Expression In Peripheral Blood Lymphocytes Research Articles

Protective effect of vitamin C on 8-hydroxy-2′-deoxyguanosine level in peripheral blood lymphocytes of chronic hemodialysis patients

This study focused on the effect of vitamin C on the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level of cellular DNA, as well as 8-oxoguanine-DNA glycosylase 1 (hOGG1) and human MutT homologue (hMTH1) gene expression in peripheral blood lymphocytes of chronic hemodialysis patients. Sixty chronic hemodialysis patients (35 men and 25 women) were recruited to participate in a randomized, placebo-controlled study. Treatment order is block-randomized with intravenous sodium ascorbate (vitamin C, 300 mg) or placebo (0.9% saline), administered postdialysis three times a week. We evaluated 8-OHdG level, intracellular reactive oxygen species (ROS) production, and gene expression of hOGG1 and hMTH1 in peripheral blood lymphocytes by using high-performance liquid chromatography (HPLC) electrochemical detection method, flow cytometric analysis, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. A total of 51 patients completed the study (26 in placebo group and 25 in vitamin C group). Mean 8-OHdG levels significantly decreased in total subjects following 8 weeks of vitamin C supplementation (22.9 vs. 18.8/10(6) dG, P < 0.01). The decrease in 8-OHdG levels after vitamin C supplementation was also noted in the patients with ferritin <500 or > or =500 microg/L and transferrin saturation (TSAT) <50 or > or =50% (P < 0.05). But 8-OHdG levels had no significant changes in total patients or in the four subgroups of patients treated with placebo as compared to their baselines. Intracellular ROS production by lymphocytes from the four subgroups of patients, either spontaneous (P < 0.05) or phorbol-12-myristate-13-acetate (PMA)-stimulated (P < 0.001), was significantly reduced after 8 weeks vitamin C supplementation. Steady-state hOGG1 mRNA levels were significantly up-regulated at 24 hours after vitamin C administration (P < 0.05), but hMTH1 mRNA levels were not. The changes in the spontaneous and PMA-stimulated ROS production, and an up-regulation of hOGG1 mRNA expression were not observed in patients treated with placebo as compared to their baselines. Vitamin C supplementation in chronic hemodialysis patients can reduce the lymphocyte 8-OHdG levels and intracellular ROS production, as well as up-regulate hOGG1 gene expression for repair. There is no compelling evidence for an in vivo pro-oxidant effect of vitamin C on lymphocyte DNA base oxidation, even in the status of increased iron stores.

Perforin expression in peripheral blood lymphocytes and skin-infiltrating cells in patients with lichen planus.

Current evidence suggests that lichen planus is a T-cell-mediated autoimmune disease in which cytotoxic mechanisms have been poorly investigated. We investigated the expression of perforin in subpopulations of peripheral blood lymphocytes (PBL) in exacerbation and remission phases of the disease as well as in skin lesions. We performed a simultaneous detection of perforin (intracellular molecule) and cell surface antigens on PBL by flow cytometry, and skin lesions were investigated by immunohistochemistry. The most interesting finding was a significant increase of perforin expression in cytotoxic T lymphocytes (CD3+ perforin+ cells) in the exacerbation phase of disease (P < 0.05), which was mostly located in the CD8+ subpopulation (CD8+ perforin+) (P < 0.01). Using immunohistochemistry we confirmed the infiltration of T lymphocytes in skin lesions, especially of CD4+ and CD8+ phenotypes, compared with uninvolved (P < 0.05) and healthy skin (P < 0.01). The expression of perforin was also significantly higher in lesional skin compared with nonlesional and healthy skin (P < 0.05). Our results clearly show the upregulation of perforin expression in peripheral blood as well as in lesions of patients with lichen planus and therefore suggest an important role for perforin in this autoimmune disease.

Loss of imprinting of insulin growth factor II gene: a potential heritable biomarker for colon neoplasia predisposition

Background & aims: Loss of genomic imprinting (LOI) of insulin-like growth factor II gene ( IGF2) involves abnormal activation of the normally silent maternally inherited allele. LOI of IGF2 has been associated with personal and family history of colorectal neoplasia (CRN), supporting a role for LOI in colorectal carcinogenesis. Whether LOI of IGF2 is associated with known environmental risk factors for CRN is unknown. Methods: We performed quantitative hot-stop PCR for imprinting analysis of IGF2 on normal peripheral blood lymphocytes (PBL) of individuals. Environmental exposures including tobacco, alcohol, NSAIDs, and nutrient consumption (calcium, folate, selenium, fiber, and fat) were correlated with LOI expression in PBL. Odds ratios (OR) and 95% CI were calculated. Results: The prevalence of LOI of IGF2 was examined in 172 individuals. Persons with CRN (adenomas/cancer) had 5.1-fold (95% CI: 1.92–13.6) increased risk of having LOI of IGF2 in PBL compared with those without CRN. In contrast, tobacco smoking (OR = 0.96, 95% CI: 0.36–2.55), alcohol consumption (OR = 1.22, 95% CI: 0.45–3.3), and NSAIDs use (OR = 1.21, 95% CI: 0.38–3.94) were not significantly associated with LOI of IGF2. Nutrient ingestion including calcium ( P = 0.61), folate ( P = 0.23), selenium ( P = 0.19), fiber ( P = 0.63), and fat ( P = 0.14) was not statistically correlated with LOI of IGF2. Conclusions: Abnormal imprinting of IGF2 gene was strongly associated with CRN but not with any of the environmental exposures examined. LOI of IGF2 does not appear to be an environmentally acquired phenomenon but rather a hereditary risk factor for CRN.

Dopamine receptor D3 mRNA expression in human lymphocytes is negatively correlated with the personality trait of persistence

It has been proposed that neurotransmitter receptor expression in peripheral immune cells reflects expression of these receptors in the brain. To test this “peripheral marker hypothesis”, we compared mRNA expression of the dopamine receptors D3 (DRD3) and D4 (DRD4) in peripheral blood lymphocytes (PBL) to personality traits assessed with the Temperament and Character Inventory (TCI) in 50 healthy and unmedicated Caucasian individuals. A shared variance of at least 17% ( p=0.016) between DRD3 mRNA expression in PBL and the personality trait of persistence was found. As personality traits have been generally assumed polygenic with a single gene accounting for rarely more than 1–2% of observed variance in a trait, this result lends further support to the peripheral marker hypothesis for DRD3 mRNA expression in PBL. It may also suggest a significant role for the DRD3 in the neurobiology of persistence and point to an interesting link between personality and functioning of the immune system.

Reduced dopamine D4 receptor mRNA expression in lymphocytes of long-term abstinent alcohol and heroin addicts.

It has been repeatedly suggested that dopamine receptor expression in peripheral blood lymphocytes reflects, to some extent, brain status. The aim of the present study was to investigate dopamine receptor expression in peripheral blood lymphocytes of long-term abstinent alcohol and heroin addicts against the background of the hypothesis, that a persisting dysfunction of the dopaminergic system contributes a biological cause to the chronic character of addiction. Dopamine D3 and D4 receptor mRNA expression in peripheral blood lymphocytes was measured by real-time polymerase chain reaction (PCR) in 19 alcohol addicts, abstinent for 6.2 +/- 4.7 months (mean +/- SD), and 20 heroin addicts, abstinent for 6.7 +/- 3.7 months (mean +/- SD), and compared to a control group of 29 age- and sex-matched individuals with no life-time history of substance abuse. One-way anova showed significant differences in D4 mRNA expression between the groups (P = 0.005): both groups of addicts showed an approximately 50% reduction in D4 receptor mRNA expression in peripheral blood lymphocytes (PBL) compared to controls. No differences were found for D3 mRNA expression between the groups. The results of the present study indicate a withdrawal-persisting dopaminergic imbalance in abstinent addicts as measured by a suggested peripheral marker.

Reduced dopamine D3 receptor expression in blood lymphocytes of smokers is negatively correlated with daily number of smoked cigarettes: a peripheral correlate of dopaminergic alterations in smokers.

The mesolimbic dopaminergic system is known to mediate rewarding effects of nicotine administration, and dysfunctions of this system may underlie failure to stop cigarette smoking. Expression of dopamine receptors in peripheral blood lymphocytes (PBLs) has been indicated as a peripheral correlate of brain status. Dopamine receptor D(3) (DRD3) and D(4) (DRD4) mRNA expression in PBLs was measured by real-time polymerase chain reaction in smokers (n=26) and former smokers (n=14), compared with nonsmoking control subjects (n=35). A significant (p=.032, Bonferroni corrected) 30% reduction of DRD3 mRNA expression in PBLs was found in smokers but not former smokers in comparison with controls. DRD3 mRNA expression in PBLs in smokers but not former smokers was negatively correlated with daily number of cigarettes consumed (Pearson correlation coefficient r=-.54, p=.005). These data suggest a selective inhibiting effect of smoking on DRD3 mRNA expression and, with the known involvement of DRD3 in reward mediation, indicates a vicious-cycle explanation for the motivation for continued smoking.

Regulation of peripheral blood and synovial fluid lymphocyte apoptosis in juvenile idiopathic arthritis

Objective: Complex regulatory mechanisms are involved in the induction of apoptosis. Their impairment may play a role in the pathogenesis of several autoimmune diseases. Recently, we have described higher incidences of spontaneous apoptosis of peripheral blood (PB) lymphocytes in children with juvenile idiopathic arthritis (JIA). This study aimed to evaluate the regulatory mechanisms that may be responsible for this phenomenon.Methods: Thirty‐four JIA children were examined and compared with 20 healthy children of similar ages. Expression of regulatory proteins p53, Bax and Bcl‐2 in lymphocytes isolated from PB and synovial fluid (SF) was assessed. Serum and SF levels of interleukin‐15 (IL‐15) were also evaluated.Results: The study showed significantly decreased Bcl‐2 expression in JIA PB lymphocytes, compared to both healthy control (p=0.03) and JIA SF lymphocytes (p=0.005). There were no significant differences found in Bax expression between groups or compartments examined. However, the Bax/Bcl‐2 ratio was nearly two‐fold higher in PB lymphocytes than in SF of JIA patients (p=0.001). p53 expression in PB lymphocytes from both JIA and control children did not statistically differ. In JIA, however, p53 was significantly higher in PB than SF lymphocytes (p=0.016). IL‐15 levels were about 20‐fold higher in JIA SF than in serum from either JIA or healthy children (p<0.0001).Conclusion: The results suggest that a higher incidence of apoptosis of PB lymphocytes observed in JIA may be associated with down‐regulation of Bcl‐2, rather than with changes in expression of Bax and p53. In contrast, the low p53 expression and elevated IL‐15 appear to provide mechanisms responsible for suppression of apoptosis in SF cells from JIA patients.

Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF.

We aimed to study T-helper 1 (Th1) and Th2 intracellular cytokine expression in peripheral blood lymphocytes of women with recurrent spontaneous abortions (RSA) or infertility with multiple implantation failures after IVF cycles. Twenty-six women with three or more RSA and 23 with two or more IVF failures (14 with no history of spontaneous abortion (SAB) and nine with more than one SAB) comprised the two study groups. Twenty-one non-pregnant healthy multiparous women served as controls. Proportions (%) of lymphocytes containing IFN-gamma, TNF-alpha, IL-4 and IL-10 and the Th1/Th2 ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, TNF-alpha/IL-4 and TNF-alpha/IL-10 in CD3+, CD3+/CD8- (T helper) and CD3+/CD8+ (T suppressor) cells were measured by 4-colour flow cytometry. RSA women demonstrated significantly higher Th1/Th2 ratios of IFN-gamma/IL-4 (P < 0.01), TNF-alpha/IL-4 and TNF-alpha/IL-10 (P < 0.05 each) in CD3+/CD8- T helper cells than those of controls. The proportion of TNF-alpha producing CD3+/CD8- cells (P < 0.05), and the Th1/Th2 ratios of TNF-alpha/IL-4 (P < 0.05) and TNF-alpha/IL-10 (P < 0.005) in CD3+/CD8- cells were significantly higher in women with multiple IVF failures without SAB as compared with those of controls. The prevalence of dominant Th1 immune responses in peripheral blood lymphocytes may reflect the systemic contribution of Th1 cytokines to RSA or multiple implantation failures in IVF cycles.

Increase of Th1 type cytokine mRNA expression in peripheral blood lymphocytes of calves experimentally infected with Cryptosporidium parvum

The expression of the messenger RNA of interleukin-12 (IL-12), interferon-γ, interleukin-4, interleukin-6, and interleukin-10 was examined by reverse-transcriptase polymerase chain reaction in peripheral blood lymphocytes of calves that were orally inoculated with Cryptosporidium parvum oocysts. In all of the calves, gene expression of interleukin-12, interleukin-6, and interferon-γ was observed at delivery and this expression was repressed within the next 24 h. In calves inoculated with C. parvum, mRNA expression of interleukin-12 and interferon-γ was noticed on day 3 post-inoculation (p.i.) and increased in the convalescent phase of the infection, whereas in non-inoculated calves no mRNA expression was detectable up to the end of the experiment. No mRNA expression of interleukin-4 or 6 was detected during the experiment. Our observations suggest that systemic Th1 type immune responses are induced in calves infected with C. parvum and may be available for evaluation of the control of the infection.

Grey and white matter changes in the aesop first-onset psychosis study: A voxel-based analysis of brain structure

Neuregulin-1 (Nrg-1)1 gene has been considered as a schizophrenia susceptibility gene. In order to observe the association of Nrg-1 gene with schizophrenia, the study was designed to investigate the effect of anti-psychotic treatment on the Nrg-1 mRNA expression in peripheral blood lymphocytes of the patients with a diagnosis of schizophrenia. The Nrg-1 mRNA expression in peripheral blood lymphocytes (PBLs) was measured by using semi-quantitative RT-PCR in 31 first-onset schizophrenia patients, 16 sibling controls and 31 no-sibship controls. Results showed that Nrg-1 mRNA expression in PBLs of patients was lower than that in other two control groups (F = 6.722, P = 0.002). However, as follow-up time extended, from the second week, Nrg-1 mRNA expression of PBLs in antipsychotic treated patients gradually increased and has obvious statistical significance compared the efficacy of taking anti-psychotic before and after therapy. These results demonstrated that Nrg-1 gene has association with schizophrenia. It is possible to select Nrg-1 mRNA expression of PBLs in schizophrenia patients as a potential therapeutic marker.

Clinical and psychophysiology correlates of a large cavum septum pellucidum in schizophrenia

Neuregulin-1 (Nrg-1)1 gene has been considered as a schizophrenia susceptibility gene. In order to observe the association of Nrg-1 gene with schizophrenia, the study was designed to investigate the effect of anti-psychotic treatment on the Nrg-1 mRNA expression in peripheral blood lymphocytes of the patients with a diagnosis of schizophrenia. The Nrg-1 mRNA expression in peripheral blood lymphocytes (PBLs) was measured by using semi-quantitative RT-PCR in 31 first-onset schizophrenia patients, 16 sibling controls and 31 no-sibship controls. Results showed that Nrg-1 mRNA expression in PBLs of patients was lower than that in other two control groups (F = 6.722, P = 0.002). However, as follow-up time extended, from the second week, Nrg-1 mRNA expression of PBLs in antipsychotic treated patients gradually increased and has obvious statistical significance compared the efficacy of taking anti-psychotic before and after therapy. These results demonstrated that Nrg-1 gene has association with schizophrenia. It is possible to select Nrg-1 mRNA expression of PBLs in schizophrenia patients as a potential therapeutic marker.

Quantitative determination of MDR1 mRNA expression in peripheral blood lymphocytes: a possible role of genetic polymorphisms in the MDR1 gene.

P-glycoprotein is a transmembrane efflux pump that extrudes a wide variety of drugs, thereby reducing their intracellular access. In humans, P-glycoprotein is encoded by the MDR1 gene. Recently, several single nucleotide polymorphisms in the MDR1 gene were identified. Moreover, it was postulated that, in addition to the full-length P-glycoprotein, a 'mini' P-glycoprotein was also present in lymphocytes. We investigated the effect of the genetic polymorphisms G2677T and C3435T in the MDR1 gene on MDR1 mRNA expression in FACS-sorted peripheral blood CD4+, CD8+, CD19+, and CD56+ cells. MDR1 mRNA expression was determined in 45 healthy individuals using a real-time quantitative RT-PCR. We detected the highest expression of MDR1 mRNA in CD56+ cells, followed by CD8+ > CD4+ > CD19+ cells. However, genetic polymorphisms of the MDR1 gene failed to affect (P > 0.05) MDR1 mRNA levels in the peripheral blood lymphocytes. Furthermore, the transcript levels for the MDR1 N-terminal half were almost two-fold lower than that of the MDR1 C-terminal half in all cell populations investigated (P < 0.0001). An almost two-fold difference in MDR1 C- and N-terminal half expressions supports the presence of mini-P-glycoprotein, an alternatively spliced form of the full-length molecule, in peripheral blood lymphocytes.

Cadmium and human health: A perspective based on recent studies in China

AbstractCadmium (Cd) occurs in low concentrations in all human diets and in cigarettes. In contaminated areas and in certain occupations, high human exposures occur. In assessing risk to human health, it is important to identify the adverse effect that occurs at the lowest exposure level, i.e., the critical effect, which is crucial for preventive action. Excessive Cd exposure may give rise to renal, pulmonary, hepatic, skeletal, reproductive effects, and cancer. Previous evaluations (e.g., by WHO) have identified renal dysfunction, occurring in long‐term Cd exposure, as the critical effect. However, skeletal and reproductive effects are also discussed as possible critical effects. For preventive action, information is important about exposure levels that give rise to the earliest (critical) effects. To gain new information on this issue, population groups in China exposed to high concentrations of Cd via rice and Cd‐exposed workers were studied for possible renal, skeletal, and male reproductive toxicity. Skeletal effects in terms of decreased bone mineral density and an increased occurrence of fractures were found in groups of the general population living in the most Cd‐exposed area. Our studies further show that renal effects measured by sensitive biomarkers, such as urinary content of beta‐2‐microglobulin, calcium, and N‐acetyl‐beta‐D‐glucosaminidase, occurs at lower cumulative exposures to Cd than those giving rise to skeletal effects and also at lower exposures than previously estimated, e.g., by WHO. The findings confirm the renal dysfunction as the critical effect of long‐term Cd exposure. Metallothionein (MT) gene expression in peripheral blood lymphocytes was measured in Cd exposed workers. A higher prevalence of renal dysfunction was found among Cd exposed workers with low MT gene expression than among those with high MT gene expression, at similar blood and urinary Cd levels. MT gene expression in PBLC thus may be a biomarker for identifying sensitive population groups. J. Trace Elem. Exp. Med. 16:307–319, 2003. © 2003 Wiley‐Liss, Inc.

Aberrant Fas Ligand Expression in Lymphocytes in Patients with Behçet’s Disease

Background: Defects in immune responses have been reported in patients with Behçet’s disease (BD). To further characterize the immune dysfunction and its contribution to the pathogenesis, we have studied Fas ligand (FasL) expression in peripheral blood lymphocytes (PBL) and mononuclear cells in the skin lesions in patients with BD. Methods: FasL expression in PBL was studied with RT-PCR and immunoblotting with rabbit anti-human FasL antibody. We studied the expression of FasL in cryostat sections of biopsy specimens of erythema nodosum lesions from 4 patients with BD and of a genital ulcer lesion in another patient using immunohistochemical staining. Apoptotic cell death was detected with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Results: We found that FasL mRNA and FasL protein expression was detected marginally in the unstimulated PBL, and was induced upon activation in normal individuals. PBL from patients with BD exhibited an enhanced expression of FasL mRNA and FasL protein without in vitro stimulation. Moreover, mitogen stimulation failed to augment FasL expression of their lymphocytes, suggesting a dysregulation of FasL expression of PBL in patients with BD. The skin biopsy specimens revealed that cells infiltrating into skin lesions expressed FasL and there were several TUNEL staining-positive cells in the lesions, suggesting that Fas/FasL-mediated apoptosis is involved in the development of the skin lesion and thus may be associated with the pathogenesis. Conclusions: We found an excessive expression of FasL in circulating as well as skin-infiltrating lymphocytes and the presence of apoptotic cells in the skin lesions, suggesting that lymphocytes expressing FasL aberrantly may play a role in the development and pathogenesis of BD.

Cytokine expression in peripheral blood lymphocytes indicates a switch to T helper cells in patients with preeclampsia

We sought to determine whether cytokine expression in peripheral blood mononuclear cells is altered in patients with preeclampsia and in patients with a history of recurrent spontaneous abortion (RSA). Twenty-four patients with preeclampsia and twenty patients with a history of RSA were included into the study. Two control groups consisted of twenty healthy pregnant and twenty healthy non-pregnant women. The intracellular expression of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-γ (IFN-γ) were determined in peripheral blood mononuclear cells (PBMCs) by flow cytometry as a measure of cytokine production. IL-2 synthesis was significantly elevated in the third trimester in preeclamptic patients in comparison with the control group. Non-pregnant women with RSA showed a significantly lower expression of IFN-γ compared to the non-pregnant control group. Our data suggest an abnormal immune response in preeclamptic patients characterised by a shift to a predominantly Th1-type immunity.

Expression of the Multidrug Resistance-associated Protein (mrp ) Gene in Chronic Lymphocytic Leukemia

In order to define more accurately the role of multidrug resistance (MDR)-related protein (mrp ) gene in chronic lymphocytic leukemia (CLL), we addressed the question of its expression pattern in isolated peripheral blood B lymphocytes in seven healthy donors and 28 patients with CLL, with respect to some laboratory and clinical parameters of the disease. For this purpose, we used a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), based on the coamplification of an internal standard not homologous to the DNA target to quantify the mrp transcription level in each studied sample. We report that the level of constitutive mrp gene's expression in peripheral blood lymphocytes is higher in CLL patients than in healthy controls. We found increased mrp gene expression levels in patients with higher white blood cells (WBC) and lymphocytes' counts as well as in more advanced disease stages according to Rai or Binet scale. Finally, mrp gene's expression was higher in patients with progressive CLL, especially in cases refractory to chemotherapy salvage. The results of the present study suggest that expression of mrp gene might be relevant in the pathogenesis of the MDR phenotype in CLL.

Apoptosis and peripheral blood lymphocyte depletion in coeliac disease.

In coeliac disease (CD) immunological abnormalities are not confined to the small bowel and it has been suggested that changes in peripheral blood lymphocytes (PBL), such as lymphopenia and increased T-cell activation, may predispose to malignant or autoimmune complications of this condition. In the light of the recent findings about the Fas-Fas ligand (FasL) system in regulating lymphocyte homeostasis, the aim of the present study was to investigate peripheral lymphocyte Fas-mediated apoptosis in CD to establish whether the homeostatic role of apoptosis in peripheral T-cell selection is maintained. Moreover, because a soluble form of Fas has been described to be functionally implicated in the Fas signalling system, suggesting a relationship between some disorders and soluble Fas function, we measured levels of soluble Fas in sera of coeliac patients and analysed the relationship between these levels and the proportions of apoptotic and Fas(+) PBL to further explore the function of the Fas-FasL pathway in this condition. Finally, we evaluated whether the increased prevalence of anticardiolipin antibodies, recently described in CD, could be related to PBL apoptosis in this condition. We demonstrated an increased apoptosis and higher levels of Fas and FasL expression in PBL isolated from untreated coeliac patients when compared to treated coeliac patients and controls. In addition, low levels of soluble Fas and a significant positive correlation between anticardiolipin antibodies and PBL apoptosis were found in untreated CD. Then, our results showed an increased susceptibility of PBL to undergo Fas-mediated apoptosis in active CD. This increased apoptosis could be responsible for both lymphopenia and immunogenic exposure of phospholipids with subsequent production of autoantibodies.

Lymphocyte cytochrome P450-CYP2E1 expression in human IDDM subjects

Cytochrome P4502E1, a phase I enzyme, has been shown to be induced in liver samples from diabetic and obese rats. One study demonstrated elevated levels of CYP2E1 in children with IDDM, using Western blot analysis. The aim of this investigation was to determine CYP2E1 expression in peripheral blood lymphocytes from eight well-controlled IDDM and eight sex- and age-matched control subjects using Western blot analysis and Phoretix image analysis. Levels of CYP2E1 were low to undetectable in human lymphocytes from healthy control subjects. However, levels of CYP2E1 were elevated in lymphocytes from IDDM subjects (mean 3.1-fold higher). The elevated levels of CYP2E1 in the IDDM subjects showed no correlation with HbA 1c nor duration of IDDM; however, there were marked inter-individual differences in levels of induction of CYP2E1 between all subjects. The results of this study suggest that in human IDDM subjects, even with good metabolic control, expression of CYP2E1 is elevated when compared to controls. CYP2E1 is known to generate ROS in vivo, the modulation of this isoform in lymphocytes from IDDM subjects, could well add to the oxidative stress associated with IDDM and the development of associated complications.

EFFECTS OF DEXAMETHASONE ON THE EXPRESSION OF FAS MOLECULES AND APOPTOSIS OF LYMPHOCYTES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Previous studies have shown that the autoimmune phenomenon could be caused by defective apoptosis of autoreactive lymphocytes. Corticosteroids used for treatment of systemic lupus erythematosus (SLE) are potent apoptosis inducers. We examined dexamethasone (DEX)-induced apoptosis and Fas expression in peripheral blood lymphocytes of SLE patients and normal subjects. Peripheral blood lymphocytes were obtained from 40 SLE patients and 18 sex- and age-matched control subjects. Percentages of apoptosis and expression of Fas molecule in lymphocytes were assessed by flow cytometry. Fas expression in lymphocytes treated with or without DEX was significantly higher in SLE patients than normal controls [median (interquartile range) of mean fluorescence intensity without DEX: 74.9 (50.7–98.0) vs 20.0 (17.7–25.0), p < 0.001; with DEX: 77.9 (56.0–130.5) vs 20.5 (18.6–24.7), P < 0.001]. DEX (0.1–5 μM) could also induce apoptosis of lymphocytes from SLE and control subjects in a dose-dependent manner. Elevation of apoptotic susceptibility was more prominent in DEX-treated SLE lymphocytes [33.9% (24.7–37.5%) vs 19.6% (13.6–26.1%), P = 0.003]. The higher apoptotic susceptibility of SLE lymphocytes upon DEX treatment in vitro may be related, at least partly, to the pharmacological action of corticosteroids.

CDNA cloning, sequencing and characterization of bovine pim-1

The cDNA clone of bovine pim-1 has been isolated from phorbol-12-myristate-13-acetate (PMA) and concanavalin A (ConA)-activated peripheral blood lymphocytes (PBLs). The full-length cDNA contains a 411 bp 5′ untranslated region (5′-UTR), followed by a 939 bp coding region and a 3′ untranslated region (3′-UTR) that contains 1403 bp. Comparison of the bovine pim-1 coding sequence with the human, rat, mouse, frog and zebrafish counterparts reveals 94, 90, 89, 67 and 40% homology at the nucleotide level, respectively. The predicted amino acid sequence of bovine Pim-1 shares 98.7, 97.1, 93.3, 68.8, and 52.4% similarity with the sequences of human, rat, mouse, frog, and zebrafish, respectively. The 5′-UTR of bovine pim-1 shares high sequence similarity to the human and mouse counterparts and is G/C-rich (75%) which may promote a high degree of secondary structure. The 3′-UTR of bovine pim-1 contains two potential polyadenylation sites and an A/T-rich motif which has been shown to decrease the stability of polyA mRNA molecules. Southern blot results indicate that a single copy of the gene exists in the bovine genome. Northern blot results show that PMA stimulation of PBLs increases the expression of the pim-1 mRNA. In addition, examination of Pim-1 protein expression in PBLs stimulated with a variety of mitogens including ConA, PMA, anti-CD3 and purified protein derivative (PPD) from Mycobacterium tuberculosis, reveals two different types of expression patterns during the course of a 24 h period of stimulation. ConA and PPD gave a biphasic pattern of expression while PMA and anti-CD3 gave single transient pattern of expression suggesting that expression is controlled by more than one signaling pathway.