting GPI-deficient monocytes and granulocytes in bone marrow specimens submitted for flow cytometric evaluation of cytopenias.
Subject C135 is one of the members of the Sydney Blood Bank Cohort, infected in 1981 through transfusion with attenuated nef/3′ long terminal repeat (LTR)-deleted HIV-1, and has maintained undetectable plasma viral load and steady CD4 cell count, in the absence of therapy. Uniquely, C135 combines five factors separately associated with control of viraemia: nef/LTR-deleted HIV-1, HLA-B57, HLA-DR13, heterozygous CCR5 Δ32 genotype and vigorous p24-stimulated peripheral blood mononuclear cell (PBMC) proliferation. Therefore, we studied in detail viral burden and immunological responses in this individual. PBMC and gut and lymph node biopsy samples were analysed for proviral HIV-1 DNA by real-time and nested PCRs, and nef/LTR alleles by nested PCR. HIV-specific antibodies were studied by Western blotting, and CD4+ and CD8+ T lymphocyte responses were measured by proliferation and cytokine production in vitro. PBMC samples from 1996, but not since, showed amplification of nef alleles with gross deletions. Infectious HIV-1 was never recovered. Proviral HIV-1 DNA was not detected in recent PBMC or gut or lymph node biopsy samples. C135 has a consistently weak antibody response and a substantial CD4+ T cell proliferative response to a previously described HLA-DR13-restricted epitope of HIV-1 p24 in vitro, which augmented a CD8+ T cell response to an immunodominant HLA-B57-restricted epitope of p24, while his T cells show reduced levels of CCR5. Subject C135's early PCR and weak antibody results are consistent with limited infection with a poorly replicating nef/LTR-deleted strain of HIV-1. With his HLA-B57-restricted gag-specific CD8 and helper HLA-DR13-restricted CD4 T cell proliferative responses, C135 appears to have cleared his HIV-1 infection 37 years after transfusion.
Ag-specific human CD4(+) memory T lymphocytes have mostly been studied using assays of proliferation in vitro. Intracellular cytokine and ELISPOT assays quantify effector cell populations but barely detect responses to certain recall Ags that elicit strong proliferative responses, e.g., tetanus toxoid, that comprise non-Th1 CD4(+) cells. We have found that culturing whole blood with Ag for 40-48 h induces specific CD4(+) T cells to simultaneously express CD25 and CD134. This new technique readily detects responses to well-described CD4(+) T cell recall Ags, including preparations of mycobacteria, CMV, HSV-1, influenza, tetanus toxoid, Candida albicans, and streptokinase, as well as HIV-1 peptides, with high specificity. The assay detects much higher levels of Ag-specific cells than intracellular cytokine assays, plus the cells retain viability and can be sorted for in vitro expansion. Furthermore, current in vitro assays for human CD4(+) memory T lymphocytes are too labor-intensive and difficult to standardize for routine diagnostic laboratories, whereas the whole-blood CD25(+)CD134(+) assay combines simplicity of setup with a straightforward cell surface flow cytometry readout. In addition to revealing the true extent of Ag-specific human CD4(+) memory T lymphocytes, its greatest use will be as a simple in vitro monitor of CD4(+) T cell responses to Ags such as tuberculosis infection or vaccines.
The fine control of T ‐cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B ‐lymphocyte‐induced maturation protein‐1 ( B limp‐1/ Prdm1 ) is highly expressed in CD 4 + T cells from chronically HIV ‐infected ( CHI ) patients compared to cells from long‐term nonprogressors or healthy controls. Stimulation through the T ‐cell receptor in the presence of IL ‐2 induces B limp‐1 protein expression. We show here that B limp‐1 levels are translationally regulated by micro RNA ‐9 (mi R ‐9). Overexpression of mi R ‐9 induces B limp‐1 repression, restoring IL ‐2 secretion in CD 4 + T cells via reduction in the binding of B limp‐1 to the il‐2 promoter. In CHI patients where IL ‐2 expression is reduced and there is generalized T ‐cell dysfunction, we show differential expression of both mi R ‐9 and B limp‐1 in CD 4 + cells compared with levels in long‐term nonprogressors. These data identify a novel mi R ‐9/ B limp‐1/ IL ‐2 axis that is dysregulated in progressive HIV infection.
Tumour-infiltrating T lymphocytes (TIL-T) have been implicated in playing a role in controlling tumour growth. We evaluated TIL-T in 55 cases of de novo diffuse large B-cell lymphoma (DLBCL) using three- or four-colour flow cytometric immunophenotyping (FCI). The percentage of TIL-T varied from 3% to 72% of total viable cellular events (mean 32 +/- 20%). The CD4:CD8 ratio varied from 0.17 to 13 (mean 2.3 +/- 2.2). Cases with >/= 20% T cells and those with CD4:CD8 ratios > or = 2.0 showed a significantly better overall survival (P = 0.017 and P = 0.034 respectively). These findings were independent of clinical stage at diagnosis. The T-cell percentage and CD4:CD8 ratio were moderately correlated (Spearman correlation coefficient = 0.47, P = 0.001) and multivariate analysis revealed that the association of the two factors with prognosis was mutually dependent. The T cells in 23 cases were studied for CD45RO. The mean percentage of total T cells expressing CD45RO was 86 +/- 10%. There was a trend towards better survival for those patients with a higher percentage of CD45RO+ T cells (P = 0.06). These results suggest that TIL-T, particularly CD4+ T cells, may play a role in the control of DLBCL, and measurement of T-cell percentage and T-cell subsets using FCI may be useful in predicting the clinical behaviour of DLBCL.
Abstract The purpose of this study was to examine the level of diabetes self‐management and its association with demographic and diabetes‐related characteristics in Chinese Americans with type 2 diabetes. A questionnaire that measured diabetes self‐management and diabetes‐related characteristics was administered to a sample of 211 Chinese Americans with type 2 diabetes living in America. The results indicated that the participants were likely to take medications but less likely to carry out diet, physical activity, self‐monitoring of blood glucose, and foot care behaviors. Associations between diabetes self‐management and demographic and diabetes‐related characteristics were observed. For example, individuals who had less education and were employed were less likely to engage in diabetes self‐management than those with higher education and who were retired, while individuals who had a longer duration of diabetes and used insulin as a treatment more frequently carried out self‐monitoring than those who had a shorter duration of diabetes and used oral hypoglycemic agents. These findings indicate that the self‐management practices among the participants are suboptimal. Research on developing culturally and linguistically appropriate interventions to promote diabetes self‐management for Chinese Americans is warranted.
We evaluated the usefulness of multiparameter flow cytometry with cluster analysis in the diagnosis of a series of 100 well-characterized small B-cell lymphomas (SBCLs). The histologic diagnoses in the 100 cases were follicular lymphoma (FL) in 58, marginal zone lymphoma (MZL) in 17, small lymphocytic lymphoma in 15, and mantle cell lymphoma (MCL) in 10. Of the 58 FLs, 57 were CD10 positive (98% sensitivity). The 1 negative case was unusual in that it occurred in the small intestine. However, architectural, cytologic, and immunohistochemical features were diagnostic of FL. Of 42 other SBCLs, 2 were CD10+ (95% specificity); 1 was a CD5+/cyclin D1+ MCL, and the other was an extranodal MZL. We found that assessment of CD10 expression using multiparameter flow cytometry with cluster analysis is highly sensitive and specific for the diagnosis of FL, validating its usefulness in situations in which adequate tissue is not available for definitive histologic diagnosis.
Abstract We report a 58-year-old man who presented with fever, pancytopenia, hepatosplenomegaly, and “sinusitis” of his right nostril. Flow cytometric analysis of his bone marrow aspirate revealed a population of cells that were CD56+ (bright), CD2+ (dim), and CD7+ (slight brightly) but negative for CD3, CD4, CD5, CD8, CD11b, CD16, CD57, and T-cell receptors, consistent with aberrant natural killer cells. Bone marrow biopsy showed an atypical lymphoid infiltrate expressing CD56 as well as Epstein-Barr virus– encoded RNA and histiocytic hyperplasia with hemophagocytosis. Subsequent biopsy of his right nasal vestibule demonstrated an atypical lymphoid infiltrate, which was cytoplasmic CD3+, CD56+, and Epstein-Barr virus–encoded RNA positive, consistent with an extranodal nasal natural killer cell lymphoma. Conventional cytogenetic studies of the bone marrow revealed isochromosome 7q10 [i(7)(q10)] as the sole chromosomal aberration. To our knowledge, this is the first report demonstrating i(7)(q10) as a primary cytogenetic abnormality in an extranodal nasal natural killer cell lymphoma.
We analyzed 53 cases of diffuse large B-cell lymphoma (DLBCL) to determine whether expression of CD10 is a relevant biologic parameter. Tumor morphologic features were assessed semiquantitatively. Bcl-2 protein expression was studied by immunohistochemical analysis. The presence or absence of CD10 by flow cytometry was correlated with clinical and pathologic characteristics. CD10+ (23 cases) and CD10- (30 cases) DLBCLs were indistinguishable based on age, sex, extranodal presentation, B symptoms, clinical stage, morphologic features, or bcl-2 expression. However, cases with a CD10+ phenotype showed a significantly lower rate of complete remission. Cases expressing bcl-2 showed trends toward a lower rate of complete remission and poorer overall survival. Examination of CD10 and bcl-2 interaction revealed that the prognostic effects for both of these antigens were due to a subset of CD10+ bcl-2-positive cases. Compared with cases expressing one or neither of these markers, patients with dual-positive tumors had a poorer complete response rate to initial therapy and strikingly worse overall survival. While CD10+ and CD10- DLBCLs are similar with regard to a variety of clinical and pathologic features, CD10 and bcl-2 coexpressing tumors are an extremely high-risk subset based on response to therapy and overall survival.
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