Reactivity Of Peripheral Blood Mononuclear Cells Research Articles

Species comparison: human and minipig PBMC reactivity under the influence of immunomodulating compounds in vitro.

Considering the similarities between swine and humans, it is a logical consequence to use swine as a translational model in research and drug development, including non-clinical safety. Here, we compared the reactivity of peripheral blood mononuclear cells (PBMCs) from humans and minipigs under the influence of different compounds in vitro. We conducted a flow cytometry-based proliferation assay that focused on the T-cell response to three different stimuli: concanavalin A (ConA), phytohemagglutinin-L (PHA-L), and staphylococcal Enterotoxin B (SEB). Furthermore, four approved immunosuppressive drugs-abatacept, belatacept, rapamycin, and tofacitinib-which are used for the treatment of rheumatoid arthritis or rejection in transplant recipients, were combined with the different stimuli. This allowed us to study the effect of suppressive drugs in comparison with the different stimuli in both species. We examined proliferating T cells (CD3+) and investigated the presence of TCR-αβ+ and TCR-γδ+ T cells. Differences in the response of T cells of the two species under these various conditions were evident. CD4+ T cells were more activated within humans, whereas CD8+ T cells were generally more abundant in swine. The effectiveness of the used humanized antibodies is most likely related to the conserved structure of CTLA-4 as abatacept induced a much stronger reduction in swine compared with belatacept. The reduction of proliferation of rapamycin and tofacitinib was highly dependent on the used stimuli. We further investigated the effect of the immunosuppressive compounds on antigen-specific restimulation of pigs immunized against porcine circovirus 2 (PCV2). Treatment with all four compounds resulted in a clear reduction of the proliferative response, with rapamycin showing the strongest effect. In conclusion, our findings indicate that the effectiveness of suppressive compounds is highly dependent on the stimuli used and must be carefully selected to ensure accurate results. The results highlight the importance of considering the response of T cells in different species when evaluating the potential of an immunomodulatory drug.

Peripheral blood mononuclear cells reactivity in recent-onset type I diabetes patients is directed against the leader peptide of preproinsulin, GAD65271-285 and GAD65431-450.

T cell reactivity against pancreatic autoantigens is considered one of the main contributors to the destruction of insulin-producing cells in type 1 diabetes (T1D). Over the years, peptide epitopes derived from these autoantigens have been described in NOD mice and in both HLA class II transgenic mice and humans. However, which ones are involved in the early onset or in the progressive phases of the disease is still unclear. In this work we have investigated, in early-onset T1D pediatric patients and HLA-matched controls from Sardinia, the potential of preproinsulin (PPI) and glutamate decarboxylase 65 (GAD65)-derived peptides to induce spontaneous T cell proliferation responses of peripheral blood mononuclear cells (PBMCs). Significant T cell responses against PPI1-18, PPI7-19 and PPI31-49, the first two belonging to the leader sequence of PPI, and GAD65271-285 and GAD65431-450, were found in HLA-DR4, -DQ8 and -DR3, -DQ2 T1D children. These data show that cryptic epitopes from the leader sequence of the PPI and GAD65271-285 and GAD65431-450 peptides might be among the critical antigenic epitopes eliciting the primary autoreactive responses in the early phases of the disease. These results may have implications in the design of immunogenic PPI and GAD65 peptides for peptide-based immunotherapy.

High-fat meal increases peripheral blood mononuclear cell pro-inflammatory cytokine expression in African-American women.

African-American (AA) women have elevated predominance of inflammatory diseases concurrent with local inflammation resulting in compromised metabolic function. The purpose of the study was 2-fold: 1) to examine the gene and protein expression of pro- and anti-inflammatory cytokine secretion by peripheral blood mononuclear cells (PBMC) obtained from AA and Caucasian-American (CA) women in response to an acute high-fat meal; and 2) to explore the influence of race (AA vs. CA) on PBMC reactivity. Ten AA and 11 CA women consumed a high-fat meal with baseline and 4h postprandial venous blood draws. PBMCs were incubated for 3 h then messenger RNA expression and supernatant protein concentration was used to examine inflammatory profiles. All women had a postprandial increase in interleukin (IL)-8 gene expression, IL-8 protein concentration, and tumor necrosis factor alpha (TNF-α) protein concentration (P< 0.05). AA women had a postprandial increase in IL-6, IL-8, and TNF-α protein concentration (P< 0.05). AA women had higher postprandial IL-1β protein concentration and IL-8 gene expression compared with CA women (P< 0.05). Our data uncovers the specific impact of race and time on pro-inflammatory PBMC (IL-1β, IL-6, IL-8, and TNF-α) expression profiles in response to an acute high-fat meal challenge. Novelty: African Americans have higher predominance of inflammatory disease. We explored the potential race impact on peripheral blood mononuclear cell reactivity in response to a meal. A pro-inflammatory response to an acute high-fat meal with race impact was observed possibly contributing to health disparities impacting African-American women.

Early suppression of peripheral mononuclear blood cells in sepsis in response to stimulation with cytomegalovirus, OKT3, and pokeweed mitogen.

Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis (n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV (P = 0.01), OKT3 (P = 0.02), and PWM (P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points (P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis.NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.

Granulocyte-augmented chemokine production induced by type II collagen containing immune complexes is mediated via TLR4 in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) patients with early elevations of antibodies against collagen type II (CII) have a distinct acute onset phenotype, associated with cytokine induction by surface‐bound anti‐CII‐containing immune complexes (ICs) and high C‐reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Polymorphonuclear granulocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) are abundant in the vicinity of CII in RA joints, and both PMN and PBMC reactivity against anti‐CII IC individually relate to early joint destruction and early elevation of CRP and ESR in RA. We searched for CII‐dependent mechanisms that might attract PMNs and PBMCs to RA joints. Human PBMCs and PMNs were stimulated with anti‐CII ICs and control ICs, either individually or in cocultures. Cocultured PMNs and PBMCs stimulated with anti‐CII ICs synergistically augmented production of the chemokines CXCL8, RANTES and MCP‐1, whereas downregulation was seen with control IC. This upregulation was unique to chemokines, as TNF‐α, IL‐1β, and GM‐CSF were downregulated in anti‐CII IC‐stimulated cocultures. The coculture‐associated chemokine upregulation depended on endogenous TLR4 ligand(s) and functionally active PMN enzymes, and was partially mediated by GM‐CSF. As anti‐CII levels peak around the time of RA diagnosis, this mechanism can attract inflammatory cells to joints in early RA and intensify the anti‐CII‐associated acute onset RA phenotype.

Effect of &lt;em&gt;Escherichia coli&lt;/em&gt; and &lt;em&gt;Staphylococcus aureus&lt;/em&gt; on the &lt;em&gt;In vitro&lt;/em&gt; Response of Peripheral Blood Mononuclear Cells Isolated from Holstein Dairy Cows had Ketosis or Milk Fever of Field Cases

The expression of cytokines mRNA, including Interleukin (IL)-2, IL-4 and Interferon (IFN)-γ and lymphocyte proliferation stimulated <em>in vitro</em> with either <em>Escherichia coli</em> (<em>E. coli</em>) or Staphylococcus aureus (S.A.) by Peripheral Blood Mononuclear Cells (PBMC) obtained from Holstein dairy cows were examined after the onset of ketosis or milk fever of field cases during the lactation period. The cows were used 50 to 150 days after calving in this study and divided into three groups; seventeen cows with milk fever (MF Group, 94.9±8.6 days after calving), ten cows with ketosis (Ketosis Group, 96.3±10.2 days after calving) and twenty-seven cows with no clinical diseases (Control Group, 89.4±5.4 days after calving). PBMC were stimulated <em>in vitro</em> by soluble antigen of <em>E. coli</em> or <em>S.A.</em> together with recombinant IL-2. The numbers of the peripheral blood T cells and monocyte were lower in the Ketosis Group in comparison with those in the Control Group and a significant difference was found in the number of $CD3^+TcR1-N12^+T$ cells. There was a significantly lower lymphocyte proliferation stimulated with <em>E. coli</em> and <em>S.A.</em> in the Ketosis Group compared to that in the Control Group. Expression of IL-2, IL-4 and IFN-γ mRNA in PBMC stimulated with <em>S.A.</em> in the Ketosis Group was significantly lower than that in the Control Group, but these cytokine expression showed no group differences when PBMC were stimulated with <em>E. coli</em>. The present study demonstrated the onset of ketosis during the lactation period decreases reactivity of PBMC at least when they were stimulated with <em>S.A in vitro</em>.

Analysis of cytokine secretion from lymphocytes of patients with hypersensitivity reactions to contaminated heparins

Beginning in 2007, anaphylactoid reactions associated with unfractionated heparin (UFH) occurred and resulted in some fatalities. These reactions were reported to be linked to the complement and contact system activation induced by certain batches of UFH containing the adulterant oversulphated chondroitin sulphate (OSCS). Drug-specific secretion of selected cytokines from peripheral blood mononuclear cells (PBMC) of patients with hypersensitivity reactions to contaminated heparin was compared with the respective in vitro cytokine pattern of individuals with or without hypersensitivity to heparin, different glycosaminoglycans or other drugs. Study individuals (n = 13) were classified as follows: patients with hypersensitivity reactions to contaminated (OSCS) heparin (n = 3), noncontaminated heparin (n = 1) or other compounds (n = 3) and patients with ongoing heparin therapy without symptoms of intolerance (n = 2). Four healthy individuals served as controls. PBMC were incubated with six different glycosaminoglycan structures. Drug-specific intracellular interleukin (IL)-5, interferon (IFN)-γ and IL-10 production was investigated by flow cytometry, while secretion of IL-5, IL-2 and IFN-γ was analysed by enzyme-linked immunosorbent assay. PBMC from individuals with hypersensitivity reactions to contaminated heparin secreted considerable amounts of IL-2 in vitro. There was a suggestion that ongoing heparin therapy and the Li-heparin in the vials may have an impact on the lymphocyte reactivity of PBMC. The in vitro lymphocyte reactivity pattern of PBMC from individuals with hypersensitivity reactions to contaminated heparins was neither typical for an immune-mediated nor for a nonimmune-mediated reaction. Possible effects of heparins in the test system itself may require consideration.

HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4+ T Cell Responses

BackgroundIdentification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines.Methodology/Principal FindingsIn the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNγ ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4+ or CD8+ T cells prior to the ELISPOT culture revealed that effectors are either CD4+ (the majority of reactivities) or CD8+ T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4+ T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay.Conclusions/SignificanceHLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4+ T cell responses restricted by HLA-II molecules.

The lipophilic hapten parthenolide induces interferon-γ and interleukin-13 production by peripheral blood-derived CD8+ T cells from contact allergic subjects in vitro

Peripheral blood mononuclear cells (PBMC) from persons with contact allergy to nickel react in vitro predominantly with nickel-induced CD4+ T cell-mediated production of both T-cell type 1 and 2 cytokines. The aim of the present study was to investigate if the contact allergen parthenolide, a sesquiterpene lactone of lipophilic character, elicits an immune response which differs from that induced by water-soluble nickel ions. Ten allergic subjects with strong (n = 6), moderate (n = 2), or weak (n = 2) patch-test reactivity to parthenolide and five patch test-negative control subjects participated in the study. PBMC from the subjects were analysed for in vitro reactivity with parthenolide by an enzyme-linked immunospot (ELISpot) assay, measuring cytokine production at the single-cell level. The allergic group, but not the control group, responded to parthenolide with increased numbers of cells producing interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5 (P < 0.05 for all) and IL-13 (P < 0.01). The responses manifested by T-cell type 1 (IFN-gamma and IL-2) and type 2 (IL-4, IL-5 and IL-13) cytokines were positively correlated between cytokines. Subjects with a strong or moderate, but not weak or negative, patch-test reaction displayed detectable in vitro responses. In contrast to the CD4+ T cell-mediated peripheral reactivity induced by nickel, cell depletion experiments identified the parthenolide-reactive IFN-gamma- and IL-13-producing cells as CD8+ T cells. The finding that the PBMC reactivity to parthenolide in humans involves a CD8+ T cell-mediated type 1 and 2 cytokine response warrants further studies on the relationship between the chemical nature of a hapten and the resulting immune response.

T helper cell-mediated interferon-gamma expression after human parvovirus B19 infection: persisting VP2-specific and transient VP1u-specific activity

Human parvovirus B19 is a small non-enveloped DNA virus with an icosahedral capsid consisting of proteins of only two species, the major protein VP2 and the minor protein VP1. VP2 is contained within VP1, which has an additional unique portion (VP1u) of 227 amino acids. We determined the ability of eukaryotically expressed parvovirus B19 virus-like particles consisting of VP1 and VP2 in the ratio recommended for vaccine use, or of VP2 alone, to stimulate, in an HLA class II restricted manner, peripheral blood mononuclear cells (PBMC) to proliferate and to secrete interferon gamma (IFN-gamma) and interleukin (IL)-10 cytokines among recently and remotely B19 infected subjects. PBMC reactivity with VP1u was determined specifically with a prokaryotically expressed VP1u antigen. In general, B19-specific IFN-gamma responses were stronger than IL-10 responses in both recent and remote infection; however, IL-10 responses were readily detectable among both groups, with the exception of patients with relapsed or persisting symptoms who showed strikingly low IL-10 responses. Whereas VP1u-specific IFN-gamma responses were very strong among the recently infected subjects, the VP1u-specific IFN-gamma and IL-10 responses were virtually absent among the remotely infected subjects. The disappearance of VP1u-specific IFN-gamma expression is surprising, as B-cell immunity against VP1u is well maintained.

An experimental model for the determination of immunomodulating effects by volatile compounds

An in vitro exposure system was developed to enable simultaneous exposure of primary cells or cell lines to defined concentrations of volatile organic compounds (VOC) without the necessity of a constant-flow exposure system. Toluene was used as model VOC and administered via the gas phase. CD3/CD28-stimulated human peripheral blood mononuclear cells (PBMC) were used as indicator cells. Vitality/proliferation of PBMC was tested using the MTT assay and their functional reactivity using cytokine ELISA for interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-13, and tumor-necrosis-factor-α (TNF-α). Chemical analysis using headspace gas chromatography confirmed that this new method guaranties reproducible VOC exposure ( R 2 = 0.995 for the correlation between external toluene concentration and toluene in the cell culture). While cytotoxic effects were not observed, dose-dependent toluene effects on functional reactivity of PBMC were found. The secretion of IFN-γ, IL-4, and IL-13 was inhibited at concentrations of 72.5 g/m 3 and above, whereas the TNF-α production was increased. Since the presented in vitro model ensures toluene exposure in concentrations comparable to the real situation, and allows the investigation of dose-dependent immunomodulatory toluene effects in concentrations without cytotoxicity, this method first described here is introduced as useful tool in analysis of VOC-triggered effects on immune cells.

Immunoregulation in onchocerciasis: predominance of Th1-type responsiveness to low molecular weight antigens of Onchocerca volvulus in exposed individuals without microfilaridermia and clinical disease.

Chronic and generalized onchocerciasis is associated with suppression of the parasite-specific cellular responsiveness, while exposed individuals without parasitological and clinical evidence of infection (endemic normals) display prominent cellular reactivity to Onchocerca volvulus antigens (OvAg). In order to identify those parasite antigens which may account for this differential cellular responsiveness, total adult worm-derived OvAg were fractionated by means of preparative SDS-PAGE and blot-elution into 22 antigen fractions of continuously decreasing molecular weight. Peripheral blood mononuclear cells (PBMC) from microfilariae (mf)-positive onchocerciasis patients (n = 18) proliferated weakly in response to all OvAg fractions. In contrast, in vitro reactivity of PBMC from endemic normals (n = 9) was depressed in response to OvAg of mol. wt 200-30 kD only, while antigens of mol. wt < 30 kD induced vigorous proliferation in these individuals compared with the microfilaridermic patients (P < 0.05). Highest proliferative reactivity of cells from endemic normals was observed in response to OvAg of mol. wt 15-11 kD. Furthermore, these low mol. wt antigen fractions induced substantial production of IL-2 and interferon-gamma (IFN-gamma) in PBMC from endemic normals, but not in those from onchocerciasis patients. Cells from individuals of both groups secreted similar amounts of IL-5 in response to all OvAg fractions, with highest production again being induced by low mol. wt OvAg. In contrast, PBMC from onchocerciasis patients clearly produced more IL-10 than did cells from endemic normals. This augmented IL-10 production by PBMC from mf-positive individuals was not only observed after stimulation with OvAg fractions, but was measured in unstimulated control cultures as well. IFN-gamma-specific mRNA in antigen-stimulated PBMC from endemic normals appeared to be more prominent than in cells from onchocerciasis patients. However, mRNA transcripts of IL-10 and IL-13 were clearly present in patients, but were absent or inconsistently observed in endemic normals. Our results suggest that vigorous Th1-type cellular responsiveness encountered in endemic normals is restricted to low mol. wt antigens of O. volvulus, while such reactivity will not be present in mf-positive individuals. Furthermore, spontaneous production of high levels of IL-10 in onchocerciasis patients is likely to suppress Th1-type immunity, and thus may favour manifestation of chronic onchocerciasis. These traits of cellular immunity may contribute to the differential outcome of O. volvulus infection, the manifestation of clinical disease, and may also regulate the build up of acquired immunity in humans.

Reactions of peripheral blood mononuclear cells (PBMC) of camels with monoclonal antibodies against ruminant leukocytes

The particular immune system of the camel has been but little investigated. In this work circulating camel peripheral blood mononuclear cells (PBMC) were studied by flow cytometry. Monoclonal antibodies (mAbs) raised against ruminant leukocytes were used for the detection of cell surface antigens. Monoclonals to T-cell markers, CD4 (CACT138A) and CD8 (CACT80C), exhibited no reactivity towards camel PBMC in contrast to their reactivity to PBMC of other ruminant species and those of cattle in particular. A relatively high percentage (29.1±8.9%) of camel PBMC reacted with a non-immunoglobulin cell surface marker, B-B2, comparable to the reactivity of bovine PBMC. The B-B7 cell marker revealed 22.4±10.0% of reactive camel PBMC while the CD45 leukocyte common antigen was identified only on 19.4±3.1% of camel PBMC as compared to 74.7±4.9% for bovine PBMC. IgM (PIg45A) was detected on 9.1±1.4% of camel PBMC and on 46.6±19.5% of the bovine PBMC. Double fluorescent labeling with two B-cell markers and an anti-ruminant λ light-chain mAb revealed 7–9% of cells bearing both B and λ L-chain markers. Light chain reactivity was also assessed using an anti-goat F(ab′) 2 antiserum. The values obtained, 14.3±5.8% for the camel and 47.8±2.7% for the cattle, are close to the values observed for surface IgM. These data suggest that camels, like other ruminants, possess L-chain bearing cells of the B-cell lineage. However, in the camel, Igs are different in that in addition to regular four chain Igs, about 65% of them possess two heavy chain Igs devoid of light chains. Because different sets of V H gene segments are used by four and two chain Igs, it is possible that there might be two lineages of B-cells each secreting a different form of antibodies.

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

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. 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. 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). 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.

The relationship between peripheral T cell reactivity to insulin, clinical remissions and cytokine production in type 1 (insulin-dependent) diabetes mellitus.

Antigenic proliferative responses of peripheral blood mononuclear cells (PBMC) to insulin were studied in 44 type 1 new-onset diabetic subjects. Of them, 14 (32%) had a stimulation index (> or =3) above the mean + 3 SD of 39 healthy controls and of 7 of 15 (47%) diabetic patients of long duration (P = 0.001). Responses to insulin were not dictated by specific major histocompatibility complex class II association and were not observed in normal subjects with diabetes-associated human leukocyte antigen-DR/DQ alleles. Whereas no relation of PBMC reactivity with insulin autoantibodies was found, there was a positive correlation with the presence of at least one of the four autoantibodies tested and with IA-2 antibody. An interesting finding was that the proportion of patients with subsequent low insulin requirement, up to 24 months, was significantly higher in patients who showed PBMC reactivity to insulin (8 of 8) than in those who did not (10 of 24, 42%; P = 0.004). The former had a higher mean stimulation index than the latter (3.3+/-2.6 vs. 1.5+/-0.6; P = 0.006). Furthermore, interleukin-4 (IL-4) production was lower in type 1 diabetic patients who proliferated to insulin than in those who did not (23+/-15 vs. 64+/-47 pg/mL; P = 0.04), but interferon-gamma, IL-2, and IL-10 productions were similar. In conclusion, these results suggest that proliferation to insulin may reflect the presence of an higher residual beta-cell mass.

Disease Activity Related Catecholamine Response of Lymphocytes from Patients with Rheumatoid Arthritis

In patients with chronic rheumatic diseases, a decreased density of beta 2-adrenergic receptors (beta 2R) on peripheral blood mononuclear cells (PBMC) could be demonstrated negatively correlating with various disease activity parameters. The aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 6 healthy blood donors (HD) were investigated. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with phytohemagglutinin (PHA) and monoclonal anti-CD3-antibodies (OKT3), respectively. The results revealed that lymphocytes of patients with RA showed a significantly reduced influence of catecholamines on PBMC function. In RA patients with high disease activity only, a shift to alpha 1-adrenergic-mediated catecholamine effects upon PBMC reactivity could be observed. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that is mediated via alpha- and beta-adrenergic receptors due to disease activity. In this respect the altered catecholamine response of PBMC from patients with RA may contribute to the pathogenic process of RA.

Gag protein epitopes recognized by CD4(+) T-helper lymphocytes from equine infectious anemia virus-infected carrier horses.

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.

Reduced catecholarnine response of lymphocytes from patients with rheumatoid arthritis

Catecholamines modulate lymphocyte function via stimulation of β2-adrenergic receptors (β2R). Previous investigations revealed a decreased density of β2R on peripheral blood mononuclear cells (PBMC) in patients with chronic rheumatic diseases. Aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 12 healthy blood donors (HD) were investigated. 㬢R were determined by a radioligand binding assay. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with pokeweed mitogen (PWM) and monoclonal anti- CD3-antibodies (OKT3), respectively. In parallel, α 1- or β-receptor antagonist were added to the culture to determine the specificity of the catecholaminergic effects. The results showed that depending on the stimulus and the catecholamine concentration employed E and NE exert inhibitory (OKT3) or stimulatory signals (PWM) on lymphocyte proliferation. Inhibitory effects could be abolished by adding β-antagonist, while stimulatory signals were diminished after addition of α 1- of β-antagonist. Patients with RA showed a significantly reduced density of β2R compared to HD paralleled by a significantly reduced influence of catecholamines on lymphocyte function. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that are mediated via α 1- and β-adrenergic receptors. In this respect the reduced catecholamine response of PBMC from RA patients may contribute to the pathogenic process of RA.

Role of immune complexes from patients with different clinical forms of schistosomiasis in the modulation of in vitro granuloma research.

Schistosomiasis is a disease whose pathology is strongly related to the granulomatous reaction formed around parasite eggs trapped in host tissues. Studies have shown that the chronic intestinal form (INT) of this infection is associated with a variety of immunoregulatory mechanisms which lead to a diminished granulomatous reaction. Using an in vitro model of granuloma reaction, we show that immune complexes (IC) isolated from sera of INT patients are able to reduce granulomatous reaction developed by peripheral blood mononuclear cells (PBMC) from acute (AC), INT and hepatosplenic (HE) patients to soluble egg antigen (SEA)-conjugated polyacrylamide beads (PB-SEA). This inhibitory activity is also observed in cell proliferation assay of PBMC from INT and HE patients stimulated with SEA and adult worm antigen (SWAP). Furthermore, IC isolated from sera of patients with different clinical forms of the disease are also able to suppress INT patients PBMC reactivity. Therefore, our results show that circulating IC present in sera of patients with different clinical forms of schistosomiasis may downregulate PBMC reactivity to parasite antigens resulting in a diminished granuloma reaction to parasite eggs.

IL-10 plays a role in the modulation of human granulomatous hypersensitivity against Schistosoma mansoni eggs induced by immune complexes.

It has been demonstrated that the chronic intestinal form of schistosomiasis is associated with the establishment and maintenance of a variety of immunoregulatory mechanisms that lead to a diminished granulomatous reaction to parasite eggs. Using an in vitro model of granuloma reaction we showed that immune complexes (IC) isolated from the sera of chronic intestinal schistosomiasis patients were able to reduce the granulomatous hypersensitivity (developed by peripheral blood mononuclear cells (PBMC) from schistosomiasis patients) to soluble egg antigen (SEA)-conjugated polyacrylamide beads (PB-SEA). This inhibitory activity, mediated by IC, was also observed in the proliferative response of PBMC stimulated with SEA and soluble worm antigen preparation (SWAP). Furthermore, we observed a decrease in TNF-alpha and an increase in IL-10 production by PBMC treated with IC in an in vitro granuloma reaction. This phenomenon was also seen in a cell proliferation assay when PBMC were treated with IC and stimulated with S. mansoni antigens. These results demonstrate that circulating IC may down-regulate PBMC reactivity to S. mansoni antigens by changing the cytokine pattern produced by these cells.