Levodopa therapy reduces DNA damage in peripheral blood cells of patients with Parkinson’s disease

Oxidative stress seems to play a major role in the pathogenesis of neurodegeneration. In Parkinson’s disease (PD) patients, the dopaminergic neurons are subjected to oxidative stress resulting from reduced levels of antioxidant defenses such as glutathione and high amount of intracellular iron. Levodopa (LD) is widely used for the symptomatic treatment of PD, but its role in oxidative damage control is still unclear. The aim of this study was to analyze the presence of DNA damage in peripheral blood lymphocytes (PBL) of PD patients, during a washout and a controlled LD dosage and to evaluate the oxidative damage fluctuation after LD intake. The standard and the Fpg-modified version of Comet assay were applied in analyzing DNA damage in PBL from blood samples of nine PD patients and nine matched controls. Due to the limited number of patients we cannot reach definite conclusions even if our data confirm the accumulation of DNA lesions in PD patients; these lesions decrease after LD intake.     

Cord blood leptin levels of healthy neonates are associated with IFN-γ production by cord blood T-cells

Leptin is a hormone synthesized by adipocytes and other tissues, including the placenta, and it regulates food intake and energy expenditure, reproductive and immune functions. To investigate the role of leptin in neonatal immunity, we measured serum leptin and cytokine (IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-12) levels in the cord blood (cb) of 510 healthy neonates, 14 small for gestational age (SGA), 312 appropriately grown for gestational age (AGA) and 184 large for gestational age (LGA). Median serum leptin concentration in the whole sample was 11 ng/ml. In 11.2% neonates (1 SGA, 32 AGA, 24 LGA), leptin levels were >90th percentile (median 39 ng/ml). In 33.3% of those (3.72% of total sample) with the highest leptin levels (median 46 ng/ml), significantly elevated levels of serum IFN-γ were also found (mean 27.11 pg/ml, range 17.5-38.5 pg/ml). In neonates with leptin levels ～50th percentile (median 12 ng/ml) or <10th percentile (median 1 ng/ml), serum IFN-γ levels were negligible. All other cytokines measured, were < the assays' detection limits. To investigate whether leptin can independently influence cytokine gene expression by cb T-cells and monocytes (Mc), we cultured cb T-cells or Mc, isolated from randomly selected AGA neonates or adult peripheral blood, with leptin. This resulted in upregulation of IL-2, IFN-γ and IL-4 gene expression in cb and adult T-cells and IL-10 expression mainly in cb-Mc. Significantly higher expression of IFN-γ occurred in female cb-T-cells cultured with leptin, compared with male cb-T-cells. In conclusion, the concurrent presence of high concentrations in both leptin and IFN-γ in cb of healthy infants, and leptin's ability to directly upregulate cytokine gene expression in cb T and Mc cells, indicate that abnormally high leptin levels can independently influence the immune system of healthy newborns, and may mediate gender differences in the development of a Th1 polarized immune response.     

Production of interleukin-1β by periodic acid-oxidized human peripheral blood mononuclear cells

Interleukin-1 (IL-1) production by periodic acid (H₅IO₆)-oxidized human peripheral blood mononuclear (PBMN) cells was assessed by the thymocyte co-mitogenesis assay. Maximum IL-1 levels ( 1.2 U/ml) in the conditioned media of PBMN cells were registered within the first 24 hrs post-oxidation, whereas no IL-1 was detected in the media from 24 hrs control cultures. Thymocyte proliferation, driven by periodic acid-induced IL-1, was abolished by an antibody to IL-1alpha and IL-1. Quantitative analysis of IL-1-containing medium by radioimmunoassay (RIA) indicated that IL-1 comprised about 80% of total IL-1. Partial characterization of H₅IO₆-induced IL-1 indicated that it was identical to IL-1 produced by lipopolysaccharide-stimulated macrophages. It is concluded that oxidation of human PBMN cells by H₅IO₆ triggers synthesis and release of IL-1, most of which was in its IL-1 form.     

Impaired activation of IFN-γCD4 T cells in peripheral blood of patients with dilated cardiomyopathy

Viral persistence and autoantibodies are pathogenic components in patients with idiopathic dilated cardiomyopathy (DCM). The aim was to evaluate T-cell function in DCM using different flow cytometry based detection techniques. Following stimulation, the frequency of IFN-γ-producing CD4⁺ T cells was significantly lower in patients compared with controls. In contrast, the frequency of IL-4 producing CD4⁺ T cells was no different. In supernatants of cultured PBMC, IFN-γ and IL-10 were significantly lower in patients. In addition, lymphocyte proliferation was significantly lower in patients compared with controls, whereas major lymphocyte subsets were not different. IFN-γ and IL-10 are key cytokines in the ability to mount protective immune responses and to maintain self-tolerance. A reduced activation of T-helper 1 (IFN-γ producing) cells and a decreased capacity to produce IL-10, found in the present study, could explain parts of the autoimmune features seen in patients with DCM.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Immune complexes from rheumatoid arthritis synovial fluid induce FcγRIIa dependent and rheumatoid factor correlated production of tumour necrosis factor-α by peripheral blood mononuclear cells

Immune complexes (ICs) can induce production of cytokines by peripheral blood mononuclear cells via Fc receptors. Rheumatoid factor (RF) develop in response to ICs in many clinical and experimental settings. We investigated whether and how polyethylene glycol (PEG) precipitated ICs from rheumatoid arthritis (RA) sera and synovial fluid (SF) can influence cytokine production by peripheral blood mononuclear cells. We also examined the relationship between RF and IC induced cytokine production. Parallel sera and SF from 47 RA patients and sera from 15 healthy control individuals were PEG precipitated. The precipitates were added to serum-free peripheral blood mononuclear cell cultures and tumour necrosis factor (TNF)-α levels were measured after 20 hours. In separate cell culture experiments FcγRIIa and FcγRIII were blocked and monocytes were depleted or enriched. RF in serum was determined by nephelometry, and IgG levels in precipitates and anti-cyclic citrullinated peptide antibodies in serum were measured using ELISA. Clinical data were collected from the patients' charts. In two separate investigations, we demonstrated a correlation between RF, PEG-precipitated IgG levels and induction of the proinflammatory cytokine TNF-α by PEG-precipitated SF ICs. No such correlation was found for serum ICs. TNF-α levels induced by SF precipitates, but not serum precipitates, correlated with the number of swollen and tender joints. Monocytes/macrophages were shown to be the main responder cells, and blockade of FcγRIIa, but not blockade of FcγRIII, inhibited TNF-α production in cultures stimulated with precipitated ICs. Anti-cyclic citrullinated peptide correlated with RF but exhibited no association with IgG content in PEG precipitates or with precipitate-induced TNF-α levels. These findings support the hypothesis that SF ICs and correlated RF production are directly linked to cytokine-dependent inflammation in RA. Suppression of monocytes/macrophages in RA joints or blockade of the primate-specific activating FcγRIIa receptor might be ways to reduce IC-induced TNF-α production in the joints of seropositive RA patients.     

Association of reduced heme oxygenase-1 with excessive Toll-like receptor 4 expression in peripheral blood mononuclear cells in Behçet's disease

Introduction

Toll-like receptors (TLRs) mediate signaling that triggers activation of the innate immune system, whereas heme oxygenase (HO)-1 (an inducible heme-degrading enzyme that is induced by various stresses) suppresses inflammatory responses. We investigated the interaction between TLR and HO-1 in an inflammatory disorder, namely Behçet's disease.

Methods

Thirty-three patients with Behçet's disease and 30 healthy control individuals were included in the study. Expression levels of HO-1, TLR2 and TLR4 mRNA were semiquantitatively analyzed using a real-time PCR technique, and HO-1 protein level was determined by immunoblotting in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes. In some experiments, cells were stimulated with lipopolysaccharide or heat shock protein-60; these proteins are known to be ligands for TLR2 and 4.

Results

Levels of expression of HO-1 mRNA were significantly reduced in PBMCs from patients with active Behçet's disease, whereas those of TLR4, but not TLR2, were increased in PBMCs, regardless of disease activity. Moreover, HO-1 expression in PBMCs from patients with Behçet's disease was repressed in the presence of either lipopolysaccharide or heat shock protein-60.

Conclusion

The results suggest that upregulated TLR4 is associated with HO-1 reduction in PBMCs from patients with Behçet's disease, leading to augmented inflammatory responses.     

Platelet-activating factor changes in phospholipid extracts from the plasma,peripheral blood mononuclear cells and bone marrow mononuclear cells of patients with acute leukemia — A <Superscript>31</Superscript>P MRS <Emphasis Type="Italic">in vitro</Emphasis> study

The aim of this investigation was to evaluate the changes in PAF concentrations in the plasma, PBMC and BMMC of patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The plasma was from 23 healthy volunteers (HV) and 44 patients with AL (16 ALL, 28 AML). The PBMC were from 15 HV and 55 patients with AL (18 ALL, 37 AML), and the BMMC from 40 patients with AL (11 ALL, 29 AML). Methanol-chloroform phospholipid extraction from 60 × 10⁶ cells (PBMC or BMMC) was performed according to a modified version of Folch’s method. ³¹P MRS data was obtained on an AMX 300 Bruker spectrometer (7.05 T). The PAF concentration in the plasma of the patients with ALL or AML was lower than that for the healthy volunteers. The PAF concentration in the plasma of the patients with ALL did not differ significantly from that of the patients with AML. In the case of both the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers.     

Increased expression of Interleukin-18 receptor in blood cells of subjects with Mild Cognitive Impairment and Alzheimer’s disease

Inflammation has been proposed as a leading force in neurodegeneration and Interleukin (IL)-18 is a pro-inflammatory cytokine which is suggested to be implicated in Alzheimer’s disease (AD). However, the meaning of the IL-18 participation in this disease is still unclear.Since IL-18 activity is mediated by its heterodimeric receptor complex IL-18Rα/β, we evaluated the presence of both IL-18R chains on peripheral blood cells of AD patients, as well as in individuals with Mild Cognitive Impairment (MCI), at increased risk to develop AD. More specifically, we compared the levels of CD14⁺ monocytes and CD3⁺ T-lymphocytes bearing IL-18Rα and β chains in the two groups of patients with those in healthy control subjects, both before and after in vitro cell treatment with lipopolysaccharide (LPS).While no differences in the levels of monocytes and T-lymphocytes bearing IL-18Rα chain were found among the three groups, either in untreated and LPS-treated conditions, the IL-18Rβ chain expression appeared differently regulated in MCI and AD patients, as compared to controls. In particular, the amount of IL-18Rβ-bearing monocytes was similar among the three groups at unstimulated conditions, while after LPS treatment it was increased in MCI vs. controls. A significant increase of IL-18Rβ-bearing T-lymphocytes was also observed in MCI and AD vs. controls, both in untreated and LPS-stimulated conditions.Our findings indicate that the expression of IL-18R complex on blood cells is perturbed in AD and even more markedly in its preclinical state of MCI, confirming that an increased peripheral activity of IL-18 may be involved in the early phase of AD pathophysiology.     

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors.MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n = 150) and RA patients (n = 40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used.ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type.ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.     

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

Abnormal activation of the Akt-GSK3β signaling pathway in peripheral blood T cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease accompanied by the activation and proliferation of T cells and B cells. In this study, we found that the distributions of lymphocytes obtained from patients with SLE or SLE with renal disease (RSLE) were reduced in the G₀/G₁ phase and were elevated in the S phase after phytohemagglutinin treatment. Increased expression of CDK2 and decreased expression of cyclin-dependent kinase inhibitors p27^Kip1 and p21^WAF1/CIP1 were observed in RSLE and SLE lymphocytes. The phosphorylation levels of Akt473 and GSK3β (ser9) were increased in lymphocytes from the patients. Moreover, inhibition of GSK3β with lithium chloride or SB216763 induced T cell proliferation, and the most significant effects were observed in RSLE lymphocytes. These results indicate that upregulation of CDKs and downregulation of p27^Kip1 and p21^WAF1/CIP1 increased the proliferation of T lymphocytes in SLE patients. Abnormal activation of the Akt–GSK3β signaling pathway increased the proliferation of lupus lymphocytes.     

Leishmania major soluble exo-antigens (LmSEAgs) protect neonatal BALB/C mice from a subsequent challenge with L. major and stimulate cytokine production by Leishmania-naïve human peripheral blood mononuclear cells

The vaccine potential and immunogenicity of soluble Leishmania major exo-antigens (LmSEAgs), a potentially novel source for vaccine candidates for leishmaniasis, were evaluated in neonatal BALB/c mice and with human peripheral blood mononuclear cells. Vaccinated neonatal BALB/c mice resisted infection with L. major, and lymphoid cells from the mice proliferated when restimulation with LmSEAgs and produced interferon-gamma and some interleukin-4. In addition, LmSEAgs stimulated human peripheral blood mononuclear cells to produce large amounts of interferon-gamma and some interleukin-5. This finding suggests that LmSEAgs may be a vaccine candidate for leishmaniasis in humans.     

Εx-vivo effect of dexamethasone on cytokine production from whole blood of septic patients: Correlation with disease severity

Background: Controversial findings of former clinical trials on the effect of low dose hydrocortisone in patients with septic shock led to investigate the effect of corticosteroids on the production of cytokines from endotoxin (LPS)-stimulated whole blood. Methods: Whole blood from 33 septic patients was sampled within 24 h alter diagnosis. Hydrocortisone was not administered during follow-up. Whole blood was stimulated with 30 ng/ml of LPS in the presence of 0.01, 0.1, 1 and 10 μM of dexamethasone. Concentrations of cytokines and of sTREM-1 were estimated in supernatants after six hours of incubation. Results: Dexamethasone inhibited LPS-stimulated release of ΤΝFα, of IL-6, of IL-8 and of IL-10 in dose-dependent manner. A dual effect on the kinetics of release of IL-1β and of sTREM-1 was shown. Release of IL-1β was either decreased, what was connected with unfavorable outcome, or it was unaffected what was connected with a favorable outcome. Release of sTREM-1 was either increased, what was connected with unfavorable outcome, or it was decreased what was connected with a favorable outcome. Conclusions: Part of the beneficiary effect of corticosteroids in sepsis may be due to an effect on the release of IL-1β and of sTREM-1. This effect does not seem to be homogeneous for all septic patients.     

Apoptosis of peripheral blood lymphocytes in patients with <Emphasis Type="Italic">LRRK2</Emphasis>-associated Parkinson’s disease

Mutations in the Leucine Reach Repeat Kinase 2 (LRRK2) gene are the most frequent cause of familial Parkinson’s disease (PD). Although the precise physiological and pathological roles of LRRK2 are unclear, a direct link between mutant LRRK2 and programmed cell death (apoptosis) has been suggested. By using flow cytometry (PI+Annexin V(FITC)), we showed an increased level of spontaneous lymphocyte apoptosis in patients with LRRK2-associated PD compared to controls after 24 h (p < 0.016) and 48 h (p < 0.031) of incubation (5% CO₂, 37°C). We found an increased FAS mRNA level in peripheral blood lymphocytes of patients with LRRK2-associated PD compared to controls (p < 0.05) and to sporadic PD (sPD) (p < 0.002). A significant difference in FAS expression between patients with LRRK2-associated PD and controls remained after 3 years and was detected after 1 and 24 h during lymphocyte incubation (p < 0.03 and 0.05, respectively). Increased spontaneous lymphocyte apoptosis, along with increased FAS expression, in patients with LRRK2-associated PD suggests that LRRK2 mutations may lead to preferable activation of extrinsic apoptotic way.     

Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile–1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 μM for GSH and GSSG, respectively). Good linearity was reached in the 0.01–20 μM range for GSH and 0.05–20 μM for GSSG. The mean recoveries of GSH and GSSG were 98.5–100.6% and 105.8–111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.