Immunological Marker Analysis of Mitogen‐Induced Proliferating Lymphocytes Using Brd U Incorporation or Screening of Metaphases

Adriaansen, H. J.; Osman, Chinar; Dongen, Jacques J.M. van; Brfsser, J. H. F. M. Wijdenes‐De; Tilborg, C. M. J. M. Kappetijn‐Van; Hooijkaas, Herbert

doi:10.1111/j.1365-3083.1990.tb03211.x

Cited by 11 publications

(9 citation statements)

References 28 publications

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“…For mitogen stimulation, 2-ml cultures were incubated without stimulant (control) or with 10 μ g/ml Con A and 10 ng/ml PMA (Con A/PMA; Sigma-Aldrich) for (randomly) 18–24 h (19, 20). For Ag stimulation, 2-ml cultures were incubated without stimulant or with 25 μ g/ml Alternaria ( Alternaria culture filtrate) or 25 μ g/ml Bermuda (both from Greer Laboratories) for (randomly) 66–72 h. Supernatants were collected following centrifugation and aliquots were frozen at −70°C until cytokine assays were performed.…”

Section: Methodsmentioning

confidence: 99%

Th1/Th2 Patterns and Balance in Cytokine Production in the Parents and Infants of a Large Birth Cohort

Halonen

Lohman

Stern

et al. 2009

The Journal of Immunology

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Regulation of human immune cell cytokine production in vivo is not well understood due in part to limitations on imposing experimental conditions. We proposed that life-imposed conditions (pregnancy, birth, age, gender), combined with large sample size, repeat sampling, and family-based recruitment would serve to reveal peripheral blood cell-derived cytokine patterns reflective of in vivo regulation regarding Th1/Th2 balance and familial correlation. Mononuclear cells were obtained from 483 trios in the Tucson Infant Immune Study: from mothers pre- and postpartum, infants at birth and at 3 mo, and fathers. Con A/PMA-stimulated supernatants were assayed by ELISA for IFN-γ, IL-4, IL-13, IL-5, and IL-10 and allergen-stimulated supernatants for IFN-γ, IL-4, and IL-13. Mitogen-stimulated prepartum samples were not globally Th2 biased, differing from postpartum only by a modestly reduced IFN-γ:IL-5 ratio. Prepartum samples actually produced less IL-10 and IL-13 although more IL-5 than paternal samples. Newborns were also not globally Th2 biased, with mitogen stimulation producing ~10-fold less IL-4, IL-5, and IFN-γ than adults but only 2- to 3-fold less IL-13 and IL-10. Despite these group differences, all cytokines showed marked positive intraindividual correlations (all p < 0.001). Allergen stimulation gave results consistent with a lack of global Th2 bias. Mitogen stimulation revealed parent-child and parent-parent correlations. Thus, rather than a global Th2 bias, cytokine production in pregnant mothers and newborns appears regulated so as to maintain a relative balance among the cytokines, with the nature of the balance differing in mothers and infants and with production influenced by familial factors that include shared environment.

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Section: Methodsmentioning

confidence: 99%

Th1/Th2 Patterns and Balance in Cytokine Production in the Parents and Infants of a Large Birth Cohort

Halonen

Lohman

Stern

et al. 2009

The Journal of Immunology

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“…The proliferative response of PBMC after CSFV in vitro re-stimulation was measured by bromodeoxyuridine incorporation [1,16]. We adapted this method for use in flow cytometry and double or triple labeling in order to evaluate the specific proliferation of B and T cell sub-populations.…”

Section: Csfv-specific Cell Proliferative Responsementioning

confidence: 99%

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

et al. 2003

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-A better understanding of cell-mediated immune responses to classical swine fever virus (CSFV) is essential for the future development of improved vaccines. We analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination. Viral infection induced high T cell responses with high primary and secondary CTL activity correlated with high IFN-g production, whereas vaccination with a live vaccine followed by infection mainly induced neutralizing antibody but low cell-mediated responses. Moreover, high IgG1 response was associated with high IFN-g response following infection whereas a weak IFN-g response was related to a good IgG2 response but a low IgG1 production. These data could reflect Th1/Th2-like balance of immune responses depending upon immunization protocols, which has not yet been described in the pig. T-cell responses to CSFV were evidenced by CSFV-specific CD25 upregulation on CD4 -CD8 + , but not on CD4 + CD8 -cells, which further illustrated the importance of CTL responses after infection. Our results indicated that generation of cell-mediated immune responses was much higher following intranasal/oral CSFV infection than after intramuscular vaccination, which implies that the capacity of new CSFV vaccines to induce higher T-cell responses should be considered. immunity / cytotoxicity / CSFV / d/d histocompatible pig

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“…anti-CD3. staphylococcal protein A (SPA) and pokeweed mitogen (PWM) activation cytospin preparations [14). These assays can be combined with phenotypic analysis, but are difficult to perform.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric analysis of the stimulatory response of T cell subsets from normal and HIV-1+ individuals to various mitogenic stimuli in vitro

Medina

Borthwick

Johnson

et al. 1994

Clinical and Experimental Immunology

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SUMMARY A novel technique is described which allows the study ofthe responses of T cell subpopulations stimulated in bulk cultures without interfering with cell-cell interactions. The number and phenotype of lymphoblasts developing following stimulation with phytohaemagglutinin (PHA), anti-CD3, staphylococcal protein A (SPA) and pokeweed mitogen (PWM) was determined in HIV-P and HlV-1' patients using a new five-parameter flow cytometric method. We found that normal T ceils responded faster to PHA than lo any of the other mitogens tested. The peak of the PHA response occurred on day 3. followed by anti-CD3 and SPA on day 4 and PWM mitogen on day 5. Although PHA and anti-CD3 stimulated up to 95% and 80% oflymphocytes, respectively, SPA and PWM stimulated only 40% and 30% of cells, respectively. A defective T cell response was observed in lymphocytes cultured from asymptomatic HlV-1' patients compared with negative controls. This loss of response was related to a selective mortality of T cells following mitogenic stimulation, referred to as activation-associated lymphocyte death (AALD). The results showed that stronger mitogens (PHA and anti-CD3) induced AALD in a larger proportion (50-60%) of T cells than weaker mitogens such as SPA and PWM (50-40%). and that AALD affected different lymphocyte subsets to different extents. AALD occurred more frequently in total CDS"*^ and CD45RO^ T ceils compared with CD4+ and CD45RA^ T cells, but memory CD4+ T cells were the population most severely affected in samples from HIV-I* donors.

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Immunological Marker Analysis of Mitogen‐Induced Proliferating Lymphocytes Using Brd U Incorporation or Screening of Metaphases

Cited by 11 publications

References 28 publications

Th1/Th2 Patterns and Balance in Cytokine Production in the Parents and Infants of a Large Birth Cohort

Th1/Th2 Patterns and Balance in Cytokine Production in the Parents and Infants of a Large Birth Cohort

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

Flow cytometric analysis of the stimulatory response of T cell subsets from normal and HIV-1+ individuals to various mitogenic stimuli in vitro

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