Taurine transporter gene expression in peripheral mononuclear blood cells of type 2 diabetic patients

Taurine acts as antioxidant, cell osmolyte, modulator of glucose metabolism, and plays a role in the retinal function. It is 10³-fold more concentrated in the intracellular than in the extracellular milieu due to a specific taurine-Na-dependent transporter (TauT), which is upregulated by hypertonicity, low extracellular taurine, or oxidative stress and acutely downregulated ‘in vitro’ by high glucose concentrations. Aim of this study was to investigate whether TauT expression was modified in mononuclear peripheral blood cells (MPC) of type 2 diabetic patients with or without micro/macrovascular complications. Plasma taurine, as well as other sulphur-containing aminoacids (assayed by HPLC) and TauT gene expression (assayed by real-time PCR analysis) were measured in MPC of 45 controls and of 81 age-and-sex matched type 2 diabetic patients with or without micro/macrovascular complications. Median value (interquartile range) of plasma taurine was significantly lower in diabetic patients than in controls [28.7 (13.7) μmol/l vs. 46.5 (20.3) μmol/l; P?<?0.05], while median TauT expression, in arbitrary units, was significantly higher in diabetics than in controls [3.8 (3.9) vs. 1 (1.3); P?<?0.05) and was related to HbA1c only in controls (r?=?0.34; P?<?0.05). Patients with retinopathy (n?=?25) had lower TauT expression than those who were unaffected [3.1 (2.8) vs. 4.1 (3.4); P?<?0.05], while persistent micro/macroalbuminuria was associated with unchanged TauT expression. A trend toward reduction in TauT expression was observed in patients with macroangiopathy [n?=?27; 3.3 (2.5) vs. 4 [3.7]; P?=?NS]. In conclusion, TauT gene is overexpressed in MPC of type 2 diabetic patients, while presence of retinopathy is specifically associated with a drop in TauT overexpression, suggesting its possible involvement in this microangiopathic lesion.     

Apoptosis and oxidative status in peripheral blood mononuclear cells of diabetic patients

We have compared the concentrations of intracellular glutathione (GSH), glutathione-dependent antioxidative enzymes, the cell death rate and immunophenotype profile of peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with insulin-dependent type I (IDDM) or non insulin-dependent type II (NIDDM) diabetes mellitus. The IDDM and NIDDM patients had above-normal absolute lymphocyte counts, whereas the percentages of CD3, CD4 and CD8 T lymphocytes were significantly reduced. In contrast, the absolute number and percentage of B lymphocytes was higher in diabetic patients than in healthy donors. The low intracellular reduced glutathione (GSH) and the unbalanced profile of key enzymes involved in GSH metabolism, gamma glutamyltransferase (-GT) and glutathione-S-transferase (GST), account for the increased oxidative status of PBMC from diabetic patients. The plasma membranes of PBMC from diabetic patients were less permeable to propidium iodide than those of PBMC from healthy donors, indicating that the apoptotic cell death rate was lower in the cells from diabetic patients. These differences are potentially useful markers of pathogenic metabolic changes which occur during clinical diabetes and if they are confirmed could be used to identify the onset of diabetes.     

Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD₆₅ or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-γ and TNF-α) and chemokines (IP-10, MCP-1, MIP-1α, MIP-1β and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-β mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-γ and MCP-1, and mRNA expression of FOXP3 and TGF-β, was detected after cryopreservation. Stimulation with GAD₆₅ induced higher levels of IL-6, IFN-γ, TNF-α and MIP-1α, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.     

High expression of tumor necrosis factor alpha receptors in peripheral blood mononuclear cells of obese type 2 diabetic women

The tumor necrosis factor system plays an important role in the pathogenesis of obesity and type 2 diabetes (DM), by a complex and only partially understood mechanism. In this study we analyze the mRNA expression levels of TNFalpha and its receptors (TNFR1 and TNFR2), in peripheral blood mononuclear cells (PBMC) from eleven, non-morbid, obese and 14, obese, type 2 DM women, by real-time quantitative PCR. We show an increase in the TNFR2 to TNFR1 ratio (mTNFR2/mTNFR1) in type 2 DM (r = 0.63; p = 0.021, after adjusting for age). Likewise, a positive correlation between mTNFR2/mTNFR1 and glucose was observed (r = 0.5; p = 0.029) in the whole group. We performed an oral glucose tolerance test with 75 g of glucose in obese, non-diabetic women in order to evaluate the effect of an acute glucose increase on the tumor necrosis factor system at 60 min and 120 min. We show that except for a positive association of mTNFR1 with body mass index at 60 min and of mTNFR2 with plasmatic triglycerids levels, no other significant differences were elicited by acute glucose in obese, non-diabetic women. These findings are in agreement with a functional role for the TNF system in obese women in obesity-linked, type 2 diabetes. Copyright John Libbey Eurotext 2003.     

Thiol-inducing and immunoregulatory effects of flavonoids in peripheral blood mononuclear cells from patients with end-stage diabetic nephropathy

The cellular thiol status and its relationship to T-cell activation and cytokine synthesis of mononuclear cells was investigated in patients with end-stage diabetic nephropathy (ESDN) undergoing dialysis treatment. The functional effects of thiol repair by in vitro and in vivo treatment with flavonoids were elucidated. The thiol status of peripheral blood lymphocytes from 30 ESDN patients on hemodialysis and healthy controls was determined by flow cytometry. T-cell activation in response to pokeweed mitogen was analyzed by CD69 expression; cytokines were determined in cell culture supernatants. In result, compared to age-matched healthy subjects, a significant thiol deficiency in ESDN patients was obvious. The lowered total intracellular thiol levels correlated directly to a significant diminished T-cell activation and an elevated synthesis of TNF-alpha in the patient group. The treatment with flavonoids led to a restoration of the thiol status within 72 h in vitro and in vivo. This effect showed a biphasic kinetic that first utilized cell surface thiols and secondly intracellular thiols. In parallel, the T-cell activation was improved substantially along with a significant decrease in TNF-alpha release. These data provide the rational for clinical trials using flavonoids in ESDN to normalize immunoregulatory defects via restoration of the cellular thiol status.     

Silica nanoparticles induce oxidative stress and inflammation of human peripheral blood mononuclear cells

In the present study, the effects of 10- or 100-nm silica oxide (SiO₂) NPs on human peripheral blood mononuclear cells (PBMC) were examined. Cytotoxic effects and oxidative stress effects, including glutathione (GSH) depletion, the formation of protein radical species, and pro-inflammatory cytokine responses, were measured. PBMC exposed to 10-nm NP concentrations from 50 to 4,000 ppm showed concentration-response increases in cell death; whereas, for 100-nm NPs, PBMC viability was not lost at <500 ppm. Interestingly, 10-nm NPs were more cytotoxic and induced more oxidative stress than 100-nm NPs. Immunoelectron micrographs show the cellular distribution of GSH and NPs. As expected based on the viability data, the 10-nm NPs disturbed cell morphology to a greater extent than did the 100-nm NPs. Antibody to the radical scavenger, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was used for Western blot analysis of proteins with radicals; more DMPO proteins were found after exposure to 10-nm NPs than 100-nm NPs. Examination of cytokines (TNF-α, IL-1ra, IL-6, IL-8, IL-1β, and IFN-γ) indicated that different ratios of cytokines were expressed and released after exposure to 10- and 100-nm NPs. IL-1β production was enhanced by 10- and 100-nm NPs;, the cytotoxicity of the NPs was associated with an increase in the IL-1β/IL-6 ratio and 100-nm NPs at concentrations that did not induce loss of cell viability enhanced IL-1β and IL-6 to an extent similar to phytohemagglutinin (PHA), a T cell mitogen. In conclusion, our results indicate that SiO₂ NPs trigger a cytokine inflammatory response and induce oxidative stress in vitro, and NPs of the same chemistry, but of different sizes, demonstrate differences in their intracellular distribution and immunomodulatory properties, especially with regard to IL-1β and IL-6 expression.     

Pravastatin sodium attenuated TREM-1-mediated inflammation in human peripheral blood mononuclear cells

Pravastatin sodium on triggering receptor expressed on myeloid cell-1 (TREM-1)-mediated inflammation in human peripheral blood mononuclear cells (PBMCs) has been poorly investigated. In this study, we isolated PBMCs from the peripheral blood samples of patients with chronic obstructive pulmonary disease, treated the cells with pravastatin sodium, and determined a concentration at which more than 90% cells could survive. Then we treated cells with 10?ng/ml of lipopolysaccharide, added with 10, 50, 100?μM of pravastatin sodium combined with or without LR-12, a known TREM-1 inhibitor. The expression of TREM-1 was determined by quantitative RT-PCR. The levels of TREM-1, IL-6, and TNF-α in cell culture supernatant were measured with ELISA. Simultaneously, NF-κB signaling pathway-related protein p-p65 and p-IκBα were detected by Western blot assay. Results demonstrated that pravastatin sodium significantly mitigated lipopolysaccharide-stimulated TREM-1 over-expression at mRNA and protein levels dose-dependently. Elevated IL-6 and TNF-α levels changed synchronously. LR-12 inhibited the TREM-1 over-expression and inflammatory factor production but did not show extra synergistic effect to pravastatin. Lipopolysaccharide induced phospho-p65 and -IκBα over-expression was weakened significantly when cells were treated with pravastatin sodium. In conclusion, pravastatin could inhibit TREM-1-medieted inflammation and NF-κB signaling pathway was involved.     

Alterations of mitochondria in peripheral blood mononuclear cells of vitiligo patients

The possible role for a defective mitochondrial functionality in the pathogenesis of vitiligo was investigated by measuring intracellular levels of reactive oxygen species and of antioxidants, the activity of Krebs cycle enzymes, as well as the effects of inhibitors of the electron transport chain, in peripheral blood mononuclear cells from patients with active or stable disease vs. normal subjects. Plasma glyoxal levels were also determined in the same groups of subjects as an index of systemic oxidative stress. In patients with vitiligo in active phase, we observed an increased intracellular production of reactive oxygen species with a consequent imbalance of the prooxidant/antioxidant equilibrium, whereas plasma did not show apparent alterations in glyoxal levels, ruling out a systemic oxidative stress. In patients with stable disease, the balance between pro-oxidants and anti-oxidants seems to be maintained. Moreover, a marked increase in the expression of mitochondrial malate dehydrogenase activity and a specific sensitivity to electron transport chain complex I inhibitor were observed. Overall, these data provide further evidence for an altered mitochondrial functionality in vitiligo patients.     

Evidence for an endothelial cell-derived factor which stimulates the growth of human peripheral blood mononuclear cells

Evidence is provided which demonstrates that conditioned media of cultured endothelial cells derived from human umbilical veins contained a factor which stimulated peripheral blood mononuclear cell [3H]thymidine uptake. A dose-dependent response in peripheral blood mononuclear cell [3H]thymidine uptake was obtained when cells were incubated with increasing concentrations of supernatant of endothelial cell cultures. Studies on temporal kinetics demonstrated that stimulatory activity was evident when mononuclear cells had been incubated with endothelial cell supernatant for 120 hr or more. Preliminary characterization showed the growth immunoregulatory factor to have a molecular weight greater than 100,000 Da.     

Detection of HCV and GBV-CHGV RNA in peripheral blood mononuclear cells of patients with chronic type C hepatitis

Hepatitis C virus (HCV) and hepatitis G virus (HGV) viraemia were investigated by RT-PCR protocols in peripheral blood mononuclear cells (PBMC) of 22 patients with chronic type C hepatitis. Samplings were at basal and 4-8 months after a 12 month period of treatment with interferon-alpha. A plus strand of HCV in PBMC was detected in 8 of 21 patients (38%) (p <0.05; chi2 test) with a lack of response to therapy; a minus strand was detected in 10% of chronic type C hepatitis and 25% of the patients harboured HCV RNA in PBMC. The association with a response was nearly significant (p <0.1; chi2 test). GBV-C/HGV RNA was detected in the serum of 9 of 21 (43%) patients and in PBMC of 20% of the patients viraemic. Genomic sequences of GBVC/HGV in PBMC were found, but further investigation is needed to assess the findings reported for HCV.     

DNA damage in peripheral blood mononuclear cells of patients undergoing anti-tuberculosis treatment

Tuberculosis (TB), a chronic infectious disease, is a major cause of morbidity and mortality worldwide. Expression of iNOS and consequent production of NO during the inflammatory process is an important defense mechanism against TB bacteria. We have tested whether pulmonary TB patients undergoing anti-tuberculosis treatment present DNA damage, and whether this damage is related to oxidative stress, by evaluating total hydrophilic antioxidant capacity and iNOS expression. DNA damage in peripheral blood mononuclear cells from patients and healthy tuberculin test (PPD) positive controls was evaluated by single-cell gel electrophoresis (comet assay), and iNOS expression was measured by qPCR. We also evaluated total hydrophilic antioxidant capacity in plasma from patients and controls. Compared to controls, pulmonary TB patients under treatment presented increased DNA damage, which diminished during treatment. Also, the antioxidant capacity of these individuals was increased at the start of treatment, and reduced during treatment. TB patients showed lower iNOS expression, but expression tended to increase during treatment. Our results indicate that pulmonary TB patients under anti-TB treatment exhibit elevated DNA damage in peripheral blood mononuclear cells. This damage was not related to nitric oxide but may be due to other free radicals.     

Cytogenetic aberrations in peripheral blood mononuclear cells in acute Lyme borreliosis patients

Cytogenetic aberrations are known to be caused by biological mutagenic factors, including both intracellular parasites, such as viruses, and some extracellular infectious agents, including bacteria, protozoa, and helminthes. The present study is aimed at investigating the cytogenetic status of acute Lyme borreliosis (LB) patients. As a result of the present study, a peripheral blood mononuclear cell culture of acute LB patients demonstrated a significantly higher frequency of lymphocytes with cytogenetic aberrations of the aneugenic type, such as polyploidy, hypoploidy, or endoreduplication, as well as of the clastogenic type, such as chromatid and chromosomal breaks accompanied by the inhibition of proliferative responses to mitogens and an increase in the elimination of genetically abnormal cells by apoptosis compared to the control.     

Brain and gut neuropeptides in peripheral blood mononuclear cells

Neuropeptides, initially thought to be common features of gut and brain, are only synthesized in immune cells and modulate immune functions. The presence and possible functions of these peptides in immune cells in both physiological or pathological conditions have been investigated in our laboratory in the last years. Some of the data obtained are reviewed here, and future developments of the field are indicated.     

Computer-aided image analysis for the differentiation of mononuclear cells in peripheral blood smears from leukemic patients

Different leukemias originating from the lymphatic system and from the myeloid tissue of the bone marrow were selected to develop and test algorithms to cytophotometrically define leukemic cells. Two of these cases were of low-grade, one of intermediate-grade and one of high-grade malignancy. The malignant cell types of each leukemic form could be identified and characterized by assessing the chromatin texture of the nucleus in the leukemic cells. With this method, normal peripheral white blood cells could be differentiated from leukemic ones. The results correlate directly with established hematologic parameters as they are described in the literature. The investigation demonstrated the usefulness of computer methods in the differentiation of closely related cell structures.     

Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells

Differences in hepatitis C virus (HCV) variants of the highly conserved 5' untranslated region (UTR) have been observed between plasma and peripheral blood mononuclear cells (PBMC). The prevalence and the mechanisms of this compartmentalization are unknown. Plasma and PBMC HCV variants were compared by single-strand conformation polymorphism (SSCP) and by cloning or by genotyping with a line probe assay (LiPA) in 116 chronically infected patients, including 44 liver transplant recipients. SSCP patterns differed between compartments in 43/109 analyzable patients (39%). Differences were significantly more frequent in patients with transplants (21/38 [55%] versus 22/71 [31%]; P < 0.01) and in those who acquired HCV through multiple transfusions before 1991 (15/20; 75%) or through drug injection (16/31; 52%) than in those infected through an unknown route (7/29; 24%) or through a single transfusion (5/29; 17%; P < 0.001). Cloning of the 5' UTR, LiPA analysis, and nonstructural region 5B sequencing revealed different genotypes in the two compartments from 10 patients (9%). In nine patients, the genotype detected in PBMC was not detected in plasma and was weak or undetectable in the liver in three cases. This genotypic compartmentalization persisted for years in three patients and after liver transplantation in two. The present study shows that a significant proportion of HCV-infected subjects harbor in their PBMC highly divergent variants which were likely acquired through superinfections.     

Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients

A 37-kDa binding polypeptide accumulates in peripheral blood mononuclear cell (PBMC) extracts from chronic fatigue syndrome (CFS) patients and is being considered as a potential diagnostic marker (De Meirleir, K., Bisbal, C., Campine, I., De Becker, P., Salehzada, T., Demettre, E., and Lebleu, B. (2000) Am. J. Med. 108, 99-105). We establish here that this low molecular weight 2-5A-binding polypeptide is a truncated form of the native 2-5A-dependent ribonuclease L (RNase L), generated by an increased proteolytic activity in CFS PBMC extracts. RNase L proteolysis in CFS PBMC extracts can be mimicked in a model system in which recombinant RNase L is treated with human leukocyte elastase. RNase L proteolysis leads to the accumulation of two major fragments with molecular masses of 37 and 30 kDa. The 37-kDa fragment includes the 2-5A binding site and the N-terminal end of native RNase L. The 30-kDa fragment includes the catalytic site in the C-terminal part of RNase L. Interestingly, RNase L remains active and 2-5A-dependent when degraded into its 30- and 37-kDa fragments by proteases of CFS PBMC extract or by purified human leukocyte elastase. The 2-5A-dependent nuclease activity of the truncated RNase L could result from the association of these digestion products, as suggested in pull down experiments.     

Use of cryopreserved peripheral mononuclear blood cells in biomonitoring

This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood cells (PMBC) obtained from donor blood. Measurements of DNA-repair, mutant frequency, and subcell content were included. Samples for large biomonitoring studies are usually taken from study groups within a short timeperiod of days/weeks and storing of study material for later analysis can be necessary. We measured the DNA repair activity as dimethylsulfate induced unscheduled DNA synthesis (UDS) in PMBC incubated with either autologous plasma or fetal bovine serum (FBS). Comparison of the hprt mutant frequency by the T cell cloning assay was made in parallel. Finally the content of B/T-lymphocytes and monocytes was measured in phytohemaglutinin (PHA)-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant correlation of frequencies was seen when comparing fresh with cryopreserved samples. Furthermore we recommend fresh human plasma used in UDS incubation media.