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PubMed2003

Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated proliferation of human peripheral blood mononuclear cells.

Cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, was recently demonstrated to stimulate proliferation of various cell types. The role of cADPR in a specific process of monocyte- and plasma-mediated activation of T-lymphocytes by lipopolysaccharide (LPS) was addressed using human mononuclear cells from peripheral blood (PBMCs). Incubation of PBMCs with 0.1 microg/ml of LPS for 24 h provided a doubling in the intracellular levels of cADPR as compared with unstimulated PBMCs. The cADPR increase was abolished either by prior removal of monocytes or by pre-incubating a whole PBMC population with a monoclonal antibody against the monocyte marker CD14. The increased concentrations of intracellular cADPR elicited by LPS stimulation were paralleled by significant increases in NAD+ levels and in the activities of ectocellular and membrane-bound fractions of ADP-ribosyl cyclase/cADPR hydrolase activities. A cytosolic ADP-ribosyl cyclase was also detectable in PBMCs and its activity was comparably enhanced by LPS stimulation. This soluble cyclase is distinguished from the membrane-bound cyclase by both substrate and inhibitor sensitivities. LPS-stimulated PBMCs showed 2-3-fold increases of intracellular calcium ([Ca2+]i), and these changes were prevented completely by the cADPR antagonist 8-Br-cADPR and by ryanodine. Both compounds, and the cyclase inhibitor nicotinamide, significantly inhibited the T-lymphocyte proliferation induced by LPS in PBMCs. These results demonstrate that cADPR plays a role of second messenger in the adaptive immune recognition process of LPS-stimulated proliferation of PBMCs.

ADP-ribosyl Cyclase↗
PubMed2000

Reduced IFNgamma production in response to IL-12 stimulation and/or reduced IL-12 production in atopic patients.

BACKGROUND: Several studies have shown that interleukin (IL)-4 and interferon-gamma (IFNgamma) are important for the regulation of IgE production and that IL-12 induces IFNgamma. OBJECTIVE: IFNgamma production in response to IL-12 stimulation and IL-12 production were investigated in peripheral blood mononuclear cells (PBMCs) of atopic patients with various levels of serum IgE. METHODS: Cytokine production from PBMCs was measured following stimulation with a nonspecific stimulator (phytohemagglutinin: PHA), a specific stimulator (Der f 1) and IL-12 in 17 healthy controls and 23 atopic patients with various serum IgE levels. RESULTS: The IFNgamma production by IL-12-stimulated PBMCs in the atopic group was lower than that in the control group. Furthermore, the serum IgE level was negatively correlated with IFNgamma production by PBMCs stimulated with IL-12 (P < 0.001), and with IL-12 production by PBMCs stimulated with Der f 1 (P < 0.001). Although the IFNgamma concentrations by PHA-stimulated PBMCs were correlated with those by IL-12-stimulated PBMCs, there were differences in several patients. CONCLUSIONS: Our results indicated that atopic patients may have some abnormality in the IL-12-IFNgamma loop. It was shown that the elevation in IgE levels in atopic patients may be due to reduced IFNgamma production in response to IL-12 stimulation and/or due to reduced IL-12 production.

Adolescent↗
PubMed2002

T-cell antigenic determinants within hepatitis C virus nonstructural protein 3 and cytokine production profiles in hepatitis C.

The aim of this study was to further investigate the role of T-helper cells in hepatitis C virus (HCV) infection, focusing on the T-cell antigenic determinants and cytokine profiles of nonstructural 3 (NS3) protein-stimulated peripheral blood mononuclear cells (PBMCs) of HCV patients. A total of 12 recombinant proteins of theNS3 region were purified and used to test T-cell proliferative response and antigenic determinants of HCV-seropositive patients. In addition, cytokines produced by antigen stimulated PBMCs were measured. Our data showed that PBMCs from 55.7% (34/61) of HCV patients proliferated to at least one antigen, but PBMCs of HCV seronegative patients did not. In addition, PBMCs from about 82.0% (32/39) HCV-seropositive patients produced significant amounts of cytokines (10 pg/mL). Interestingly, PBMCs from 66% of patients produced TH2-related cytokines such as interleukin (IL)-4 and IL-5. In mappingexperiments, the data showed multiple T-cell antigenic determinants. Our data demonstrated that NS3 antigen-stimulated PBMCs of HCV patients recognized multiple T-cell antigenic determinants and produced significant amounts of TH0 or TH2-related cytokines, which might play a critical role in the chronicity of HCV infection.

Cytokines↗
PubMed2001

Effects of dialyzer membrane on interleukin-1beta (IL-1beta) and IL-1beta-converting enzyme in mononuclear cells.

BACKGROUND: In vitro stimulation of mononuclear cells (peripheral blood mononuclear cells; PBMCs) with an endotoxin (lipopolysaccharide; LPS) revealed elevated cell-associated levels of interleukin-1beta (IL-1beta) in end-stage renal disease (ESRD) patients on Cuprophan hemodialysis (HD), suggesting a defect in the process of IL-1beta's release from activated cells. IL-1beta is initially synthesized as an inactive precursor called proIL-1beta. ProIL-1beta is processed into the biologically active mature form of IL-1beta (mIL-1beta) requiring the specific IL-1beta-converting enzyme (ICE). METHODS: Using specific immunoassays (enzyme-linked immunosorbent assays), we measured cell-associated and extracellular proIL-1beta as well as mIL-1beta in LPS-stimulated PBMCs to test whether ICE-dependent processing of proIL-1beta and/or secretion of mIL-1beta was impaired in ESRD patients compared with healthy controls. PBMCs of healthy controls (N = 9), of ESRD patients on peritoneal dialysis (PD, N = 10), and of those patients on intermittent HD (N = 8) were studied. The same HD patients were studied three times with low-flux Cuprophan (GFS 12), low-flux polysulfon (F6 HPS), and high-flux polysulfon (F60S) in randomized order. PBMCs were separated from whole blood by Ficoll-Hypaque centrifugation and incubated in vitro for 18 hours in the presence LPS (10 ng/mL). Extracellular (PBMC culture supernatants) and cell-associated (cell lysates) levels of proIL-1beta and mIL-1beta were measured. RESULTS: The total production (cell-associated plus extracellular) of LPS-induced IL-1beta (proIL-1beta plus mIL-1beta) was similar in healthy controls (25.96 +/- 0.84 ng/2.5 x 10(6) PBMC), PD patients (29.53 +/- 1.31 ng/2.5 x 106 PBMC), and in Cuprophan-treated HD patients (23.28 +/- 1.24 ng/2.5 x 10(6) PBMC). In normal controls, 43.6% of the total IL-1beta was processed into mIL-1beta, which was significantly more than that in PD patients (27.3%, P < 0.02) but was similar to that in Cuprophan-treated HD patients (37.1%). Comparing cell-associated and extracellular concentrations of mIL-1beta, PBMCs of normal controls secreted 82.2% of mIL-1beta; this was significantly more than that in PD patients (59.4%, P < 0.01) and that in Cuprophan HD patients (54.2%, P < 0.01). When HD patients were switched from Cuprophan to F6 HPS or F60S, neither total IL-1beta production nor processing of IL-1beta changed. However, secretion of mIL-1beta increased significantly with F6 HPS (80.6%, P < 0.01) as well as with F60S (76.6%, P < 0.02) compared with Cuprophan. CONCLUSION: We conclude that the ability of PBMCs to produce IL-1beta in response to LPS is normal in PD patients as well as in HD patients. ICE-dependent processing of inactive proIL-1beta into biologically active mIL-1beta is reduced in PD patients, but not in HD patients. Secretion of mIL-1beta is impaired in PD and HD patients treated with Cuprophan. This impaired ability to secrete active mIL-1beta seems to be independent of ICE activity and is normalized when HD-patients are switched from Cuprophan to low- or high-flux polysulfon. Increased cell-associated levels of biologically active mIL-1beta in circulating PBMCs represent a state of inflammation that may contribute to chronic inflammatory diseases such as beta2-microglobulin amyloidosis. Replacement of Cuprophan by synthetic membranes normalizes PBMC function and reduces the state of inflammation in ESRD patients.

Adult↗
PubMed2002

Arachidonate cascade, apoptosis, and vitamin E in peripheral blood mononuclear cells from hemodialysis patients.

BACKGROUND: Lipid peroxidation and oxidative stress are enhanced in peripheral blood mononuclear cells (PBMCs) from hemodialysis (HD) patients because of upregulation of the 5-lipoxygenase pathway of the arachidonate cascade. 5-Lipoxygenase activity is specifically inhibited by vitamin E both in vitro and in vivo regardless of its administration route. METHODS: The effect of arachidonate cascade enzymes and vitamin E on oxidative stress and apoptosis was investigated in PBMCs from 16 maintenance HD patients treated for at least 6 months with cuprammonium rayon membranes in a two-step crossover study: after a 4-week treatment with vitamin E-coated cuprammonium rayon membranes and again after a 4-week treatment with oral vitamin E. Control PBMCs were obtained from 16 healthy volunteers. RESULTS: Membrane lipoperoxidation, cellular luminescence, membrane fluidity, and leukotriene B(4) content were significantly greater in PBMCs from HD patients; lipoxygenase was upregulated, but prostaglandin H synthase (PHS) was not affected. Regardless of administration route, vitamin E partially controlled lipid peroxidation and oxidative stress through direct inhibition of 5-lipoxygenase. Cultured PBMCs from HD patients showed a significant increase in apoptotic cells compared with controls. Vitamin E markedly reduced cell luminescence, membrane fluidity, and apoptosis, whereas the PHS inhibitor indomethacin was ineffective. Similar results were obtained with control PBMCs induced to apoptosis by hydrogen peroxide. CONCLUSION: Reported data suggest that the 5-lipoxygenase branch of the arachidonate cascade is only responsible for membrane peroxidation, oxidative stress, and apoptosis of PBMCs of HD patients, and administration of vitamin E may be helpful in the control of oxidative stress-related disease in these subjects.

Apoptosis↗
PubMed2001

Visual demonstration of hepatitis C virus-specific memory CD8(+) T-cell expansion in patients with acute hepatitis C.

Hepatitis C virus (HCV)-specific CD8(+) T cells in peripheral blood mononuclear cells (PBMCs) from patients infected with HCV were quantitatively analyzed by flow cytometry using an HLA-B*3501-HCV epitope tetrameric complex. In chronic hepatitis C, tetramer(+)CD8(+) T cells were detected at frequencies ranging from 0.05% to 0.12% of total CD8(+) T cells. The number of tetramer(+)CD8(+) T cells in acute phase PBMCs from patients with acute hepatitis C was about 3 to 5 times higher than in recovery phase PBMCs from the same patients and in PBMCs from patients with chronic hepatitis C. Expanding tetramer(+)CD8(+) T cells in PBMCs from patients with acute hepatitis C express a CD28(+)CD45RA(-) memory T-cell phenotype. In contrast, tetramer(+)CD8(+) T cells in PBMCs from patients with chronic hepatitis C did not predominantly express this phenotype. These tetramer(+)CD8(+) T cells did not have perforin in their cytoplasma. The present study visually showed that a high number of circulating HCV-specific CD8(+) T cells in acute phase PBMCs from patients with acute hepatitis C are mostly memory T cells.

Acute Disease↗
PubMed2002

Inhibitory effect of lidocaine on T cells from patients with allergic asthma.

BACKGROUND: Nebulized lidocaine enables dosages of oral corticosteroids to be tapered in the treatment of severe asthma. OBJECTIVE: We sought to investigate the effect of lidocaine on T cells from patients with allergic asthma. METHODS: PBMCs and CD4(+) T cells were isolated from 6 patients with asthma and house dust mite allergy. PBMCs were cultured with lidocaine for up to 7 days and then stained with propidium iodide to evaluate the involvement of apoptosis. In addition, the viability of CD4(+) T cells when cultured with lidocaine was investigated. Effects of lidocaine on proliferative response, mRNA expression, and protein production of IL-5 and IFN-gamma by PBMCs were investigated after stimulation with Dermatophagoides farinae, purified protein derivative, and phorbol 12-myristate 13-acetate plus calcium ionophore. The effects of lidocaine on the proliferative response of steroid-insensitive PBMCs from 6 nonallergic donors induced by preculture with IL-2 and IL-4 were also investigated. RESULTS: No significant increase in the staining of PBMCs with propidium iodide was observed in the presence of 100 micromol/L lidocaine. The viability of CD4(+) T cells was not significantly affected by culture with lidocaine at this concentration. However, lidocaine inhibited, in a dose-dependent manner, the proliferative response and mRNA expression and protein production of IL-5 and IFN-gamma of PBMCs stimulated with D farinae, purified protein derivatives, or phorbol 12-myristate 13-acetate plus calcium ionophore. Preincubation of PBMCs with IL-2 and IL-4 significantly decreased the inhibitory effects of both corticosteroids and lidocaine compared with that after preincubation with medium alone. CONCLUSION: Lidocaine has immunoregulatory effects on T cells. Therefore, lidocaine might be studied as an anti-inflammatory agent in the treatment of severe steroid-dependent asthma.

Allergens↗
PubMed2003

Chlamydia pneumoniae DNA in patients with symptomatic carotid atherosclerotic disease.

OBJECTIVE: We evaluated the correlation between the presence of Chlamydia pneumoniae in atherosclerotic carotid plaques, lymph nodes, and peripheral blood mononuclear cells (PBMCs), and symptomatic carotid atherosclerotic disease. METHODS: Fifty-one consecutive patients undergoing carotid endarterectomy were enrolled; 18 of the 51 patients had symptomatic disease, whereas 33 had asymptomatic disease. Detection of C pneumoniae DNA in atherosclerotic carotid plaques, lymph nodes, and PBMCs was performed with polymerase chain reaction (PCR). C pneumoniae antibodies were measured with a microimmunofluorescence test. RESULTS: C pneumoniae DNA in atherosclerotic carotid plaques was detected in 44.4% of patients with symptomatic disease and in 30.3% of those with asymptomatic disease (P =.48). C pneumoniae DNA in lymph nodes was detected in 33.3% of patients with symptomatic disease and 18.2% of those with asymptomatic disease (P =.19). Prevalence of C pneumoniae DNA in PBMCs was significantly higher in patients with symptomatic disease (72.2%) compared with those with asymptomatic disease (30.3%) (P =.01). Serologic results support the association between C pneumoniae DNA in PBMCs and symptomatic carotid atherosclerotic disease, because seropositivity for anti-chlamydial immunoglobulin (Ig) A antibodies was significantly higher in PCR-positive PBMCs in patients with symptomatic disease compared with patients with asymptomatic disease (P =.02). CONCLUSIONS: Detection of C pneumoniae in PBMCs shows that C pneumoniae may be associated with symptomatic carotid atherosclerotic disease. Although we believe there is need for standardization of PCR methods and for assessing sensitivity, specificity, and predictive values of the tests, we suggest that presence of C pneumoniae DNA in PBMCs is a valid surrogate marker of risk for endovascular chlamydial infection. Only long-term cohort studies and interventional trials will clarify the etiopathogenic role of C pneumoniae.

Aged↗
PubMed2006

Equal amounts of intracellular and virion-enclosed hepatitis C virus RNA are associated with peripheral-blood mononuclear cells in vivo.

BACKGROUND: Hepatitis C virus (HCV) replicating in peripheral-blood mononuclear cells (PBMCs) may represent an extrahepatic viral reservoir. Quantitation of HCV RNA with regard to its subcellular distribution and longitudinal course is needed for better understanding of the largely unexplored in vivo dynamics and potential pathogenetic significance of HCV in PBMCs. METHODS: Plasma and PBMCs from 30 patients coinfected with HCV and human immunodeficiency virus were evaluated in cross-sectional and longitudinal analyses, for up to 40 months. Differential extraction of virion-enclosed HCV RNA associated with cells was performed in parallel with extraction of total cellular HCV RNA. HCV RNA of either orientation was quantified by real-time polymerase chain reaction. RESULTS: HCV RNA was detected only in PBMCs from patients with viremia and at relatively stable quantities over time. Intracellular HCV RNA corresponding to ~60% of total cellular HCV RNA was strongly correlated with virion-enclosed HCV RNA but was only weakly associated with viral loads in plasma. In contrast, the ratio of HCV RNA load in PBMCs versus that in plasma was patient specific and stable over time. CONCLUSIONS: The substantial and patient-specific amounts of intracellular HCV RNA found by the present study support a concept of low-level replication in PBMCs. There was no evidence for persistent HCV infection in PBMCs after clearance of viremia in plasma.

Adult↗
PubMed2000

Substantial correlation between HIV type 1 drug-associated resistance mutations in plasma and peripheral blood mononuclear cells in treatment-experienced patients.

The correlation of detectable HIV-1 drug resistance mutations in plasma and PBMCs in patients extensively treated with all antiretroviral drug classes has not been fully elucidated. The detection of mutations in PBMCs may reveal resistant HIV-1 associated with past therapies. In addition, these measures in PBMCs may have a practical value when plasma virus is at low levels that are difficult to detect with current assays. The reverse transcriptase (RT) and protease (P) genes were analyzed for drug resistance, using an in-house method carried out on 36 paired samples of plasma and PBMCs from 12 treatment-experienced patients in order to investigate resistance in the two compartments. When viruses in plasma and PBMCs were analyzed by patient, the mean of the Cohen kappa values was 0.728 (substantial agreement). When viruses were analyzed by codon the mean of the Cohen kappa values was 0.715 (substantial agreement). Baseline samples were concordant at 280 of 288 (97%) codons analyzed. This study shows that a minority of mutations associated with previous therapy can be detected in PBMCs and not in plasma. Overall, mutations in plasma and PBMCs showed a substantial correlation in extensively treated patients, suggesting that either compartment is suitable for the detection of mutations as a virological guide for clinical care.

Anti-HIV Agents↗
PubMed2000

Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients after tumor peptide vaccination.

BACKGROUND: Monitoring the immune response to epitope-specific vaccination in cancer patients is important for vaccine development. The traditional method, in which the in vitro sensitization of peripheral blood mononuclear cells (PBMCs) with epitope is compared before and after vaccination, is time-consuming and allows only a qualitative assessment of the response. We used a rapid, quantitative, real-time polymerase chain reaction (PCR) assay to directly measure the immune reactivity of patients' PBMCs to the vaccine epitope. METHODS: PBMCs were obtained from melanoma patients before and after two rounds of vaccination with either g209-2M, a peptide derived from melanoma protein gp100 (n = 24), or ESg209-2M, a modified version of this peptide (n = 20). PBMCs were tested for immune reactivity by assaying interferon gamma (IFN gamma) protein release after in vitro sensitization with the epitope or for IFN gamma messenger RNA expression by real-time PCR. A twofold or more increase in IFN gamma protein release or a 1.5-fold or more increase in IFN gamma transcript accumulation in PBMCs after vaccination was considered to be evidence of a specific response. Correlation between the two methods was tested by use of the Spearman correlation coefficient after the results were ranked as positive or negative. All statistical tests were two-sided. RESULTS: The results obtained with the two methods were strongly correlated (Spearman's rho = 0.72; P =.0006). The g209-2M and Esg209-2M peptides resulted in similar percentages of vaccine-specific reactivity in PBMCs after in vitro sensitization (63% and 65% of patients, respectively; Fisher's exact test P =.6 for comparison of the two groups). The PCR method could detect vaccine-specific reactivity in a subset of patients (38% and 35% of patients, respectively; Fisher's exact test P =.7 for comparison of the two groups). CONCLUSION: Vaccination induces circulating antitumor lymphocytes, albeit in low frequencies, capable of directly reacting with tumor antigen. PBMCs of vaccinated individuals can respond to a vaccine-specific stimulus in a direct assay that does not require prolonged in vitro manipulations.

CD8 Antigens↗
PubMed2000

Injury induces deficient interleukin-12 production, but interleukin-12 therapy after injury restores resistance to infection.

OBJECTIVE: To assess at serial intervals the production of interleukin-12 (IL-12) by monocytes/macrophages from the peripheral blood of injured patients and control subjects, and using a mouse model to confirm human findings and explore the effectiveness of low-dose IL-12 therapy in restoring resistance to infection after injury. SUMMARY BACKGROUND DATA: Serious injury is associated with loss of function of the T helper 1 lymphocyte phenotype, but little is known about IL-12 production in injured patients. The authors previously reported that early, moderate-dose IL-12 therapy in a mouse model of burn injury restored resistance to a later infectious challenge (cecal ligation and puncture, CLP). However, the efficacy of clinically relevant low-dose IL-12 therapy carried out to or beyond the time of septic challenge remains to be tested. METHODS: Peripheral blood mononuclear cells (PBMCs) and adherent cells were obtained from 27 patients with major burns or traumatic injury and 18 healthy persons and were studied at serial intervals for IL-12 production stimulated by bacterial lipopolysacharide (LPS). PBMCs from 18 of the same patients were studied for IL-10 production as well. IL-12 production by adherent cells from the spleens of burn or sham burn mice was studied at serial intervals after injury to confirm the human findings. Low-dose IL-12 or vehicle was given every other day to groups of burn and sham burn mice, which were then challenged with CLP on day 10, and survival was determined. Finally, spleens were harvested from burn or sham burn animals receiving low-dose IL-12 or vehicle after CLP. After splenic cellularity was determined by hemocytometer, splenocytes were cultured and production of tumor necrosis factor-alpha, interferon-gamma, and IL-10 were assessed by immunoassay. RESULTS: Adherent cells from patients' PBMCs produced significantly less IL-12 than normal PBMCs after injury, reaching a nadir 8 to 14 days after injury. Stimulation of whole PBMCs by LPS indicated that at 8 to 14 days after injury, IL-12 production by PBMCs was significantly lower and IL-10 production was significantly higher than that of PBMCs from healthy persons. Low-dose IL-12 therapy significantly increased survival after CLP. Splenocytes from burn mice treated with IL-12 had significantly increased production of TNF-alpha and IF-beta, both before and after CLP, when compared with vehicle-treated burn animals. IL-10 production by bum splenocytes remained high after IL-12 treatment. Splenic cellularity increased after IL-12 treatment in burn mice. CONCLUSION: The capacity to produce IL-12 by adherent cells of the monocyte/macrophage lineage is significantly reduced after serious injury in humans and in a mouse burn model. In humans, there is a reciprocal relation between diminished IL-12 production and increased IL-10 production at approximately 1 week after injury. Low-dose IL-12 therapy in the mouse burn model markedly increased survival after a septic challenge, even when treatment was carried beyond the onset of sepsis. Low-dose IL-12 treatment in the mouse increased production of proinflammatory mediators important in host defense and at the same time maintained or increased production of IL-10, an important antiinflammatory cytokine.

Adult↗
PubMedDate not supplied

Identification of endogenous HLA-A2-restricted reactivity against shared melanoma antigens in patients using the quantitative real-time polymerase chain reaction.

This study was conducted to determine whether reactivity to melanoma cells of pretreatment peripheral blood mononuclear cells (PBMCs) from patients with metastatic melanoma correlated with subsequent response to treatment with interleukin-2 (IL-2). The sensitivity of the quantitative real-time polymerase chain reaction (PCR) assay was optimized, including the total number of cells used (3 x 10(6) in 1 mL), the responder-to-stimulator cell ratio (5:1), the optimal time to incubate PBMCs with tumor (2 h), the appropriate tumor stimulators (melanoma cell lines differing only in the expression of histocompatibility leukocyte antigen [HLA-A2]), the duration of recovery in the culture of PBMCs after cryopreservation (18-24 h), and the medium used (Iscove, 10% human AB serum). Using this optimized assay to detect HLA-A2-restricted antitumor reactivity in the pretreatment PBMCs from patients with melanoma, positive reactive responses were detected in 7 of 28 patients with an objective clinical response to IL-2 therapy compared with 6 of 21 positive reactive responses in nonresponding patients. None of 12 healthy donors were positive in this study. Thus, there was no significant difference in the reactivity of pretreatment PBMCs when responders were compared with nonresponders, although the melanoma patients had an increased incidence of response compared with healthy donors (p = 0.05). The PBMCs from 11 of the 13 melanoma patients with pretreatment HLA-A2-restricted antimelanoma reactivity were tested against a panel of transfectants expressing known shared melanoma antigens. Anti-MART-1 reactivity was detected in the pretreatment PBMCs of three patients. It thus appears that some melanoma patients are immunologically primed to antigens expressed on the tumor surface, although the HLA-A2-restricted antimelanoma activity detected in this real-time PCR assay was not predictive of patients' responses to IL-2 therapy.

Antigens, Neoplasm↗
PubMed1999

Mibefradil-induced inhibition of proliferation of human peripheral blood mononuclear cells.

To evaluate the role of intracellular calcium and particularly Ca2+-uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels, on the proliferation of human peripheral blood mononuclear cells (PBMCs) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of [3H]thymidine incorporation into control and concanavalin A-stimulated PBMCs in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 microM), and of the intracellular calcium antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; 1, 10, 25, or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures also was determined in mibefradil- or nifedipine-treated control or stimulated cells. Restoration of the proliferative response in mibefradil- or nifedipine-treated cells was investigated by addition of exogenous interleukin-2. Interleukin-2-receptor expression in the cells was monitored by using anti-activated T-cell antigen (Tac) antibody, and the interleukin-2 production in the cell supernatants of the cultures was determined by an enzyme-amplified sensitive immunoassay. Mibefradil and nifedipine concentration-dependently reduced the cell number and the [3H]thymidine incorporation or the de novo DNA synthesis in control and concanavalin A-stimulated human PBMCs. Mibefradil exhibited a more pronounced inhibition of the proliferation of human PBMCs than did nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent on the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine was added 1-7 h after cell stimulation. However, regardless of time of addition, TMB-8 caused a persistent inhibition of the proliferation of human PBMCs. The inhibitory effect of mibefradil or nifedipine on the proliferation of human PBMCs is nearly abolished by addition of the calcium channel activator Bay K 8644. The proliferative response of mibefradil- or nifedipine-treated cells is restored by addition of exogenous interleukin-2. The normal expression of interleukin-2 receptors was preserved, whereas the interleukin-2 production was blocked in the presence of mibefradil or nifedipine. Our data show that mibefradil has a more pronounced inhibitory effect on the proliferation of human PBMCs than nifedipine and that this inhibitory effect on DNA synthesis is dependent on the timing of treatment with both drugs.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

Increased production of vascular endothelial growth factor by peripheral blood mononuclear cells in patients with inflammatory bowel disease.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent angiogenic, vascular permeability-enhancing cytokine with overexpression in various pathological disorders, including tumour growth, chronic inflammation and tissue repair. Recent studies have shown significantly increased serum levels of VEGF in patients with inflammatory bowel disease. The origin of the circulating VEGF is still unknown. The present investigation examines the VEGF production by peripheral blood mononuclear cells (PBMCs) in patients with inflammatory bowel disease. METHODS: VEGF levels were measured in culture supernatants of unstimulated PBMCs of 27 patients with inflammatory bowel disease and 10 healthy volunteers using a solid phase ELISA. In addition, VEGF serum levels were determined. RESULTS: PBMCs of both active Crohn's disease patients (1142.6+/-483.9 pg/ml, P < 0.001, n = 12) and active ulcerative colitis patients (748.0+/-637.6 pg/ml, P = 0.006, n = 4) produced significantly higher amounts of VEGF compared with PBMCs of healthy volunteers (113.4+/-101.8 pg/ml, n = 10). In addition, there was a significantly increased VEGF production by PBMCs of patients with active disease compared with PBMCs of patients with quiescent Crohn's disease (261.6+/-254.8 pg/ml, P < 0.001, n = 7) and inactive ulcerative colitis (147.7+/-100.3 pg/ml, P = 0.02, n = 4). There was no significant difference in VEGF release between patients with inactive inflammatory bowel disease and healthy controls. CONCLUSIONS: Significantly increased VEGF production by PBMCs was found in patients with active Crohn's disease and active ulcerative colitis. The study helps to clarify one of the origins of the significantly enhanced VEGF serum levels in patients with active inflammatory bowel disease observed in recent studies.

Adult↗
PubMed1992

Demonstration of woodchuck hepatitis virus infection of peripheral blood mononuclear cells by flow cytometry and polymerase chain reaction.

Peripheral blood mononuclear cells (PBMCs) from 10 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of WHV surface (WHs) and core (WHc) antigens (WHsAg and WHcAg) by cytofluorometry using fluorescein isothiocyanate-conjugated anti-WHs and anti-HBc-purified immunoglobulins from woodchuck and human sera. The presence of viral DNA and RNA was detected in the serum and PBMCs from the same blood samples by polymerase chain reaction (PCR) with two primer sets located in the S and C genes of the WHV genome. Seven animals were found positive for both WHsAg and WHcAg on the surface of PBMCs: four WHV-chronic carriers, two WHsAg-positive animals with acute WHV infection, and one woodchuck which was bled during the incubation phase of WHV infection and which became WHsAg-positive only 1 month later. Sixteen to 71% of the studied leukocyte population expressed WHsAg with a low density of expression whereas 7 to 72% expressed WHcAg with a high density of expression. Only two cases were positive for WHsAg without WHcAg on PBMCs, one WHV chronic carrier and one anti-WHs-positive animal. All woodchucks positive for WHcAg and/or WHsAg by cytofluorometry were positive also for WHV DNA and RNA in PBMCs by PCR. The tenth animal was found negative for both viral antigens as well as for WHV DNA and RNA in PBMCs despite the presence of persistent viral DNA in the serum as detected by PCR. Five healthy woodchucks devoid of WHV serological markers served as negative controls. These results obtained with a novel approach further confirm, in the woodchuck model, that a significant proportion of PBMCs are probably permissive for WHV replication. The possible immunopathogenic implications of the phenomenon are discussed.

Animals↗
PubMed1997

Comparison of hepatitis B virus core promoter sequences in peripheral blood mononuclear cells and serum from patients with hepatitis B.

It has been reported that hepatitis B virus (HBV) DNA is present in peripheral blood mononuclear cells (PBMCs), although it is unclear whether it actually replicates there or is adsorbed from serum. HBV DNA sequences from the core promoter and precore regions were amplified from PBMCs and serum taken from 13 patients with hepatitis B infection. Analysis by single strand conformation polymorphism, direct sequencing and cloning revealed identical HBV DNA sequences in both PBMCs and serum from five patients with acute hepatitis and in five out of eight patients with chronic hepatitis. However, in the remaining three chronic hepatitis cases, HBV DNA sequences in both PBMCs and serum were different: two patients harboured HBV DNA sequences from their PBMCs with deletions/insertions in the core promoter region and one patient harboured HBV DNA sequences from their PBMCs with two nucleotide substitutions. These findings point to a possible presence of independent HBV DNA replication in PBMCs.

Base Sequence↗
PubMed2002

Circulating and cell-bound antibodies increase coxsackievirus B4-induced production of IFN-alpha by peripheral blood mononuclear cells from patients with type 1 diabetes.

Increased levels of IFN-alpha have been found in patients with type 1 diabetes who have detectable levels of coxsackievirus B4 (CVB4) RNA in their blood. The IFN-alpha-inducing activity of CVB4 in vitro is weak but can be enhanced by human IgGs. Therefore, it was investigated in vitro whether a preferential IFN-alpha response of peripheral blood mononuclear cells (PBMCs) to CVB4 exists in patients with type 1 diabetes (n=56) compared with healthy subjects (n=20) and whether antibodies play a role. In patients, the levels of IFN-alpha obtained after stimulation by PBMCs with CVB4 were higher (P=0.008), an individual IFN-alpha response by PBMCs to CVB4 was more frequent (P=0.0004) and increased levels of IFN-alpha were observed in CVB4-infected whole blood cultures. The IFN-alpha-inducing activity of patients plasma and IgGs mixed with CVB4 and then added to PBMCs was high in comparison with healthy subjects (P<0.001) and was inhibited by preincubating the cells with anti-FcgammaRII, anti-FcgammaRIII and anti-CAR (coxsackievirus and adenovirus receptor) antibodies. The strong IFN-alpha responsiveness of PBMCs to CVB4 suggested that IgGs bound to the cell surface might play a role. A short 56 degrees C incubation of PBMCs from patients responsive to CVB4 generated supernatants, which, when added to cells, exhibited IFN-alpha-enhancing activity in combination with CVB4, whereas those of controls did not. Specific antibodies for FcgammaRI, FcgammaRII and CAR inhibited this activity. These studies demonstrate that CVB4, through interactions with circulating and/or cell-bound IgGs, can strongly induce the production of IFN-alpha by PBMCs from patients with type 1 diabetes.

Adenoviridae↗