Concanavalin A-stimulated Peripheral Blood Mononuclear Cells Research Articles

Peripheral blood mononuclear cell activation sustains seizure activity.

The influx of immune cells and serum proteins from the periphery into the brain due to a dysfunctional blood-brain barrier (BBB) has been proposed to contribute to the pathogenesis of seizures in various forms of epilepsy and encephalitis. We evaluated the pathophysiological impact of activated peripheral blood mononuclear cells (PBMCs) and serum albumin on neuronal excitability in an in vitro brain preparation. A condition of mild endothelial activation induced by arterial perfusion of lipopolysaccharide (LPS) was induced in the whole brain preparation of guinea pigs maintained in vitro by arterial perfusion. We analyzed the effects of co-perfusion of human recombinant serum albumin with human PBMCs activated with concanavalin A on neuronal excitability, BBB permeability (measured by FITC-albumin extravasation), and microglial activation. Bioplex analysis in supernatants of concanavalin A-stimulated PBMCs revealed increased levels of several inflammatory mediators, in particular interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (INF)-γ, IL-6, IL-10, IL-17A, and MIP3α. LPS and human albumin arterially co-perfused with either concanavalin A-activated PBMCs or the cytokine-enriched supernatant of activated PBMCs (1) modulated calcium-calmodulin-dependent protein kinase II at excitatory synapses, (2) enhanced BBB permeability, (3) induced microglial activation, and (4) promoted seizure-like events. Separate perfusions of either nonactivated PBMCs or concanavalin A-activated PBMCs without LPS/human albumin (hALB) failed to induce inflammatory and excitability changes. Activated peripheral immune cells, such as PBMCs, and the extravasation of serum proteins in a condition of BBB impairment contribute to seizure generation.

Suppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells of rheumatoid arthritis patients.

To investigate the immunosuppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients. Concanavalin A-stimulated PBMC culture procedure was used to evaluate the pharmacodynamics of vitamin K2 in vitro. Methotrexate was set up as the positive control. The proliferation of PBMCs was detected by MTT assay. Relationship between IC50 values of drugs on PBMC proliferation and patient-related factors including laboratory data was analyzed by nonparametric Spearman correlation test. Vitamin K2 inhibited the proliferation of mitogen-activated PBMCs of RA patients with an IC50 value of 3,288.47±4,910.02 ng/mL (mean±SD). There was a significant correlation between IC50 values of vitamin K2 and patient-related factors of RA patients (p<0.05), such as C-reactive protein (CRP), rheumatoid factor, anti-cyclic citrullinated peptide antibody (ACPA), matrix metalloproteinase-3, Pre-DAS-28 (CRP), and ∆DAS-28 (CRP). It would be possible to predict the pharmacodynamics of vitamin K2 in RA patients according to the above factors. Methotrexate inhibited the proliferation of mitogen-activated PBMCs of RA patients with a IC50 value of 22.83±12.47 ng/mL (mean±SD). IC50 values of methotrexate only showed significant correlation with ACPA (p=0.0158, r=0.6905), which suggests that ACPA might be a suitable predictor of the pharmacodynamics of methotrexate. The above information suggests that vitamin K2 could provide a benefit for the treatment of RA patients via its immunosuppressive function.

Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells.

Mitogens are diverse compounds of plant and microbial origin, widely employed to test immunocompetence in animals. The blastogenic response of bovine Peripheral Blood Mononuclear Cells (PBMC) to lypopolysaccharides (LPS) has been investigated in our laboratories for a long time. In particular, a possible correlation between blastogenic response to LPS and disease resistance of periparturient dairy cows had been observed in previous studies. Most important, low responder cows presented a higher frequency of disease cases after calving, compared with high responder animals. Owing to the above, different aspects of the blastogenic response to LPS were investigated on PBMC of healthy Friesian cows, using a 72-hour Bromodeoxyuridin (BrDU) cell proliferation assay. Stimulation with LPS induced little if any replication of bovine PBMC over 72 hours despite consistent BrDU detection in all the PBMC samples under study. Poor replication of LPS-stimulated PBMC was confirmed by cell cycle and cell growth flow cytometry analyses. In particular, LPS stimulation gave rise to very low percentages of S phase cells, sometimes lower than in control, unstimulated cells, as opposed to Concanavalin A-stimulated PBMC. Magnetic separation and analysis of BrDU-treated bovine PBMC after exposure to LPS showed that both B and CD4 T cells are involved in the blastogenic response to LPS, in contrast with current data based on human and murine models. Finally, LPS caused an early, specific up-regulation of TNF-α and TLR4 genes in bovine PBMC, and significant correlations were shown between the expression of inflammatory cytokine and Indoleamine-pyrrole 2,3-dioxygenase (IDO1) genes. On the whole, our data indicate that differences in the blastogenic response to LPS could be partly accounted for by heterogenicity of responding cells (B and T lymphocytes), which might also have an impact on induction and regulation of inflammatory responses and endotoxin tolerance.

The role of the ovarian cycle and the effects of mitogen-induced cytokines on human prolactin gene expression in peripheral blood mononuclear cells

ABSTRACT Background: little is known on the influences of normal menstrual cycle on prolactin gene expression in immune cells. Aim of the study: to determine the effects of the ovarian cycle on prolactin and its receptor expression. Methods: peripheral blood mononuclear cells (PBMC) were obtained from twenty-six normal menstruating women at different intervals of their menstrual cycle. The PBMC were incubated during 24 h in the presence or absence of Concanavalin-A (Con-A) and the gene expression of PRL, PRLR and cytokines was evaluated by qPCR. Prolactin, IL-2 and cAMP were determined in each culture by specific immunoassays. Results: neither PRL nor its receptor expression in PBMC changed significantly among groups, including the cytokines (IL-2, IL-10, and IFNG) studied. Similar results, among groups, were obtained, when PRL expression was stimulated by PGE2 or 8-Br-cAMP. Concanavalin A-stimulated PBMC expressed significantly less prolactin and a significant negative correlation between secreted IL-2 and PRL expression was found. The presence of anti-IL-2 antibodies in Con-A stimulated-cultures significantly increased PRL expression when compared to control cells regardless the hormonal status. Conclusions: these data suggest that the menstrual cycle does not significantly modulate or influence prolactin and cytokines gene expression in PBMC, and indicate that IL-2 may be involved in the Con-A regulation of PRL expression in immune cells.

Effects of insulin on pharmacodynamics of immunosuppressive drugs against mitogen-activated human peripheral blood mononuclear cells

Context: Diabetes mellitus is one of the most common causes of chronic renal failure. Immunosuppressive efficacies of glucocorticoids, calcineurin inhibitors, and mycophenolic acid are possibly affected by insulin after renal transplantation in these patients.Objectives: We investigated the effects of insulin on responses of mitogen-activated human peripheral blood mononuclear cells (PBMCs) to several immunosuppressive drugs.Materials and methods: Antiproliferative efficacies of prednisolone, hydrocortisone, cyclosporine, tacrolimus, and mycophenolic acid against concanavalin A-stimulated PBMCs were evaluated in the presence of physiological (5 μunits/mL) and super physiological (50 μunits/mL) concentrations of insulin. Insulin-receptor expressions on PBMCs were evaluated by flow cytometry.Results: Insulin itself had no effects on the mitogen-induced proliferation of PBMCs. The IC50 values of cyclosporine against the mitogen-activated PBMCs in the presence of 5 or 50 μunits/mL insulin were significantly higher than those of cyclosporine without insulin (p < 0.05). The IC50 values of mycophenolic acid significantly increased by 50 μunits/mL insulin (p < 0.01). Insulin receptors were detected on the mitogen-activated CD4+/CD14+ cells in PBMCs.Discussion and conclusions: These results indicate that insulin at even physiological concentration attenuates suppressive efficacies of several immunosuppressive drugs against mitogen-activated proliferation of human PBMCs, possibly via insulin receptors. Insulin used in dialysis patients accompanying diabetes mellitus is suggested to attenuate efficacies of immunosuppressive drugs after renal transplantation.

Lactobacillus helveticus Lafti L10 supplementation reduces respiratory infection duration in a cohort of elite athletes: a randomized, double-blind, placebo-controlled trial.

A randomized, double-blind, placebo-controlled study was conducted to evaluate if Lactobacillus helveticus Lafti L10 (Lallemand Health Solutions, Montreal, Que., Canada) supplementation during 14 weeks in winter can influence the duration, severity, and incidence of upper respiratory tract illness (URTI), as well as to monitor different immune parameters in the population of elite athletes. Before and after the treatment, cardiopulmonary testing and self-rated state of moods evaluation (by Profile of Mood States questionnaire) were performed and blood samples were collected. Thirty-nine elite athletes were randomized either to the placebo (n = 19) or the probiotic (n = 20) group. The probiotic group received L. helveticus Lafti L10, 2 × 10(10) Colony Forming Units. Lafti L10 significantly shortened the URTI episode duration (7.25 ± 2.90 vs. 10.64 ± 4.67 days, p = 0.047) and decreased the number of symptoms in the probiotic group (4.92 ± 1.96 vs. 6.91 ± 1.22, p = 0.035). Severity and incidence of URTI did not differ between the treatments. There were no significant changes in leukocyte subpopulation abundance, transforming growth factor-β serum levels, level of interleukin-10 secreted from peptidoglican stimulated peripheral blood mononuclear cells (PBMCs), interferon-γ level secreted from concanavalin A-stimulated PBMCs or viability/proliferation of PBMCs upon antigen stimulation. Group effect for CD4+/CD8+ ratio was significant (F[1,37] = 6.99, p = 0.020, η(2) = 0.350); this difference was not significant at baseline, but was evident after 14 weeks (p = 0.02). A significant interaction effect was noted for self-rated sense of vigor (F[1,37] = 11.76, p = 0.009, η(2) = 0.595). Self-rated sense of vigor increased in the probiotic group (18.5 ± 4.1 vs. 21.0 ± 2.6, p = 0.012). Probiotic strain Lafti L10 can be a beneficial nutritional supplement for the reduction of URTI length in elite athletes.

Suppressive effects of peptide antibiotics against proliferation and cytokine production in mitogen-activated human peripheral-blood mononuclear cells

Certain kinds of peptide antibiotics are suggested to have immunomodulatory effects; however, few studies have been carried out systemically to evaluate the antiproliferative effects of peptide antibiotics in human lymphoid cells. The suppressive efficacies of nine peptide antibiotics and seven non-antibiotic peptides against proliferation of human peripheral-blood mononuclear cells (PBMCs) stimulated with T cell mitogen were examined in vitro. Nigericin (CAS 28643-80-3), valinomycin (CAS 2001-95-8), gramicidin D (CAS 1405-97-6), and tyrothricin (CAS 1404-88-2) strongly inhibited the proliferation of concanavalin A-stimulated PBMCs with IC50 values of 0.15-11.2 ng/ml, while these antibiotics did not show cytotoxicity at 10 000 ng/ml. The IC50 value of the immunosuppressant cyclosporine (CAS 59865-13-3) was 5.2 ng/ml. Virginiamycin (CAS 11006-76-1) and gramicidin S (CAS 113-73-5) moderately inhibited PBMC-proliferation with IC50 values of 1000 and 1900 ng/ml, respectively. On the other hand, bacitracin (CAS 1405-87-4), capreomycin (CAS 11003-38-6), polymyxin B (1404-26-8), angiotensin II antipeptide (CAS 121379-63-3), angiotensin III antipeptide (CAS 133605-55-7), fibrinogen binding inhibitor peptide (CAS 89105-94-2), LH-RH (CAS 71447-49-9), pepstatin A (CAS 26305-03-3), oxytocin (CAS 50-56-6), and vasopressin (CAS 16679-58-6) showed little or no suppressive effect on PBMC-proliferation. Nigericin and valinomycin decreased the concentrations of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, and IL-17 in the culture medium with IC50 values less than 0.01 ng/ml. Nigericin also decreased the concentrations of IL-4 and IL-6 with IC50 values of less than 1 ng/ml. The results show that peptide antibiotics such as nigericin and valinomycin efficiently suppress the production of several cytokines and proliferation in mitogen-stimulated human PBMCs.

Liposome-incorporated dexamethasone palmitate inhibits in-vitro lymphocyte response to mitogen.

The use of liposomes for the pulmonary delivery of corticosteroid is an area that is under active investigation. We have recently developed a novel liposomal corticosteroid preparation based on the incorporation of dexamethasone palmitate (DMP) within the bilayer of small unilamellar vesicles (SUVs) made of egg yolk phosphatidylcholine (EPC) and cholesterol; molar ratio EPCC:cholesterol: DMP, 4:3:0.3. In the present study, the biological activity of DMP-SUVs was evaluated using the lymphocyte transformation test with peripheral blood mononuclear cells (PBMCs) and a gamma-interferon production assay. Results showed that DMP-SUVs (but not empty SUVs) inhibited [3H]thymidine uptake and gamma-interferon production by concanavalin A-stimulated PBMCs by 94 and 96%, respectively, at a concentration corresponding to 10(-6) M dexamethasone. The inhibition by DMP-SUVs was found to require a 24-h pre-incubation with unstimulated PBMCs, suggesting that interaction of SUVs with lymphocytes may be altered by mitogen stimulation. We conclude that our DMP liposomal preparation is biologically active and may be considered a promising alternative to conventional local glucocorticoid therapy.

Comparative study of the effects of betamethasone butyrate propionate, vitamin D3 derivatives, and cyclosporine on human lymphocyte-proliferation stimulated with a hemolytic streptococci-derived superantigen

Bacterial infection might influence the clinical response of patients with immunological disorders including psoriasis to the therapeutic efficacies of immunosuppressive drugs, but few studies have been carried out to investigate the implication of bacterial superantigens. We evaluated the suppressive efficacies of betamethasone butyrate propionate, vitamin D3 derivatives, and cyclosporine against concanavalin A- or superantigen-induced proliferation of peripheral-blood mononuclear cells obtained from 18 healthy subjects. In vitro drug concentrations giving 50% inhibition (IC50s) of peripheral-blood mononuclear cell-proliferation stimulated with concanavalin A or streptococcal pyrogenic enterotoxin A were estimated. Concentrations of tumor necrosis factor-alpha, interferon-gamma, interleukin-2, -4, -5, or -10, in a peripheral-blood mononuclear cell-culture medium were measured with beads-array procedures. The median (range) IC50 value for betamethasone butyrate propionate evaluated in the streptococcal pyrogenic enterotoxin A-stimulated peripheral-blood mononuclear cells was 291.6 (0.001-1171.5) ng/ml, which was significantly higher than the value 0.072 (0.01-222.5) ng/ml found in concanavalin A-stimulated peripheral-blood mononuclear cells (P=0.0245). However, the median (range) IC50 value for calcipotriol in the streptococcal pyrogenic enterotoxin A-stimulated peripheral-blood mononuclear cells was 0.3 (0.24-1357.4) ng/ml, which was significantly lower than the value 128.6 (0.1-776.9) ng/ml found in concanavalin A-stimulated peripheral-blood mononuclear cells (P=0.0323). The IC50 value for cyclosporine was not significantly different between the concanavalin A- and streptococcal pyrogenic enterotoxin A-stimulated PBMCs. Concentration for none of the cytokines was significantly different between concanavalin A- and streptococcal pyrogenic enterotoxin A-stimulated peripheral-blood mononuclear cell cultures. The effects of betamethasone butyrate propionate to suppress these cytokine productions were rather stronger than those of calcipotriol. Streptococcal pyrogenic enterotoxin A induced by hemolytic streptococci colonization is suggested to attenuate the therapeutic efficacy of betamethasone butyrate propionate. While the mechanistic background of calcipotriol to suppress streptococcal pyrogenic enterotoxin A-induced peripheral-blood mononuclear cell-proliferation remains to be elucidated, vitamin D3 derivatives appears to be effective in suppressing anomalistic immunity in patients having hemolytic streptococci colonization.

CERVINE (CERVUS ELAPHUS) CYTOKINE mRNA QUANTIFICATION BY REAL-TIME POLYMERASE CHAIN REACTION

It has been difficult to perform cytokine studies for many wildlife and nontraditional species because of a lack of immunologic reagents at the protein level. Recently, simple and rapid assays for quantifying mRNA expression by real-time reverse transcription-polymerase chain reaction (RT-PCR) have been used for analysis of cytokine profiles in humans and other mammalian species. This report describes the development and application of real time RT-PCR to measure the expression of several important elk (Cervus elaphus) cytokine mRNAs, including interleukin (IL)-2, IL-4, IL-10, IL-12p40, interferon-gamma, tumor necrosis factor (TNF)-alpha, and the enzyme-inducible nitric oxide synthase, all of which are involved in immune responses and regulation. For the broadest potential application of the assay, primers and probes were designed using consensus sequences from several species of interest. To obtain standardized quantitative results, external controls consisting of a DNA template for each target gene were used to generate linear standard curves over a 6 to 8 log range with detection of as few as 10 copies of amplicon per reaction. Sample-to-sample variation in the efficiency of the RT, as well as in the quantity and quality of the starting RNA, was compensated for by normalizing the results to the endogenous housekeeping gene beta(2)-microglobulin. The assay was evaluated by monitoring the kinetics of cytokine mRNA synthesis induced by mitogenic and antigenic stimulation of peripheral blood mononuclear cells (PBMCs) from Mycobacterium bovis-infected elk. Concanavalin A-stimulated PBMCs demonstrated a rapid but transient increase in cytokine mRNA expression following in vitro mitogenic activation with optimal mRNA induction observed after 4 to 16 hr. The PBMCs stimulated with the mycobacterial recall antigen, bovine-purified protein derivative (PPD-bovis), demonstrated variable mRNA induction kinetics for each cytokine. Whereas PPD-bovis optimally induced IL-2 mRNA after 8 hr of in vitro stimulation, longer in vitro stimulation times were necessary for the optimal induction of IL-4 and TNF-alpha mRNA (up to 48 hr). We demonstrate real-time RT-PCR to be a rapid, sensitive, and reproducible technique, which will make it a valuable tool in the study of immunologic responses and cytokine profiles of cervids and other nontraditional livestock and wildlife species.

Cortisone counteracts apoptosis-inducing effect of cortisol in human peripheral-blood mononuclear cells

Glucocorticoids (GCs) have been considered to regulate immune cell systems through induction of apoptosis in thymocytes and mature peripheral-blood lymphocytes. Here we report that apoptosis induced by cortisol in mitogen-activated peripheral-blood mononuclear cells (PBMC) is suppressed by cortisone, an oxidized metabolite of cortisol. Apoptosis in PBMCs is quantified by a cell death ELISA procedure, which can specifically detect fragmented DNA. Cortisol induced PBMC-apoptosis at concentrations more than 10 ng/ml (28 nM) in concanavalin A-stimulated PBMCs and cortisone suppressed this apoptosis at a concentration range of 1–10,000 ng/ml (2.8–28,000 nM) dose-dependently. Prednisone, a synthetic oxidized-GC, also suppressed the apoptosis-inducing effect of cortisol in a dose-dependent manner. Suppression of cortisol-induced apoptosis by cortisone was consistently observed in PBMCs derived from 16 healthy subjects. Examination for inhibitory activities of the steroids against [ 3H]dexamethasone binding to PBMCs suggested that cortisone can bind cellular GC-receptors (GC-Rs), but the affinity of cortisone to GCRs is 1/30 or less than that of cortisol. The results raised a possible role of cortisone in cortisol-mediated regulation of apoptosis in activated human PBMCs. The counteracting action of cortisone against cortisol-induced apoptosis may take place partially through intervention of GC-receptors (GC-Rs), but may also be due to unknown pathway(s) different from those mediated by cellular GC-Rs.

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.

Proliferation of human peripheral blood mononuclear cells during calcium entry blockade. Role of protein kinase C.

To evaluate the role of protein kinase C (PKC) and intracellular calcium and particularly Ca(2+)-uptake in the initiation of lymphocyte mitogenesis, the proliferation of human peripheral blood mononuclear cells (PBMC) was investigated during calcium entry blockade with nifedipine (an L-type calcium channel blocker) and mibefradil (an L- and T-type calcium channel blocker with a higher selectivity for T-type channels). The rate of [3H]-thymidine, [3H]-uridine and [3H]-leucine incorporation into control and concanavalin A-stimulated PBMC cultured for 3 days in the presence or absence of the calcium channel blockers nifedipine or mibefradil (1, 10 or 50 microM) is assayed. Nifedipine and mibefradil concentration-dependently reduced cell number and [3H]-thymidine incorporation or de novo DNA synthesis in control and concanavalin A-stimulated PBMC, as well as de novo RNA and protein synthesis. The proliferative response of nifedipine- or mibefradil-treated cells was restored by addition of phorbol-12-myristate-13-acetate (PMA), an exogenous PKC activator. Our data show that PBMC treated with the Ca2+ channel blockers nifedipine or mibefradil are still capable of proliferating in response to PMA. However, in PKC-depleted cells, the proliferative response of PBMC was suppressed.

Proliferation of human peripheral blood mononuclear cells during calcium channel blockade

To evaluate the role of intracellular calcium and particularly Ca 2+ 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 (PBMC) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of 3H-thymidine, 3H-uridine, and 3H-leucine incorporation into control and concanavalin A-stimulated PBMC in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 μmol/L), and of the intracellular calcium antagonist TMB-8 or the calmodulin antagonist W-7 (1, 10, 25, or 50 μmol/L) was assayed in cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures was also determined in mibefradil- or nifedipine-treated control or stimulated cells. Mibefradil and nifedipine reduced the cell number and the 3H-thymidine, 3H-uridine, or 3H-leucine incorporation or the de novo DNA, RNA, or protein synthesis in control and concanavalin A-stimulated human PBMC in a concentration-dependent manner. Mibefradil exhibited a more pronounced inhibition than nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent upon 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 were added 1 to 7 h after cell stimulation. However, regardless of time of addition, TMB-8 and W-7 caused a persistent inhibition of the proliferation of human PBMC. Our data show that mibefradil had a more pronounced inhibitory effect on the proliferation of human PBMC than nifedipine and that this inhibitory effect on de novo DNA synthesis was dependent upon the timing of treatment with both drugs. Mibefradil and nifedipine also reduce RNA and protein synthesis in human PBMC. Therefore, administration of these calcium channel blockers to inhibit cellular proliferation might be most beneficial at anatomic sites where cellular proliferation is not already an active process, while being ineffective in the presence of ongoing active proliferation, as suggested by some prospective studies.

Dehydroepiandrosterone may be one of the regulators of cytokine production in atopic dermatitis.

Previous studies in mice have shown that dehydroepiandrosterone (DHEA) increases the production of Th1-associated lymphokines, and of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), by lymphocytes. However, there are no reports concerning the effect of DHEA on the production of Th2-associated lymphokines, IL-4 and IL-5, by lymphocytes in humans. We examined serum DHEA levels in patients with atopic dermatitis (AD), which is thought to be associated with a higher activity of Th2 cells than of Th1 cells. We also studied the effects of DHEA on the production of IL-4 and IL-5 by human lymphocytes. Serum DHEA concentrations in 47 adult male patients with AD aged 19-30 years were significantly lower than those of 53 age-matched healthy male controls. Preincubation of peripheral blood mononuclear cells (PBMCs) with DHEA reduced the IL-4 production by concanavalin A-stimulated PBMCs. Their IL-5 production also showed a tendency to decrease. These results suggest that DHEA may be one of the regulators of IgE synthesis and eosinophil proliferation in patients with AD and it may act by controlling IL-4, IL-5 and IL-2 production by lymphocytes.

Oral cow milk challenge abolishes antigen‐specific interferon‐γ production in the peripheral blood of children with atopic dermatitis and cow milk allergy

A major feature of atopic dermatitis (AD) is the propensity to generate IgE to environmental antigens. Despite extensive information on IgE dysregulation in AD, the nature of immune responses to ingested allergens is poorly characterized. To determine the clinical and immunological responses to food in AD patients. To characterize the type and timing of clinical reactions to oral cow milk, 83 AD patients aged 2 to 60 months were subjected to double-blind placebo-controlled food challenge (DBPCFC). IFN-gamma and IL-4 production by their peripheral blood mononuclear cells (PBMC) was determined before and after DBPCFC. Of 50 patients positive to DBPCFC. 46% manifested exanthematous-type immediate-onset reactions and 54% eczematous-type late-onset reactions. In either group, the production of IL-4 and IFN-gamma by Concanavalin A-stimulated PBMC was comparable before and after DBPCFC. For immediate-reacting patients, the median IFN-gamma production by milk-stimulated PBMC was 11.5 (4.2-17.2) pg/ml as against 2.3 (0.2-5.7) pg/ml by unstimulated PBMC, P = 0.0008 before DBPCFC, and 4.6 (2.8-10.3) pg/ml. vs 4.2 (1.7-9.0) pg/mL, p = 0.40, correspondingly after DBPCFC. Before DBPCFC, immediate-reacting but not late-reacting patients were found to be capable of allergen-specific IFN-gamma production in vitro, indicating the heterogeneity in AD patients. After DBPCFC, the IFN-gamma generation abolished, indicating the effect of oral allergen exposure on IFN-gamma producing responses of AD patients.

In vitro effects of live and killed Brucella abortus on bovine cytokine and prostaglandin E 2 production

Live and γ-irradiated-killed Brucella abortus strain 2308 increased interleukin 1 (IL-1), but not interleukin 2 (IL-2), interferon-γ (IFN-γ), or prostaglandin E 2 (PGE 2) production when incubated with normal bovine peripheral blood mononuclear cells (PBMC). Live B. abortus was more effective than killed B. abortus in stimulating IL-1 production by normal PBMC. Both live and killed B. abortus were equally effective in suppressing IL-2 and IFN-γ production by Concanavalin A-stimulated PBMC. Incubation of PBMC with the cyclo-oxygenase inhibitor, indomethacin, blocked PGE 2 synthesis, but did not further enhance IL-1 production or prevent suppressed IL-2 and IFN-γ production that was induced by live and killed B. abortus. These results suggest that B. abortus-induced suppression of IL-2 and IFN-γ production did not appear to be mediated by the suppressive prostaglandin, PGE 2, or other cyclo-oxygenase metabolites.

Detection of feline immunodeficiency virus (FIV) nucleic acids in FIV-seronegative cats.

A study was undertaken to determine the rate of viral transmission among naive specific-pathogen-free (SPF) cats living in close contact with feline immunodeficiency virus (FIV)-infected cats. Twenty SPF cats were housed in the same rooms with experimentally FIV-infected seropositive and virus culture-positive cats for 2 to 4 years and were monitored for the presence of FIV nucleic acids and antibodies. Only 1 of the 20 cats became seropositive and virus culture positive and developed signs of disease. Genomic DNA from bone marrow and peripheral blood mononuclear cells (PBMC) of 10 of 19 healthy-appearing seronegative cats became positive for FIV DNA by the polymerase chain reaction. Twenty-eight SPF cats housed as groups in separate quarters and never exposed to FIV-infected cats were uniformly negative for FIV DNA. FIV RNA transcripts were detected in concanavalin A-stimulated PBMC cultures from 4 of 10 FIV DNA-positive, seronegative cats by in situ hybridization. PBMC from three of four naive SPF cats acquired FIV nucleic acids after the cats were transfused with blood and bone marrow from FIV genome-positive, seronegative donors. Three of five FIV-seronegative cats housed for years with naturally FIV-infected cats in a private household were also found to harbor FIV DNA, indicating that the same phenomenon occurred in the field. These findings demonstrate that cats living in close contact with FIV-infected seropositive cats can acquire FIV nucleic acids without developing detectable levels of serum antibodies or disease.

Role of lipid peroxidation in the inhibition of mononuclear cell proliferation by normal lipoproteins

Stimulated peripheral blood mononuclear cells (PBMC) can oxidize normal lipoproteins, and sufficiently oxidized lipoproteins are cytotoxic. However, the role of lipid peroxidation in the inhibition of mitogen-stimulated PBMC proliferation by physiologic concentrations of normal lipoproteins is unclear. In the present investigation, normal low density lipoprotein (LDL) and very low density lipoprotein (VLDL) suppressed [3H]thymidine incorporation and gamma interferon production in concanavalin A-stimulated PBMC without causing cell death. This suppression was accompanied by parallel increases in lipid peroxidation products measured as thiobarbituric acid reactive substances (TBARS). In contrast, high density lipoprotein (HDL) failed to inhibit PBMC and TBARS remains low. Differences between the PBMC suppression from LDL, VLDL, and HDL were best accounted for by normalizing the lipoprotein concentrations by their total lipid content. Moreover, the antioxidants superoxide dismutase and butylated hydroxytoluene each substantially ameliorated the inhibition of PBMC caused by LDL, and reduced the levels of lipid peroxidation products that were generated. Altogether, these results suggest that reactive oxygen species generated by stimulated PMBC may cause oxidative alterations of normal lipoproteins that may, in turn, account for much of the previously reported inhibition of PBMC by normal lipoproteins.