Measurement of Cytokine Secretion, Intracellular Protein Expression, and mRNA in Resting and Stimulated Peripheral Blood Mononuclear Cells

Sullivan, Kathleen E.; Cutilli, Joie; Piliero, Lisa M.; Ghavimi-Alagha, Darlene; Starr, Stuart E.; Campbell, Donald E.; Douglas, Steven D.

doi:10.1128/cdli.7.6.920-924.2000

Cited by 103 publications

(73 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Published studies have shown that ELISAs are generally reproducible, with a typical interassay variation of approximately 50% (2). The lower limit of detection for ELISA is 3 to 8 pg/ml (18).…”

Section: Methodsmentioning

confidence: 99%

“…Phorbol myristate acetate (PMA) (Sigma, St. Louis, Mo.) was used at a final concentration of 100 ng/ml for ELISA and RNA analysis, previously demonstrated to provide optimal conditions for the assay (18).…”

Section: Methodsmentioning

confidence: 99%

“…One of the technical limitations of our data, a limitation shared by others working in this area, is the use of bulk PMA or phytohemagglutinin (PHA) stimulation assays. During the course of these investigations, we measured cytokine secretion and intracellular protein expression in mRNA in resting and stimulated PBMCs in a group of subjects in order to examine the effects of various stimuli (18). The limitations of bulk (PMA or PHA) stimulation of PBMCs relates to the existing cellular makeup of the PBMC population (18).…”

Section: Vol 10 2003 Cytokines In Hiv-positive and -Negative Adolesmentioning

confidence: 99%

See 2 more Smart Citations

TH1 and TH2 Cytokine mRNA and Protein Levels in Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Youths

Douglas

Durako

Sullivan

et al. 2003

Clin Vaccine Immunol

Self Cite

View full text Add to dashboard Cite

The roles of cytokines in the progression of human immunodeficiency virus (HIV)-associated disease are controversial. The patterns of innate cytokine production have been postulated to shift from TH1-to TH2-type cytokines with the progression of HIV-associated disease. Although there have been studies of cytokines in children and adults, no data are available on cytokine production in healthy or HIV-infected adolescents. We analyzed and characterized cytokine mRNA and protein levels for gamma interferon, interleukin 2 (IL-2), IL-4, and tumor necrosis factor alpha and protein levels of IL-6 in both stimulated and unstimulated peripheral blood mononuclear cells obtained from a large longitudinal, observational cohort study of HIV-seropositive and -seronegative adolescents. We correlated cytokine results with viral load and CD4 ؉ -T-cell counts as critical markers of disease progression in HIV-infected adolescents. These data were used to examine hypotheses related to the TH1-to-TH2 cytokine shift in a sample of HIV-infected adolescents. Five hundred twenty subjects participating in the REACH (Reaching for Excellence in Adolescent Care and Health) Project of the Adolescent Medicine HIV/AIDS Research Network contributed blood samples. Samples selected for the cross-sectional data set analyzed had to meet selection criteria developed to minimize the potential confounding effects of acute intercurrent illnesses or infections, recent vaccination for hepatitis, and altered hormone status and to optimize congruence of cytokine measurements with assays of viral load and CD4؉ -T-cell counts. Group differences in the proportions of subjects with detectable levels of each cytokine marker were compared. In the subset of subjects with detectable cytokine values, differences in detected values were compared across subgroups defined by HIV serostatus and among HIV-seropositive subjects by three viral load classifications. The study sample was 65% HIV seropositive, 71% African-American, and 75% female with a mean age of 17.4 years. HIV-seropositive subjects were relatively healthy with mean and median CD4؉ -T-cell counts of 534 and 499 cells/mm 3 , respectively. Only 8.1% of subjects had CD4 ؉ -T-cell counts below 200 cells/mm 3 , and 25% had viral loads that were below the threshold of detection (<400 copies/ml). Detailed analyses of these data indicate that there were no differences in cytokines detected in HIV-seropositive and HIV-seronegative adolescents, and there was no apparent relationship between the cytokine measurements and the viral load or CD4؉ -T-cell categorization, the parameters selected as markers of HIV-associated disease status. These adolescents, including the HIV-seropositive subjects, were relatively healthy, and the HIV-infected subjects were at an early stage in the course of their HIV-associated disease. On the basis of our data, we conclude that, early in the course of HIV-associated disease in adolescents, there are no detectable shifts from TH1 to TH2 cytokine production.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Vol 10 2003 Cytokines In Hiv-positive and -Negative Adolesmentioning

confidence: 99%

See 1 more Smart Citation

TH1 and TH2 Cytokine mRNA and Protein Levels in Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Youths

Douglas

Durako

Sullivan

et al. 2003

Clin Vaccine Immunol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Spontaneous ex vivo cytokine production by PBMCs has been reported not to occur in healthy individuals (2,4,7,21,22) or in persons with various disorders, including allergic asthma (4), active and inactive systemic lupus erythematosus (18), and sarcoidosis (6), and those recovering from vascular surgery (1). We ourselves have rarely, if ever, noted spontaneous cytokine production in PBMCs of healthy U.S. blood donors (reference 7 and unpublished data).…”

mentioning

confidence: 99%

Spontaneous Cytokine Production and Its Effect on Induced Production

Walker

Jason

Wallace

et al. 2002

Clin Vaccine Immunol

View full text Add to dashboard Cite

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-␣), and gamma interferon (IFN-␥) by peripheral-blood B lymphocytes, T cells, CD8 We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-␣ production occurred most frequently, followed in descending order by IFN-␥, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-␣; for 12 of these patients, TNF-␣ was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-␣ and IFN-␥, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.

show abstract

“…It also serves as a valuable tool to monitor disease progression or inflammation (120,353). Some conventional methods available for cytokine measurement include radioimmunoassay (RIA) and ELISA.…”

Section: 347-352) the Effect Of Opioid Peptides On The Regulatimentioning

confidence: 99%

The immunomodulation of dynorphin 1-17 and its biotransformation fragments on inflammatory signalling pathways

Rahiman¹,

Sarah²

View full text Add to dashboard Cite

Inflammation is a complex physiological process that involves host defense mechanisms in response to the intrusion of harmful external stimuli. Uncoordinated inflammatory responses, however, are the underlying pathophysiological cause of many chronic diseases. Inflammation is characterised by the burgeoning migration of leukocytes to the point of injury and subsequent release of pro-nociceptive mediators, generating inflammatory pain. Dynorphin 1-17 (DYN 1-17) is endogenously produced and released from leukocytes upon stimulation by local inflammatory factors in the inflamed area. This opioid peptide primarily binds to kappa-opioid receptor (KOR) to produce analgesia. Under inflammatory milieu, DYN 1-17 undergoes spontaneous degradation, yielding a variety of opioid and non-opioid fragments that may have significant implications in inflammation. The underlying cellular effects of these fragments remain unclear. This thesis seeks to explore potential mechanistic insights into selected major DYN 1-17 fragments, identified from a previous biotransformation study of DYN 1-17 in rodent inflamed tissue. This thesis examines the DYN 1-17 fragments modulation of intracellular signals associated with inflammation and thereby, gain an insight into novel therapeutic targets through exploring these endogenous mechanisms.Utilising THP-1 macrophages as an in vitro model of inflammation, this thesis begins with a semiquantitative assessment of nuclear factor-kappa B/p65 (NF-κB/p65) translocation, a major transcription factor that regulates genes responsible for immune and inflammatory responses. The findings presented in Chapter Three of this thesis demonstrated that DYN 1-17 and selected major fragments (DYN 1-6, 1-7, 1-9, 1-10, 1-11, 3-14, 6-12, 2-17 and 7-17) significantly attenuated NF-κB/p65 nuclear translocation induced by lipopolysaccharide (LPS), with the greatest reduction observed with DYN 1-7 at 10 nM. A selective KOR antagonist, ML-190, was used to examine KOR involvement in this inhibitory action. ML-190 significantly reversed the inhibition of NF-κB/p65 translocation produced by DYN 1-17, DYN 1-6, DYN 1-7 and DYN 1-9, but not DYN 1-10 and DYN 1-11.Cytokine production is linked as a downstream process to NF-κB activation; hence it is necessary to evaluate the ability of DYN 1-17 and selected biotransformation fragments in the modulation of IL-1β and TNF-α release in differentiated THP-1 cells, as presented in Chapter Four, to gain an insight into the effects on major pro-inflammatory cytokines. DYN 1-17 and the fragments, demonstrated differential modulatory effects on LPS-induced release of IL-1β and TNF-α. DYN 1-7 and DYN 1-6, inhibited and elevated the secretion of both cytokines, respectively, in a concentration-dependent manner (10 -11 to 10 -7 M). DYN 1-17, however, only inhibited IL-1β release from differentiated iii THP-1 cells and had no effect on TNF-α secretion. Intriguingly, DYN 3-14 at 10 -17 to 10 -11 M demonstrated significant inhibition on IL-1β release and paradoxically increased TNF-α lev...

show abstract

Measurement of Cytokine Secretion, Intracellular Protein Expression, and mRNA in Resting and Stimulated Peripheral Blood Mononuclear Cells

Cited by 103 publications

References 23 publications

TH1 and TH2 Cytokine mRNA and Protein Levels in Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Youths

TH1 and TH2 Cytokine mRNA and Protein Levels in Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Youths

Spontaneous Cytokine Production and Its Effect on Induced Production

The immunomodulation of dynorphin 1-17 and its biotransformation fragments on inflammatory signalling pathways

Contact Info

Product

Resources

About