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.     

The protective role of metformin in autophagic status in peripheral blood mononuclear cells of type 2 diabetic patients

Autophagy plays an important role in the pathophysiology of type 2 diabetes (T2D). Metformin is the most common antidiabetic drug. The main objective of this study was to explore the molecular mechanism of metformin in starvation‐induced autophagy in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients. PBMCs were isolated from 10 diabetic patients and 7 non‐diabetic healthy volunteers. The autophagic puncta and markers were measured with the help of monodansylcadaverine staining and western blot. Additionally, transmission electron microscopy was also performed. No significant changes were observed in the initial autophagy marker protein levels in PBMCs of T2D after metformin treatment though diabetic PBMCs showed a high level of phospho‐mammalian target of rapamycin, p62 and reduced expression of phospho‐AMP‐activated protein kinase and lysosomal membrane‐associated protein 2, indicating a defect in autophagy. Also, induction of autophagy by tunicamycin resulted in apoptosis in diabetic PBMCs as observed by caspase‐3 cleavage and reduced expression of Bcl2. Inhibition of autophagy by bafilomycin rendered consistent expression of p62 indicating a defect in the final process of autophagy. Further, electron microscopic studies also confirmed massive vacuole overload and a sign of apoptotic cell death in PBMCs of diabetic patients, whereas metformin treatment reduced the number of autophagic vacuoles perhaps by lysosomal fusion. Thus, our results indicate that defective autophagy in T2D is associated with the fusion process of lysosomes which could be overcome by metformin.     

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.     

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.     

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.     

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.     

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.     

Regulation of autophagy following ex vivo heating in peripheral blood mononuclear cells from young adults

Under conditions of extreme heat stress, the process of autophagy has previously been shown to protect human cells, but the exact body temperature at which autophagic activation occurs is largely unknown. Further, the interplay between autophagy, the heat shock response (HSR), inflammation, and apoptosis have yet to be examined together under temperature conditions representative of human internal body temperatures at rest (37 °C) or under severe heat stress conditions (41 °C). Thus, the purpose of this study was to examine threshold changes in autophagy, the HSR, inflammation, and apoptosis to increasing levels of ex vivo heat stress. Whole blood was collected from 20 young (23 ± 4 years; 10 men, 10 women) physically active participants. Peripheral blood mononuclear cells (PBMCs) were isolated immediately (baseline) and after 90-min of whole blood heating in 37, 39, and 41 °C water baths, representative of normal resting (non-heat stress) as well as moderate and severe heat stress conditions in humans, respectively. At 37 °C, increased autophagic activity was demonstrated, with no change in the HSR, and inflammation. Subsequently, responses of autophagy, the HSR, and inflammation increased with a moderate heat stress (39 °C), with further increases in only autophagy and the HSR under a severe heat stress of 41 °C. We observed no increase in apoptosis under any temperature condition. Our findings show that in human PBMCs, the autophagy and HSR systems may act cooperatively to suppress apoptotic signaling following heat stress, which may in part be mediated by an acute inflammatory response.     

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.     

Chemiluminescence in peripheral blood mononuclear cells of solid tumor cancer patients

Summary Levels of chemiluminescence were measured in peripheral blood mononuclear cells (PBMC) from normal subjects and from solid tumor cancer patients. Patients with advanced malignant disease were found to have significantly elevated baseline chemiluminescence activity in their resting PBMC as compared to normal subjects or to cancer patients with, at most, minimum residual disease. Patients with either advanced disease or minimum residual disease, however, were found to exhibit significantly elevated activation of chemiluminescence by treatment of cells with phorbol myristic acetate (PMA). Treatment of surgically resected stage I lung cancer patients with Freund's complete adjuvant alone or emulsified with extracted lung cancer antigens was found to elevate chemiluminescence levels in patient PBMC. Serum from those vaccinated patients was found to elevate chemiluminescence levels of resting PBMC from normal subjects. That serum activity did net correlate with levels of immune complexes measurable in the Clq or Raji cell assay.     

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.     

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.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Cytokines profile and peripheral blood mononuclear cells morphology in Rett and autistic patients

A potential role for immune dysfunction in autism spectrum disorders (ASD) has been well established. However, immunological features of Rett syndrome (RTT), a genetic neurodevelopmental disorder closely related to autism, have not been well addressed yet.By using multiplex Luminex technology, a panel of 27 cytokines and chemokines was evaluated in serum from 10 RTT patients with confirmed diagnosis of MECP2 mutation (typical RTT), 12 children affected by classic autistic disorder and 8 control subjects. The cytokine/chemokine gene expression was assessed by real time PCR on mRNA of isolated peripheral blood mononuclear cells (PBMCs). Moreover, ultrastructural analysis of PBMCs was performed using transmission electron microscopy (TEM).Significantly higher serum levels of interleukin-8 (IL-8), IL-9, IL-13 were detected in RTT compared to control subjects, and IL-15 shows a trend toward the upregulation in RTT. In addition, IL-1β and VEGF were the only down-regulated cytokines in autistic patients with respect to RTT. No difference in cytokine/chemokine profile between autistic and control groups was detected. These data were also confirmed by ELISA real time PCR. At the ultrastructural level, the most severe morphological abnormalities were observed in mitochondria of both RTT and autistic PBMCs.In conclusion, our study shows a deregulated cytokine/chemokine profile together with morphologically altered immune cells in RTT. Such abnormalities were not quite as evident in autistic subjects. These findings indicate a possible role of immune dysfunction in RTT making the clinical features of this pathology related also to the immunology aspects, suggesting, therefore, novel possible therapeutic interventions for this disorder.     

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.