Altered cytokine release in peripheral blood mononuclear cell cultures from patients with the chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is an idiopathic illness associated with a variety of immunologic abnormalities. To investigate potential pathogenetic mechanisms, we evaluated serum levels and peripheral blood mononuclear cell (PBMC) production of selected cytokines and immunoglobulins. Serum bioactive transforming growth factor beta (TGF-beta) levels were higher (P less than 0.01) in patients with CFS (290 +/- 46 pg/mL) than in control subjects (104 +/- 18 pg/mL), but levels of other cytokines tested were not different. Lipopolysaccharide-stimulated release of interleukin 1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha was increased (P less than 0.05) in PBMC cultures from patients with CFS versus control subjects; enhanced (P less than 0.01) IL-6 release to phytohemagglutinin was also observed. In contrast, TGF-beta release in response to lipopolysaccharide was depressed (P less than 0.01) in PBMC cultures derived from patients with CFS. No differences in IL-2 and IL-4 or immunoglobulin production were observed. The enhanced release of inflammatory cytokines by stimulated PBMC from patients with CFS suggests that these cells are primed for an increased response to immune stimuli. These data also suggest an association between abnormal regulation of TGF-beta production in vivo and in vitro with the immunologic consequence of CFS.     

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.     

Assessment of a 44 gene classifier for the evaluation of chronic fatigue syndrome from peripheral blood mononuclear cell gene expression

Chronic fatigue syndrome (CFS) is a clinically defined illness estimated to affect millions of people worldwide causing significant morbidity and an annual cost of billions of dollars. Currently there are no laboratory-based diagnostic methods for CFS. However, differences in gene expression profiles between CFS patients and healthy persons have been reported in the literature. Using mRNA relative quantities for 44 previously identified reporter genes taken from a large dataset comprising both CFS patients and healthy volunteers, we derived a gene profile scoring metric to accurately classify CFS and healthy samples. This metric out-performed any of the reporter genes used individually as a classifier of CFS.To determine whether the reporter genes were robust across populations, we applied this metric to classify a separate blind dataset of mRNA relative quantities from a new population of CFS patients and healthy persons with limited success. Although the metric was able to successfully classify roughly two-thirds of both CFS and healthy samples correctly, the level of misclassification was high. We conclude many of the previously identified reporter genes are study-specific and thus cannot be used as a broad CFS diagnostic.     

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.     

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.     

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.     

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.     

Prevalence of human herpesvirus 8 DNA in peripheral blood mononuclear cells of acute and chronic leukemia patients in Taiwan

Human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma and several other human malignancies. The prevalence of HHV-8 DNA in peripheral blood mononuclear cells (PBMCs) in Taiwanese leukemia populations has not been investigated. In this study, HHV-8 DNA was extracted from PBMCs, and detected in 10.29% of the leukemia cases and 8.94% of the relatives' cases. In addition, the prevalence of HHV-8 DNA in PBMCs was nonsignificantly associated with gender, age and leukemia subtypes. The study examines the prevalence of HHV-8 DNA in PBMCs in Taiwanese leukemia and can be applied in further epidemiological studies.     

Suppressor cell activity of peripheral mononuclear cells from patients undergoing chronic haemodialysis

The enhanced in vitro proliferative response of peripheral mononuclear cells (PMNC) when pre-cultured for 24 hours prior to the addition of Concanavalin-A has been used as an indirect parameter of suppressor cell activity in healthy subjects and in patients undergoing chronic haemodialysis. The proliferative response of PMNC from haemodialysis patients is lower than that of control subjects when Con-A is added at the initiation of culture but approximates to that of normal subjects when the addition of Con-A was deferred for 24 hours. The Suppressor Indices of PMNC from haemodialysis patients were at least as great and sometimes greater than those of control subjects but the absolute T-cell counts were lower in haemodialysis patients than in controls. These results suggest that the relative energy of haemodialysis patients is partly attributable to T-cell depletion but this is accompanied by retention, and in some cases augmentation, of suppressor cell activity.     

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 time period 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.     

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.     

High prevalence of Mycoplasma infections among European chronic fatigue syndrome patients. Examination of four Mycoplasma species in blood of chronic fatigue syndrome patients

Prevalence of Mycoplasma species infections in chronic fatigue syndrome (CFS) has been extensively reported in the scientific literature. However, all previous reports highlighted the presence of Mycoplasmas in American patients. In this prospective study, the presence of Mycoplasma fermentans, M. penetrans, M. pneumoniae and M. hominis in the blood of 261 European CFS patients and 36 healthy volunteers was examined using forensic polymerase chain reaction. One hundred and seventy-nine (68.6%) patients were infected by at least one species of Mycoplasma, compared to two out of 36 (5.6%) in the control sample (P<0.001). Among Mycoplasma-infected patients, M. hominis was the most frequently observed infection (n=96; 36.8% of the overall sample), followed by M. pneumoniae and M. fermentans infections (equal frequencies; n=67; 25.7%). M. penetrans infections were not found. Multiple mycoplasmal infections were detected in 45 patients (17.2%). Compared to American CFS patients (M. pneumoniae>M. hominis>M. penetrans), a slightly different pattern of mycoplasmal infections was found in European CFS patients (M. hominis>M. pneumoniae, M. fermentansz.Gt;M. penetrans).     

Hepatitis B core antigen-specific IFN-gamma production of peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the specificity of cellular immune response to hepatitis B virus (HBV) Ag in patients with chronic HBV infection, we have measured IFN-gamma production and proliferation of PBMC of 16 patients with chronic active hepatitis (CAH), 17 asymptomatic carriers of HBV (ASC), 6 anti-hepatitis B surface (HBs)-positive subjects, and 6 control individuals with ELISA procedure and [3H]thymidine incorporation. There was no significant increase in the mean proliferative response to recombinant HB surface and core Ag (rHBsAg and rHBcAg), nor was IFN-gamma production elicited with rHBsAg in any group. In contrast, PBMC of HBeAg-positive and anti-HBe-positive CAH patients, and anti-hepatitis B "e" Ag (HBe)-positive ASC showed significantly enhanced IFN-gamma production in response to HBcAg, whereas those of HBeAg-positive ASC and anti-HBs-positive subjects did not respond to HBcAg. The maximal response was observed in a 5-day culture with 500 ng/ml of rHBcAg when assessed by stimulation index value. Monocytes did not demonstrate an increased suppressor or helper activity for IFN-gamma production in these patients. T cell subset fractionation revealed that CD4+ cells were main population of IFN-gamma production specific for HBcAg and CD8+ cells did not suppress IFN-gamma production of CD4+ cells. Furthermore, CD4+ cells of HBeAg-positive ASC generated lesser amounts of IFN-gamma than HBeAg-positive CAH patients did. These results show that the measurement of IFN-gamma production is useful to determine cellular immune response to HBV Ag and suggest that IFN-gamma production depends on the helper activity of CD4+ T cells sensitized to HBcAg.