Bruce Blais

Bio: Bruce Blais is an academic researcher from University of Massachusetts Amherst. The author has contributed to research in topics: Peripheral blood mononuclear cell & T cell. The author has an hindex of 3, co-authored 4 publications receiving 166 citations.

Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization

Abstract: The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4+ and CD8+ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8+ T lymphocytes

Abstract: We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174–179, 2000. © 2000 Wiley-Liss, Inc.

Standardizing immunophenotyping for the Human Immunology Project

Abstract: The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described Their comprehensive elucidation has been termed the 'Human Immunology Project' The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project

Lymphocyte subsets in healthy children from birth through 18 years of age : The pediatric AIDS clinical trials group P1009 study

Abstract: Background Peripheral blood lymphocyte subsets need to be determined in a large, urban, minority-predominant cohort of healthy children to serve as suitable control subjects for the interpretation of the appearance of these cells in several disease conditions, notably pediatric HIV-1 infection. Objective We sought to determine the distribution of lymphocyte subsets in healthy urban-dwelling infants, children, and adolescents in the United States. Methods Lymphocyte subsets were determined by means of 3-color flow cytometry in a cross-sectional study of 807 HIV-unexposed children from birth through 18 years of age. Results Cell-surface marker analysis demonstrated that age was an extremely important variable in 24 lymphocyte subset distributions measured as percentages or absolute counts—eg, the CD4 (helper) T cell, CD8 (cytotoxic) T cell, CD19 B cell, CD4CD45RACD62L (naive helper) T cell, CD3CD4CD45RO (memory helper) T cell, CD8HLA-DRCD38 (activated cytotoxic) T cell, and CD8CD28 (activation primed cytotoxic) T cell. The testing laboratory proved to be an important variable, indicating the need for using the same laboratory or group of laboratories to assay an individual's blood over time and to assay control and ill or treated populations. Sex and race-ethnicity were much less important. Conclusion The results of this study provide a control population for assessment of the effects of HIV infection on the normal development and distribution of lymphocyte subsets in children of both sexes, all races, and all ethnic backgrounds from birth through 18 years of age in an urban population. This study's findings will also prove invaluable in interpreting the immune changes in children with many other chronic diseases, such as primary immunodeficiency, malignancy, rheumatoid arthritis, and asthma.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Abstract: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Cellular Receptors and Viral Glycoproteins Involved in Retrovirus Entry

Abstract: The initial steps in virus infection involve the binding of virions to specific cell surface receptors. Following the attachment of enveloped viruses, further events lead to membrane fusion either at the cell surface or in endocytic vesicles that deliver the core of the virus to the cytoplasm (Marsh and Helenius, 1989). For retroviruses, the outer surface (SU) envelope glycoproteins bear epitopes for specific receptor recognition. Binding and conformational changes in the envelope spikes lead to the exposure of hydrophobic domains on the transmembrane (TM) envelope protein that are believed to effect membrane fusion by embedding in the plasma membrane. The individual envelope (env) proteins (p) and glycoproteins (gp) are named after their estimated molecular weight. Thus for the human immunodeficiency virus type 1 (HIV-1) the SU protein, gp120, is approximately 120 kDa, and the TM protein, gp41, is 41 kDa. The retroviral TM/SU spikes are trimers or tetramers (Einfeld and Hunter, 1988; Schawaller et al., 1989; Earl et al., 1990, 1992; C. D. Weiss et al., 1990). With HIV-1, a tetrameric complex may assemble via dimerization of TM dimers (Pinter et al., 1989; Rey et al., 1990).

Microbial translocation induces persistent macrophage activation unrelated to HIV-1 levels or T cell activation following therapy

Abstract: OBJECTIVE HIV-1 replication and microbial translocation occur concomitant with systemic immune activation. This study delineates mechanisms of immune activation and CD4 T-cell decline in pediatric HIV-1 infection. DESIGN Cross-sectional and longitudinal cellular and soluble plasma markers for inflammation were evaluated in 14 healthy and 33 perinatally HIV-1-infected pediatric study volunteers prior to and over 96 weeks of protease-inhibitor-containing combination antiretroviral therapy (ART). All HIV-1-infected patients reconstituted CD4 T cells either with suppression of viremia or rebound of drug-resistant virus. METHODS Systemic immune activation was determined by polychromatic flow cytometry of blood lymphocytes and ELISA for plasma soluble CD27, soluble CD14, and tumor necrosis factor. Microbial translocation was evaluated by limulus amebocyte lysate assay to detect bacterial lipopolysaccharide (LPS) and ELISA for antiendotoxin core antigen immunoglobulin M (IgM) antibodies. Immune activation markers were compared with viral load, CD4 cell percentage, and LPS by regression models. Comparisons between healthy and HIV-1-infected or between different viral outcome groups were performed by nonparametric rank sum. RESULTS Microbial translocation was detected in healthy infants but resolved with age (P < 0.05). LPS and soluble CD14 levels were elevated in all HIV-1-infected patients (P < 0.05 and P < 0.0001, respectively) and persisted even if CD4 T cells were fully reconstituted, virus optimally suppressed, and lymphocyte activation resolved by ART. Children with CD4 T-cell reconstitution but viral rebound following ART continued to display high levels of soluble CD27. CONCLUSION Microbial translocation in pediatric HIV-1 infection is associated with persistent monocyte/macrophage activation independent of viral replication or T-cell activation.