Influence of Specimen Age and Use of Different Negative Controls in Determination of Intracytoplasmic Levels of Cytokines after Whole-Blood Culture Assay

Schultz, Christian; Rott, Christina; Temming, Petra; Puttkammer, Julia von; Bucsky, Peter

doi:10.1128/cdli.9.2.295-298.2002

Cited by 20 publications

(30 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Flow cytometric analysis of cytokine production was done as described earlier (22,23). Briefly, heparinized whole blood was suspended in RPMI 1640 supplemented with 1% penicillin/streptomycin, 2 mM of glutamine, 1 mM of pyruvate, and nonessential amino acids at a concentration of 2 ϫ 10 6 leukocytes/mL.…”

Section: Methodsmentioning

confidence: 99%

Differential Maturation of the Innate Immune Response in Human Fetuses

et al. 2004

Self Cite

View full text Add to dashboard Cite

Newborns and especially preterm infants show a unique susceptibility to severe bacterial infections that cause significant morbidity and mortality. As very few data are available on innate immune functions in human fetuses, we conducted a comprehensive study to investigate the expression of several adhesion molecules essentially involved in migration (CD11a, CD11b, CD11c, CD18, and CD62L). Furthermore, phagocytic activity, generation of respiratory burst products, and production of several proinflammatory cytokines were assessed. Various functions of the fetal innate immune system were demonstrated to be essentially different from those observed in term neonates or adults. Expression of several surface markers was significantly diminished on fetal granulocytes. Furthermore, a significantly reduced phagocytic activity of fetal granulocytes and monocytes was found, contrasted by an enhanced generation of reactive oxygen products. In addition, we demonstrate that significant numbers of fetal monocytes are capable of the production of proinflammatory cytokines in response to stimulation. However, the pattern of cytokine production is different from the more mature individuals: the number of IL-6 -and tumor necrosis factor-␣-positive monocytes were significantly diminished, whereas more IL-8 -producing monocytes were found compared with adults. The results of our study add significantly to our understanding of the maturation and impairment of the innate immune response. Neonates, especially preterm infants, are prone to severe and overwhelming bacterial infection with substantial morbidity and mortality. Despite intensive supportive care and early use of antibiotics, the rate of morbidity and mortality caused by infections remains high (1). Although multiple factors contribute to this susceptibility, the immaturity of the innate immune system, especially production of phagocytes and several phagocyte functions (e.g. chemotaxis, phagocytosis, respiratory burst, cytokine production) are of paramount importance (2,3). Evidence of this is provided by the pattern of infections closely resembling that seen in profound neutropenia (4). Depleted neutrophil storage pools and neutropenia were described to be associated with neonatal sepsis and are negative predictors of outcome (5,6). Several qualitative deficiencies of phagocyte function were demonstrated earlier in preterm infants and stressed or septic neonates. The functional deficiencies comprised migration, phagocytosis, and bactericidal potency, for example, release of bactericidal/permeabilityincreasing protein (7-15). However, normal or even enhanced spontaneous neutrophil respiratory burst activity in term and preterm neonates was reported in other studies (16,17). In contrast, little is known to date about the phagocytic and oxidative capacity as well as the adhesion molecule expression of fetal neutrophils and monocytes. The expression of several adhesion molecules essentially involved in migration was reported to be significantly impaired in neonates and to contri...

show abstract

Section: Methodsmentioning

confidence: 99%

Differential Maturation of the Innate Immune Response in Human Fetuses

et al. 2004

Self Cite

View full text Add to dashboard Cite

show abstract

“…51). It has always been the user's responsibility to verify the manufacturer's expected antibody binding characteristics (even for FDA/CE approved kits where comparison with previous lots is required) and thus all, rather than only one, of the aforementioned controls may need to be incorporated in the development of a cell labeling protocol to reliably call a cell population truly positive (52). Equally important in establishing a reliable antibody labeling protocol is verification by other technologies.…”

Section: Establish Antibody Specificitymentioning

confidence: 99%

“…The potentially different nonspecific binding characteristics of an isotype antibody, the possible pitfalls in the interpretation of the data (41) and the notion that it is virtually impossible to manufacture the perfectly matched isotype antibody, have lead to the reconsideration of earlier recommendations (54,55). Over the last decade or so, it has become more widely accepted that isotype controls are of little value in distinguishing positive from negative (6,30,41,44,52,56) and should therefore not be used to set positive gating regions for test antibodies. Isotype controls are not recommended for use in quantitative (cell counting) assays to determine nonspecific binding (7)(8)(9)38,43,57).…”

Section: Understand and Minimize Undesirable Antibody Bindingmentioning

confidence: 99%

Considerations for the control of background fluorescence in clinical flow cytometry

Hulspas¹,

O’Gorman

Wood

et al. 2009

Cytometry Part B Clinical

210

190

View full text Add to dashboard Cite

“…Aliquots of whole blood were preincubated without or with vitamin C in appropriate concentrations (1,5,10,20, 50 m M ) for 2 h in multiwell plates at 37 ° C, 5% CO 2 . To induce IL-1 ␣ , IL-6, IL-8 and TNF-␣ production in monocytes, preincubated whole blood cultures were stimulated with 100 ng/ml LPS for 4 h. Similarly, cytokine production (IL-2, interferon gamma [IFN-␥ ], TNF-␣ ) in lymphocytes was stimulated with 3 g/ml PMA and 3 M ionomycin for 4 h. Cells were exposed to 3 M monensin (Sigma) during the whole stimulation period, followed by fixation with 4% paraformaldehyde (Riedel de Haen, Seelze, Germany), as described previously [11,12] . An unstimulated control was added in each experiment.…”

Section: Culture and Stimulation Of Cellsmentioning

confidence: 99%

“…Isotype-specific antibodies were used to detect irrelevant specificity for surface molecule staining. Flow cytometric analysis was performed as previously described [11] .…”

Section: Intracellular Staining Of Cytokinesmentioning

confidence: 99%

Immunomodulatory Effect of Vitamin C on Intracytoplasmic Cytokine Production in Neonatal Cord Blood Cells

et al. 2006

Self Cite

View full text Add to dashboard Cite

Background: Vitamin C (ascorbic acid) is an essential water-soluble antioxidant in cells and plasma. Besides metabolic functions, vitamin C is also known to contribute to immune homeostasis. Recently, it has been demonstrated that vitamin C has an inhibitory effect on the expression of pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in adult whole blood cells in vitro. It has been postulated that vitamin C might be an interesting compound for modulation of an over-exuberant immune response, e.g., in patient cohorts susceptible for the development of systemic inflammatory response syndrome such as neonates. It was the aim of this study to investigate the modulatory effects of vitamin C on the production of inflammatory mediators in neonatal cord blood cells. Methods: The intracytoplasmic production of pro-inflammatory cytokines in neonatal cord blood cells stimulated with lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin was assessed by flow-cytometry. Results: In contrast to our previous observations from adult whole blood cells, 20 mM vitamin C mildly stimulated the percentage of neonatal monocytes producing IL-6 after lipopolysaccharide stimulation (e.g., 11.3% increase compared to control, p = 0.005). In the presence of 20 mM vitamin C, even a stronger stimulatory effect was noted for the percentage of IL-8 (e.g., 46.7% increase, p < 0.001) and TNF-α producing neonatal monocytes (e.g., 69.2% increase, p = 0.004; n = 20). In accordance with adult data, the percentage of neonatal lymphocytes producing IL-2 after phorbol 12-myristate 13-acetate/ionomycin stimulation was dose-dependently reduced (e.g., 41.3% inhibition, p = 0.001, 20 mM vitamin C), while the percentage of TNF-α producing lymphocytes was mildly stimulated (e.g., 20.8% increase, p = 0.003, 20 mM vitamin C). Conclusions: Interestingly, vitamin C was demonstrated to enhance pro-inflammatory responses in CD14⁺ cord blood cells while only intracellular IL-2 production in CD3⁺ cells was diminished. These data suggest that vitamin C differentially influences intracytoplasmic cytokine production in adults and neonates, and further studies are needed to elucidate the underlying mechanisms of this selective immunomodulation.

show abstract

Influence of Specimen Age and Use of Different Negative Controls in Determination of Intracytoplasmic Levels of Cytokines after Whole-Blood Culture Assay

Cited by 20 publications

References 16 publications

Differential Maturation of the Innate Immune Response in Human Fetuses

Differential Maturation of the Innate Immune Response in Human Fetuses

Considerations for the control of background fluorescence in clinical flow cytometry

Immunomodulatory Effect of Vitamin C on Intracytoplasmic Cytokine Production in Neonatal Cord Blood Cells

Contact Info

Product

Resources

About