Peripheral Blood Mononuclear Cells Of Healthy Controls Research Articles

Antigenemia, RNAemia, and innate immunity in children with acute rotavirus diarrhea

Antigenemia is commonly detected in children with acute rotavirus diarrhea, but the prevalence of viremia has not been clearly defined. We examined antigenemia in plasma and RNAemia in peripheral blood mononuclear cells (PBMC) of children with acute diarrhea by EIA, RT-PCR, and Southern hybridization, using primers and a probe specific to rotavirus NSP4 gene. We detected the presence of rotavirus antigen in 33.3% and almost full-length NSP4 gene in 70.8% of the acute-phase plasma and PBMC, respectively. In contrast, antigenemia and RNAemia were detected in 0% and 4.2% of the convalescent-phase plasma and PBMC, respectively, which were similar to antigenemia (0%) and RNAemia (7.7%) in healthy controls. We demonstrated an increase in the proportions of activated myeloid dendritic cells (mDC) and activated plasmacytoid DC (pDC) in acute-phase PBMC of patients when compared to those in convalescent phase of patients and in PBMC of healthy controls. The activation of mDC peaked on days 2-4 after illness onset, and the activation of acute-phase pDC appeared to correlate with levels of antigenemia. High prevalence of NSP4 gene in acute-phase PBMC indicates possible rotavirus replication in white blood cells, and extraintestinal spread and the activation of DC may have implications for the prevention of rotavirus disease in children.

Activated FVII-Antithrombin Complex and Tissue Factor mRNA As Potential Markers of Severe Thrombo-Hemorrhagic Syndrome in APL Patients

Abstract 1195▪▪This icon denotes a clinically relevant abstract Introduction:Tissue Factor (TF) expressed by acute promyelocytic leukemia (APL) cells may have an important role in the pathogenesis of the life-threatening coagulopathy typical of the onset of this disease. In APL patients, the remission induction differentiating therapy with all-trans-retinoic acid (ATRA) downregulates TF expression by APL blasts and produces a simultaneous correction of the hypercoagulation and thrombo-hemorrhagic syndrome (THS). However the rate of early deaths due to the THS is still relevant in this disease. Therefore characterizing the coagulopathy and identifying predictive markers remain a critical issue. Aim: In this study, in newly diagnosed APL patients, we prospectively evaluated the rate of hemorrhage and thrombosis, in the first month after diagnosis, in relation to the plasma levels of activated factor VII-antithrombin complex (FVIIa-AT), a new parameter which may reflect the degree of TF exposure and activity. TF mRNA in the peripheral blood APL cells was also evaluated as well as the plasma levels of thrombin-antithrombin complex (TAT) and D-dimer, as additional coagulation markers. Methods: Fifty-four consecutive APL patients receiving ATRA+Idarubicin for the remission induction (GIMEMA protocol AIDA2000) were enrolled. Blood samples from 26 of these patients (F/M= 10/16) were obtained at the onset (T0) of the disease, and on days 7, 14 and 28 (T7, T14, T28) of remission induction therapy. Twenty-five healthy subjects acted as a control group. Plasma FVIIa-AT, TAT, and D-dimer were measured by ELISA. TF mRNA expression by peripheral blood mononuclear cells (PBMC) was evaluated by real time-polymerase chain reaction. Results: At the disease onset 8/54 patients had early major hemorrhages (15%), including 3 (5.5%) fatal intracranial bleeding and 5 non-fatal major bleeding (9.2%); 3/54 had thrombosis (1 fatal Budd-Chiari syndrome, and 2 non-fatal events). Two more patients developed deep vein thrombosis at 7 and 14 days of induction therapy (3.7%), respectively. Of the 26 patients included in the laboratory study, 3 had thrombosis at the diagnosis of APL. At T0, before starting therapy, the levels of FVIIa-AT as well as those of TAT and D-dimer were significantly higher in APL patients compared to controls (p<0.05). However, while the levels of FVIIa-AT complex were lower in patients with thrombosis than in those without (120±50 vs 332±75 ng/ml), differently the levels of TAT and D-dimer were not different between two groups, indicating that the FVIIa-AT is more sensitive to the consumption coagulopathy compared to the other thrombotic markers. During ATRA therapy the levels of FVIIa-AT were progressively decreased at T7 and T14, and dropped significantly at T28 (p<0.05 vs T0); TAT and D-dimer levels were also decreased starting from T7 to T28 (p<0.05 vs T0). In addition, in APL PBMC from 9 patients, the levels of TF mRNA, initially elevated compared to healthy control PBMC (p<0.05), were significantly decreased by 68% and 90%, at T7 and T28, respectively. Conclusions: These results confirm a significant rate of early deaths in APL due to the coagulopathy. FVIIa-AT complex levels paralleled TF mRNA expression and may therefore be a useful surrogate tool for the TF activity measurement in the peripheral blood. In addition, the study shows that, among hypercoagulation markers, which are overall elevated at the onset of APL, the ‘low' levels of plasma FVIIa-AT complex is the only parameter able to distinguish patients at highest risk of severe THS. Although the small size of the study did not allow to calculate the predictive value of this marker, FVIIA-AT complex may be a promising simple candidate biomarker to test for the predictive risk of lethal/severe THS in large prospective studies of APL patients. Disclosures:No relevant conflicts of interest to declare.

Low and dysregulated production of follistatin in immune cells of relapsing–remitting multiple sclerosis patients

One of the mechanisms known to play a key role in neuronal and oligodendroglial fate specification of neural stem cells (NSCs) is restriction of bone morphogenic proteins (BMP) signaling by BMP antagonists. Here, we demonstrate that follistatin mRNA and protein secreted levels in peripheral blood mononuclear cells (PBMCs) of relapsing–remitting multiple sclerosis (RR-MS) patients are significantly reduced compared to healthy controls (HC). We also observed a different profile of regulation mechanisms. Follistatin was similarly expressed and secreted by T lymphocytes and monocytes among the PBMCs of HC, and follistatin upregulation of HC was subjected to stimulation with both LPS and TNF-α. Among PBMCs of RR-MS patients, however, follistatin was found to be downregulated in their monocytes and unresponsive to stimulation with either LPS or TNF-α. Our results may shed some light on the mechanisms involved in remyelination failure in MS, which may be related to the inability of RR-MS patients' immune cells to provide a sufficient pro-neurogenic and oligodendrogenic niche, by expressing and secreting follistatin, in addition to the previously described noggin reduced expression. Our results indicate that the low expression of follistatin in immune cells of patients with RR-MS is a result of the altered immunoregulation of monocytes in these patients.

Role of IL-17 in the Th1 systemic defects in rheumatoid arthritis through selective IL-12Rβ2 inhibition

BackgroundPatients with rheumatoid arthritis (RA) have a systemic Th1 defect associated with inflammation.ObjectiveTo examine the hypothesis that interleukin 17 (IL-17) contributes to this defect.MethodsIL-17 effects on Th1 markers were examined...

Expression and subcellular localization of APOBEC3G in peripheral blood mononuclear cells and liver tissues of chronic HBV patients

To study the expression level and intracellular localization of APOBEC3G in peripheral blood mononuclear cells (PBMCs) and liver tissues of chronic HBV patients. The expression level and intracellular localization of APOBEC3G in PBMCs and liver tissues were detected using the western blot and confocal laser scanning microscope (CLSM). Western-blot showed that the expression level of APOBEC3G in PBMCs of healthy controls was very low. The relative expression levels of APOBEC3G in PBMC of patients with chronic hepatitis B, chronic severe hepatitis, liver cirrhosis, or liver cancer were 4.12+/-0.21, 4.07+/-0.28, 4.16+/-0.36 or 4.21+/-0.39 respectively, which were higher than that in the healthy controls. However, there was no significant difference in APOBEC3G expression among different chronic HBV patients (q = 0.931, 0.744, 1.675, 1.675, 2.606 or 0.931, respectively, all P values more than 0.05). In addition, there was no significant difference on APOBEC3G in liver tissues between chronic hepatitis B patients and hepatocellular carcinoma patients (4.40+/-0.34 vs 4.34+/-0.43, q = 0.588, P more than 0.05). CLSM indicated that the localization of APOBEC3G protein was in cytoplasm of PBMCs and hepatocytes. APOBEC3G is upregulated in the PBMCs of chronic hepatitis B patients.

Abnormalities at the level of E3 ubiquitin ligases in peripheral blood T cells from Systemic Lupus Erythematosus patients

Objective It is now unanimously accepted that autoimmunity could be defined as a breakdown of mechanisms responsible for self-tolerance. Among peripheral tolerance mechanisms, T cells anergy plays an important role. Previously, in patients with Systemic Lupus Erythematosus (SLE) it was demonstrated that peripheral T cells are resistant to anergy induction. Since some of E3 ubiquitin ligases (Cbl-b, GRAIL) are involved in T cells tolerance we initiated a study in order to establish the role of these ligases in SLE T cells anergy. Methods Our study included fifty eight SLE patients with active disease, forty nine with inactive disease and fifty healthy controls (HC). The patients were selected and characterized in Bucharest hospitals according to the American Rheumatism Association criteria. Cbl-b and GRAIL protein expression levels in peripheral blood mononuclear cells (PBMCs)/T cells were determined by western blotting using Cbl-b and GRAIL specific antibodies. The expression level of cbl-b mRNA in PBMCs/T cells was evaluated by RT-PCR. Cbl-b expression and distribution in T cells as well as its co-localization with lipid rafts was analyzed by confocal microscopy. Results Both Cbl-b and GRAIL protein expression levels were significantly reduced in SLE, especially in patients with active disease, than in HC PBMCs. Also, a reduced level of Cbl-b mRNA was identified in active and inactive SLE T cells by comparison with HC. Confocal microscopy analysis confirmed these results showing that in SLE T cells Cbl-b has a reduced and diffuse expression and did not co-localize with preformed large lipid raft domains. Conclusions Our results suggested that abnormal expression of some E3 ligases could be involved in enhanced response of SLE T cells even if these are stimulated under anergic conditions.

Dysfunction of Toll-Like Receptor-2, −4, and −6: Is There a Role in the Pathogenesis of Immunodeficiency in Multiple Myeloma?.

Infections are a major cause of death in multiple myeloma (MM). There is limited information for the possible role of innate immunity in the pathogenesis of immunodeficiency in MM. Innate immune detection of pathogens relies on specific classes of microbial sensors, such as Toll-like receptors (TLRs) that detect structural patterns, which do not exist in the host. The aim of this study was to evaluate the expression and function of TLRs in newly diagnosed patients with MM. According to our knowledge, such information is not available in the literature. Thirty-two MM patients at diagnosis (18M/14F; median age 69 years), 6 MGUS patients and 14 healthy, age- and gender-matched controls were studied. Seven patients had stage 1, 14 stage 2 and 11 stage 3 myeloma, according to ISS. After the collection of peripheral blood, mononuclear cells (PBMCs) were isolated by Ficoll centrifugation (Histopaque-1077; Sigma-Aldrich). These cells were measured for the expression of TLRs (antibodies from eBioscience) using fluorescence activated flow cytometry (FC 500, Beckman Coulter). In addition, 1x106 cells/ml were cultured in 5% FCS 1% pen/strep RPMI in the presence or absence of various TLR ligands and supernatants collected after 20h. These were examined for the presence of inflammatory cytokines (tumor necrosis-alpha, TNF-α; and interleukin-6, IL-6) by ELISA (Becton Dickinson). We found that although patients with MM express TLRs in PBMCs, their response to certain TLR ligands is defective when compared to healthy controls. TLR2, TLR4 and TLR6 of PBMCs of healthy controls reacted to the presence of their respective ligands PAM3CYS (gram positive and negative bacterial), LPS (gram negative) and FLT-1 (gram positive) producing TNF-α at a median value of 2.4 ng/mL (range: 1–3 ng/ml), while their action in patients with MM was significantly reduced (median value of TNF-α: 190 pg/ml; range 100–500 pg/ml; p<0.001). The reduced response of TLR4 in MM patients was independent of the LPS concentration used. On the contrary, TLR7 and TLR8 from MM reacted normally to their ligand R-848 (Imiquimob) to secrete high levels of TNF-α (median value for patients and controls: 4.5 and 4.2 ng/ml, respectively; p=NS). NOD1, another pattern recognition receptor that recognizes bacterial peptidoglycans also reacted normally in MM. Similar observations have been made for IL-6 expression. There was no difference in terms of TLRs function between MGUS patients and controls and between myeloma patients of different disease stages. In seven MM patients, cells were pre-treated with bortezomib for 30 min before addition of the TLR ligand. Bortezomib administration abrogated TLR 7/8 response in PBMCs even at very low concentrations (1nM). This study suggests that there is a significant defect in TLR function in MM, especially of these involved in immunity against bacterial infections. Thus the immune system fails to receive early priming signal, which may contribute to the increased rate of infections observed in MM. The restoration of function of TLRs to their normal levels has the potential to improve bacterial immunity in MM.

Immune responses of liver-infiltrating lymphocytes and peripheral blood mononuclear cells to hepatitis C virus core and NS3 antigens

Th1/Th2 cytokine balance is thought to play an important role in antiviral immunity and pathogenesis in viral infection. Ex vivo hepatitis C virus (HCV) antigen-specific T-cell responses were investigated. Using enzyme-linked immunospot assay, HCV core and NS3 antigen-specific interferon-gamma-, interleukin-4- and interleukin-10-secreting cells were enumerated in peripheral blood mononuclear cells (PBMCs) from 30 chronic hepatitis C patients and 16 healthy controls, and in liver-infiltrating lymphocytes (LILs) from 17 of the 30 patients. IFN-gamma- and IL-10-secreting cells in response to stimulation with HCV core and NS3 antigen were detectable in both PBMCs and LILs from patients with chronic hepatitis C, although frequencies of the cytokine-secreting cells were much higher in LILs than PBMCs. They were not detectable in PBMCs of healthy controls except for IL-10-secreting cells in response to HCV NS3 antigen stimulation. IL-4-secreting cells were hardly detectable in both PBMC and LIL in both the patients and the healthy controls. Frequencies of HCV NS3 antigen-specific IFN-gamma- and IL-10-secreting cells in PBMCs correlated with those in LILs (rho=0.599, p=0.044 and rho=0.716, p=0.004, respectively). These data provide further evidence of the immunomodulatory role of the CD4(+)CD25(+) regulatory T lymphocytes in chronic HCV infection.

Anti‐HBV effects of CpG oligodeoxynucleotide‐activated peripheral blood mononuclear cells from patients with chronic hepatitis B

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are known as a potent Th1-like immune enhancer in vertebrates. Chronic hepatitis B is the immunocompromising condition. We therefore investigated the effects of CpG ODN on cultured cells from chronic hepatitis B patients and healthy controls. The inhibitory effects of CpG ODN on hepatitis B virus (HBV) were also studied. The secretion of IFN-alpha by CpG ODN-activated peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B patients and healthy controls was significantly increased when compared with PBMCs alone or GpC ODN-stimulated PBMCs. After activation with CpG ODN, the IFN-alpha secretion by chronically HBV-infected patient PBMCs is less than that by healthy control PBMCs. Treatment of HepG2 2.2.15 cells with culture supernatants of PBMCs activated by CpG ODN can significantly suppress the secretion of HBsAg, HBeAg and HBV DNA as compared with that of PBMCs without CpG ODN activation under the same conditions. No inhibitory effect on the replication of HBV was found for CpG ODN treatment alone. Our results indicated that CpG ODN could efficiently enhance the immune response of chronic hepatitis B patients. Moreover, the CpG ODN-activated PBMCs from chronic hepatitis B patients were able to significantly inhibit HBV replication in vitro, suggesting that CpG ODN may be a potential immunoregulator against HBV infection in the future.

Coordinate overexpression of interferon‐α–induced genes in systemic lupus erythematosus

To study the contribution of interferon-alpha (IFNalpha) and IFNgamma to the IFN gene expression signature that has been observed in microarray screens of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE). Quantitative real-time polymerase chain reaction analysis of healthy control PBMCs was used to determine the relative induction of a panel of IFN-inducible genes (IFIGs) by IFNalpha and IFNgamma. PBMCs from 77 SLE patients were compared with those from 22 disease controls and 28 healthy donors for expression of IFIGs. Expression of IFNalpha-inducible genes was significantly higher in SLE PBMCs than in those from disease controls or healthy donors. The level of expression of all IFIGs in PBMCs from SLE patients with IFNalpha pathway activation correlated highly with the inherent responsiveness of those genes to IFNalpha, suggesting coordinate activation of that cytokine pathway. Expression of genes preferentially induced by IFNgamma was not significantly increased in SLE PBMCs compared with control PBMCs. IFNalpha-regulated gene-inducing activity was detected in some SLE plasma samples. The coordinate activation of IFNalpha-induced genes is a characteristic of PBMCs from many SLE patients, supporting the hypothesis that IFNalpha is the predominant stimulus for IFIG expression in lupus.

Expression of the markers BDCA-2 and BDCA-4 and production of interferon-alpha by plasmacytoid dendritic cells in systemic lupus erythematosus.

To study the expression of blood dendritic cell antigen 2 (BDCA-2) and BDCA-4 molecules by plasmacytoid dendritic cells (PDCs) in the blood of patients with systemic lupus erythematosus (SLE), and to study PDC production of interferon-alpha (IFN alpha) and its inhibition by anti-BDCA-2 and anti-BDCA-4 antibodies. Peripheral blood mononuclear cells (PBMCs) from SLE patients (SLE PBMCs) and from healthy controls were induced to produce IFN alpha in vitro by SLE serum containing an endogenous IFN alpha-inducing factor (SLE-IIF) or by herpes simplex virus type 1 (HSV-1). The frequencies and numbers of BDCA-2-, BDCA-3-, and BDCA-4-expressing cells were analyzed by flow cytometry, and the effects of anti-BDCA-2 and anti-BDCA-4 monoclonal antibodies (mAb) on IFN alpha production were investigated. IFN alpha production by SLE PBMCs induced by SLE-IIF or HSV-1 was decreased compared with that of healthy control PBMCs (P = 0.002 and P = 0.0007, respectively). The proportions of BDCA-2- and BDCA-3-expressing cells in SLE PBMCs were reduced compared with those in PBMCs from healthy controls (P = 0.01 and P = 0.004, respectively). IFN alpha producers in culture, especially among SLE PBMCs, displayed reduced BDCA-2 expression and constituted only a minority of the BDCA-2-positive cells, at least in healthy control PBMCs (median 18%). IFN alpha production by both SLE and healthy control PBMCs stimulated by SLE-IIF or HSV-1 was markedly reduced by anti-BDCA-2 mAb (median 81-98% inhibition). Anti-BDCA-4 mAb only partially inhibited SLE-IIF-induced IFN alpha production. SLE patients had a reduced number of BDCA-2-expressing PDCs, also termed natural IFN alpha-producing cells, and their IFN alpha production could be inhibited by anti-BDCA-2/4 mAb. Such mAb may be a therapeutic option for inhibiting the ongoing IFN alpha production in SLE patients.

Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome.

A 2',5'-oligoadenylate (2-5A)-dependent 37-kDa form of RNase L has been reported in extracts of peripheral blood mononuclear cells (PBMC) from individuals with chronic fatigue syndrome (CFS). In the current study, analytic gel permeation FPLC, azido photoaffinity labeling, two-dimensional (2-D) gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been used to examine the biochemical relationship between the 80-kDa RNase L in healthy control PBMC and the 37-kDa RNase L in PBMC from individuals with CFS. Like the 80-kDa RNase L, the 37-kDa RNase L is present as a catalytically inactive heterodimer complex with the RNase L inhibitor (RLI). Formation of a 37-kDa RNase L-RLI complex indicates that the 37-kDa RNase L is structurally similar to the 80-kDa RNase L at the N-terminus, which contains the 2-5A binding domain. The enzymatically active monomer form of 37-kDa RNase L resolved by 2-D gel electrophoresis has a pI of 6.1. RT-PCR and Southern blot analyses demonstrated that the 37-kDa RNase L is not formed by alternative splicing. In-gel tryptic digestion of the 37-kDa RNase L that was excised from 2-D gels and subsequent MALDI-MS analysis identified three peptide masses that are identical to three predicted peptide masses in the 80-kDa RNase L. The electrophoretic mobility of 2-5A azido photolabeled/immunoprecipitated 37-kDa RNase L was the same under reducing and nonreducing conditions. The results presented show that the 37-kDa form of RNase L in PBMC shares structural and functional features with the native 80-kDa RNase L, in particular in the 2-5A binding and catalytic domains.

Shift from Th2 to Th1 response in immunotherapy with venoms.

Cytokines released from T lymphocytes regulate antibody production by B lymphocytes and releasability of mast cells and basophils. Immune tolerance in specific allergen (Ag) immunotherapy (SIT) might be a consequence of decreased Th2 or increased Th1 response of Ag specific T lymphocytes. We compared function of T lymphocytes in 11 patients with Hymenoptera venom allergy and in 7 healthy volunteers. We followed function of T lymphocytes in 6 patients during SIT. We measured released IL-2, IFN-γ, IL-4 and IL-5 from Ag and mitogens (either with phorbol-miristate-acetate (PMA) and ionomycine or anti-CD3 antibodies) stimulated peripheral blood mononuclear cells (PBMC). After stimulation with Ag PBMC of patients released 86 ± 133 pg/ml IFN-γ and 19.7 ± 20.1 pg/ml IL-4 (Th2 response), PBMCs of healthy controls released 184 ± 116 pg/ml IFN-γ and 4.8 ± 8 pg/ml IL-4 (Th1 response). Spontaneous release of IFN-γ in cultures of PBMC of patients increased after rush SIT (before: 25 ± 31 pg/ml, after 241 ±281 pg/ml, p<0.05). After 6 months of SIT response of PBMCs of patients became similar to response of PBMCs of healthy controls. Time pattern of cytokines secreted from PBMCs after stimulation with PMA and ionomycine was different than after stimulation through T-cell receptor/CD3 complex.

Endotoxin inhibits heat shock protein 70 (HSP70) expression in peripheral blood mononuclear cells of patients with severe sepsis.

To investigate the ex vivo endotoxin-inducible heat shock protein 70 (HSP70) expression in the peripheral blood mononuclear cells (PBMC) of patients with severe sepsis in order to assess the capacity of this potentially protective response during systemic inflammation. Prospective observational study in consecutive patients with severe sepsis and healthy blood donors. Surgical intensive care unit in a university hospital. Eleven patients with the diagnosis of severe sepsis, one patient who had recovered from severe sepsis and 13 healthy blood donors. None. We studied the inducibility of HSP70 expression in the PBMC of patients with severe sepsis and healthy blood donors ex vivo. Human whole blood was incubated with variable lipopolysaccharide (LPS from Salmonella minnesota Re 595) concentrations (0; 0.1; 10; 100 ng/ml) for different periods of time (0.5; 2; 4; 10 h). The PBMC were separated by Ficoll density gradient and then disrupted by hypotonic lysis. HSP70 was measured by means of enzyme-linked immunosorbent assay (ELISA). We found a LPS dose- and time-dependent inhibition of ex vivo HSP70 expression in the PBMC of both patients with severe sepsis and healthy individuals. However, the levels of HSP70 expression in patients were significantly lower compared to those of healthy individuals at all LPS concentrations and incubation times. On average, HSP70 expression in the PBMC of healthy controls was 2.8 (range 1.2-3.9) times higher than in patients. HSP70 expression was inducible by thermal heat shock in the PBMC of both patients and healthy individuals. Endotoxin inhibits HSP70 expression in PBMC ex vivo. In vivo, the suppression of HSP70 expression induced by endotoxin and high levels of proinflammatory cytokines may contribute to the cellular dysfunction of immunocompetent cells concerning antigen presentation, phagocytosis and antibody production associated with decreased resistance to infectious insults during severe sepsis.

Patients with Systemic Lupus Erythematosus have Reduced Numbers of Circulating Natural Interferon-α- Producing Cells

Systemic lupus erythematosus (SLE) patients often have continuous production of interferon-α (IFN-α), but production of in vitro IFN-α by peripheral blood mononuclear cells (PBMC) may be varyingly reduced. We here report that IFN-α production induced by Herpes simplex virus (HSV) in PBMC resembling immature dendritic cells and designated natural IFN-α producing cells (NIPC), was much more affected than that induced by sendai virus (SV) in monocytes. At the cell level, the frequency of HSV-activated NIPC was reduced 70-fold, but residual NIPC produced normal amounts of IFN-α (1–2U/cell). The NIPC frequency increased 10-fold in SLE-PBMC, but not in control PBMC, when co-stimulated by the combination IFN-α-γ and GM- CSF. No spontaneous IFN-α production by PBMCs was detected in SLE patients. While no SLE serum factor inhibiting IFN-α production was seen, sera of four out of 11 SLE patients induced IFN-α production in healthy control PBMC. We propose that the number of NIPC in SLE are reduced in blood because of recruitment to tissues and activation by an endogenous IFN-α inducer, as well as because of lack of co-stimulatory cytokines. IFN-α produced in SLE could be of pathogenic significance, because autoimmune diseases develop in patients with infections or tumours during IFN-α therapy.

Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome.

Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.

T-Lymphocyte cytokine mRNA expression in cystic echinococcosis

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Effect of interferon-alpha on immunoglobulin production by peripheral blood mononuclear cells in multiple myeloma.

To test a hypothesis that interferon-alpha (IFN) treatment might restore normal immunoglobulin (Ig) production in multiple myeloma (MM), the effect of IFN on Ig isotype (IgG and IgA) production by peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) in MM patients was analyzed by ELISA. IFN at a concentration of 1000 U/ml was found to enhance IgA production by PB MNCs in IgA-MM and had a trend to stimulate IgG production in IgG-MM. The effect of IFN on nonparaprotein Ig isotype production was more variable, with mostly neutral or inhibitory effects being seen in both the MM subtypes. In contrast to the influences observed in MM patients, IFN at the same concentration inhibited both IgG and IgA production by PB MNCs in healthy controls. In studying BM cells, IFN was found to reduce IgA production in IgA-MM, but had a neutral effect on IgG production in IgG-MM. In the controls, the production of both the IgG and the IgA isotypes by BM MNCs was decreased by IFN. On the basis of these results it seems that the disease itself somehow affects the Ig-producing cells in MM, when measured as different responses of the cells to exogenous IFN in vitro. The results do not support the hypothesis that IFN treatment could restore normal Ig production in MM patients.

Enhanced production and gene expression of interleukin-5 in patients with bronchial asthma: possible management of atopic diseases by down-regulation of interleukin-5 gene transcription.

Interleukin (IL)-5 was produced in vitro by peripheral blood mononuclear cells (PBMCs) of mite-sensitive atopic patients upon challenge with specific allergen, while PBMCs of healthy controls produced essentially no IL-5. Stimuli delivered by the combination of phorbol ester (PMA) and CA2+ ionophore (ionomycin) induced marked IL-5 production by PBMCs obtained from atopic as well as nonatopic asthmatics. CD2- or CD4-bearing-cell depletion almost completely removed IL-5 production and gene transcription while CD8 depletion did not. These findings indicated that CD4+ T cells are the principal source of IL-5 in asthmatic PBMCs. The capacity of PBMCs of atopic asthmatics, nonatopic asthmatics and healthy controls to produce IL-2, IL-3, IL-4, interferon-gamma and granulocyte/macrophage-colony-stimulating factor was comparable among the three groups. FK506 suppressed IL-5 production and gene expression in vitro in a dose-dependent manner.

Interferon Production in Patients Infected with HIV-1

The production of interferon (IFN) from cultured peripheral blood mononuclear cells (PBMC) after virus or mitogen stimulation was evaluated in 141 patients positive for antibodies to human immunodeficiency virus type 1 (HIV-1). IFN-alpha production by PBMC of patients at Centers for Disease Control (CDC) stage II (Walter Reed [WR] 1) was comparable to that produced by PBMC of healthy controls. However, cells of patients at early CDC stage III (WR 2) produced significantly lower titers of IFN-alpha (P less than .001), and IFN-alpha was almost absent at CDC stage IV (WR 6) (P less than .001). IFN-gamma production was altered in patients at late CDC stage III (WR 4-5) and CDC IV (WR 6). A strong correlation between the disappearance of antibodies to core proteins and low IFN-alpha level was observed. IFN-alpha levels were significantly diminished in patients positive for HIV antigen. Reduced IFN-alpha production paralleled the HIV-1-related depletion of CD4+ lymphocytes and might serve as an additional parameter in defining the stage of HIV-1 infection.