mRNA Expression In Lymphocytes Research Articles

Activation of the Adenosine A2A Receptor Promotes the Development of Donor-Derived T Regulatory Cells in a Graft-Versus Host Disease Mouse Model.

Abstract 1339Poster Board I-361 IntroductionGraft versus host disease (GVHD) remains a significant complication of allogenic stem cell transplantation and is a considerable cause of transplant morbidity and mortality. The recognition of the role of Foxp3+ regulatory T cells in immunomodulation has given rise to interest in using these cells to abrogate or modify the severity of graft versus host disease as well as in methods to increase their development during transplant. A less well known avenue of research is the targeting of the adenosine A2A receptor (A2AR). In ischemia models, activation of the Gs-coupled adenosine receptors play a role in terminating inflammation and improving survival of damaged and/or transplanted organs by directly down regulating the activity of the receptor bound T cell. We have shown previously that the use of a specific A2AR agonist known as ATL146e decreases the incidence and severity of GVHD as well as improves survival of mice in a GVHD transplant model (paper in submission). MethodsIn order to further understand the role of the agonist in GVHD abrogation we performed studies looking at the possible role of T regulatory cells in relation to the use of the agonist. Using a parental into irradiated F1 offspring transplant model (C57BL/6J [B6, H-2b] → B6D2F1/J [BDF1, H-2b/d]) we can induce GVHD manifested by weight loss and mortality in 100% of mice by infusing an additional 10 million donor T cells into mice previously engrafted with 10 million bone marrow donor cells and 800cGy of radiation. We administered the ATL146e or a PBS control by osmotic mini pumps resulting in continuous subcutaneous infusion for 14days starting one day before the donor T cell infusion. Mice that received only the congenic donor bone marrow transplant and no donor T cells did not develop GVHD and served as an additional control. Post transplant, splenocytes and peripheral blood cells were collected and stained for CD4, CD25 and FoxP3 and were analyzed by flow cytometry. The level of Foxp3 mRNA expression in lymphocytes was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and the concentration of IL-10 in serum was measured by enzyme-linked immunosorbent assay (ELISA). To identify the origin of activated T regulatory cells, we also performed transplants used a B6.PL-Thy1/CyJ (B6-Thy1.1, H-2b) mouse strain as the donor. ResultsFrom these studies we confirmed that the A2AR agonist, ATL146e, inhibited the weight loss and mortality associated with acute GVHD progression and seen in the PBS treated controls. More notably, treatment with ATL146e resulted in a 7 fold increase in CD4+CD25+FoxP3+ T regulatory cells in both the spleen and peripheral blood compared to our PBS treated group at days 14 to 20 after hematopoietic stem cell transplantation. From our Thy-1 disparate transplants we determined that the increased T regulatory cells were of donor origin. We also found that the expression of Foxp3 mRNA in splenocytes and the level of IL-10 in the serum was increased 3 fold and 2.4 fold respectively in the ATL146e treated mice. ATL146e agonist activity is very specific to A2AR. When using an alternate adenosine agonist less specific to A2AR, we did not see the same increases of Foxp3 or IL-10, nor did we see any decrease in the severity of the graft versus host disease. ConclusionsThus we believe that the specific activation of A2AR inhibits acute GVHD through the increase of donor-derived CD4+ CD25+ FoxP3+ immunosuppressive T regulatory cells. Our observation provides an additional mechanistic basis for the anti-inflammatory capacity of A2AR agonist in acute GVHD. Additional studies are ongoing to elucidate further the mechanism of the agonist's ability to increase the T regulatory population as well as the effects of combination therapies. DisclosuresNo relevant conflicts of interest to declare.

Myocardial and Peripheral Lymphocytic Transcriptomic Dissociation of β-adrenoceptors and G Protein–coupled Receptor Kinases in Heart Transplantation

The genetic expression of adrenergic receptors plays an important pathophysiologic role in heart failure. G protein-coupled receptor kinases (GRKs) desensitize the beta-receptor to catecholaminergic stimulation. It has been suggested that their mRNA expression in peripheral lymphocytes could mirror the changes in their myocardial expression in the failing heart, but this relationship between the myocyte and lymphocyte has not been studied in heart transplantation (HT). The objective of this study was to analyze adrenergic receptor and GRK mRNA expression in myocardium and lymphocytes and their correlation. Twenty-three HT patients without evidence of acute rejection or echocardiographic dysfunction were assessed. Myocardial biopsy samples and peripheral blood lymphocytes were obtained, and alpha(1)- and beta-adrenoceptor subtype and GRK subtype mRNA was analyzed using reverse transcript-polymerase chain reaction (RT-PCR). Mean age was 45 +/- 15 years, with a median of time since HT of 205 (351) days. In biopsies, the beta(1)/beta(2)-adrenoceptor ratio was 57%/42%, and GRK5 was the most commonly expressed, followed by GRK2. In lymphocytes, the beta(1)/beta(2) ratio was 3%/96%, whereas GRK2 mRNA expression was greater than that of other subtypes. There was no correlation between myocardial and lymphocyte parameters. There were no correlations with clinical variables, but lymphocyte beta(2) and GRK2 were increased with time since HT. In the transplanted heart, there is no correlation between mRNA expression of adrenoceptors and GRKs in myocardium and peripheral lymphocytes. With time since transplant, mRNA expression of lymphocyte but not myocardial beta(2)-adrenoceptor and GRK2 increases. Therefore, this dissociation between myocardial and lymphocyte mRNA expression limits the potential use of peripheral blood samples for diagnosis of graft dysfunction.

Role of anandamide transporter in regulating calcitonin gene-related peptide production and blood pressure in hypertension

To explore the role of anandamide (AEA) transporter in regulating calcitonin gene-related peptide (CGRP) production and blood pressure. Plasma levels of AEA, CGRP, asymmetric dimethylarginine (ADMA) and nitric oxide in patients with essential hypertension, spontaneously hypertensive rats (SHRs) and 2 kidney 1 clip hypertensive rats and the CGRP mRNA expression in dorsal root ganglion of rats were measured. Peripheral blood lymphocytes were isolated to examine the AEA transporter activity, the role of AEA transporter in regulating CGRP mRNA expression or the effect of exogenous ADMA on AEA transporter activity. In both hypertensive patients and SHRs, the plasma level of AEA was elevated, but the AEA transporter activity was attenuated concomitantly with decreased CGRP production. Moreover, plasma ADMA level in SHRs was elevated accompanied by decreased nitric oxide level. By contrast, the plasma AEA level was elevated accompanied by increased CGRP production in 2 kidney 1 clip hypertensive rats, and there were no significant changes in plasma levels of ADMA, nitric oxide and the AEA transporter activity. In vitro, exogenous administration of AEA upregulated CGRP mRNA expression in lymphocytes, which was inhibited by AEA transporter blocker, AM404, and the AEA transporter activity was reduced by ADMA. Decreased plasma CGRP level in patients with essential hypertension or SHRs is likely due to the reduced AEA transporter activity, and the increased ADMA level may account for the reduced AEA transporter activity.

The high risk allele of a six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with favorable outcome in multiple cancers

11073 Background: Recently, two polymorphisms in CASP8 were independently identified as factors influencing cancer susceptibility. A non-synonymous single nucleotide polymorphism (SNP) in exon 9 (D302H, rs1045485) has been associated with breast cancer risk and an insertion-deletion promoter polymorphism (-652 6N del, rs3834129) has been associated with susceptibility to multiple cancers, e.g. breast and colorectal cancer. The deletion variant of this polymorphism disrupts a Sp1 binding site and is associated with decreased mRNA expression in lymphocytes of healthy volunteers. Methods: 500 healthy blood donors and 436 patients with different malignancies (breast cancer (n=199), colorectal cancer (n=140), B-CLL (n=97)) were retrospectively genotyped for both polymorphisms. Polymorphisms were analysed for linkage equilibrium and possible association of genotypes as well as haplotypes with risk for cancer. CASP8 expression in the cDNA of 180 snap-frozen tumor samples was analysed by quantitative Real-Time-PCR. Both genotypes and haplotypes were correlated with CASP8 expression and the patients’ survival. Results: Kaplan-Meier curves revealed a significant association of the -652 6N del polymorphism with survival in breast cancer patients (p=0.002), colorectal cancer patients (p=0.042), and patients with B-CLL (p=0.046). Additionally, CASP8 expression was genotype-dependent significantly different in tumor specimens of the three malignancies: breast cancer (p=0.010), colorectal cancer (p=0.015), and B-CLL (p=0.041), respectively. -652 6N del and the second polymorphism D302H were in strong linkage disequilibrium (D´=1) and Kaplan-Meier curves revealed no independent or cooperative effect of the D302H SNP. Conclusions: The CASP8 -652 6N del polymorphism is a potential survival prognosticator as well as indicator of high risk groups within different cancers. Additionally, these results suggest that, although the exon 9 SNP is non-synonymous, only the -625 6N del polymorphism seems to be functional and of further interest. No significant financial relationships to disclose.

G allele of the SNP -1149G/T of the Extrapituitary prolactin promoter is involved in the SLE pathogenesis

Objective Prolactin (PRL) is a polypeptide hormone produced by pituitary lactotrophs and extrapituitary tissues including immune cells. PRL acts as a cytokine and influences immune cells maturation and differentiation. Association between high serum PRL levels and systemic lupus erythematosus (SLE) has been demonstrated. Extrapituitary PRL synthesis is directed by an alternative promoter, which contains single nucleotide polymorphism (SNP) -1149 G/T. Higher PRL mRNA expression in lymphocytes has been associated with G allele. We investigated the role of this G allele in relation to clinical and laboratory features of SLE. Methods We investigated -1149 G/T SNP in 156 SLE patients and 123 healthy individuals (control group). SLE patients: 134 (85.9%) females and 22 (14.1%) males, average age 43.4 years. Control group: 40 (32.5%) females and 83 (67.5%) males, average age 38.7. PCR-RFLP methodology was used for -1149 G/T SNP detection. PCR: The 137 base pairs (bp) region of the PRL extrapituitary promoter was amplified by employing the following primers: forward 5’-GCAGGTCAAGATAACCTGGA and reverse 5’-CATCTCAGAGTTGAATTTATTTCCTT. RFLP: ApoI restriction endonucleasis was used. The genotypes we identified were:TT homozygote characterized by 120bp+17bp, GG homozygote by 85bp+35bp+17 bp and GT heterozygote by 120bp+85bp+35bp+17bp DNA fragments. Results were evaluated by χ 2 test with Bonferroni correction. Results In the SLE group there was no difference in genotype and allele frequencies compared to healthy individuals. With respect to specific organ manifestation of SLE we detected an association between G allele and arthritis (Pc=0.0086;OR 2.56,CI 1.51-4.33). According to age when SLE was diagnosed we observed GG genotype frequency in 21-to-40-years subgroup in 44.8% compared to 15.8% and 24.0% in the <20 years and >40 years subgroup, respectively (Pc=0.023;OR 2.94,CI 1.43-5.96). Conclusion The presence of G allele and GG genotype of the PRL extrapituitary promoter -1149 G/T SNP is associated with certain clinical features of SLE namely arthritis and age of SLE onset.

Thyroglobulin mRNA expression in peripheral blood lymphocytes of healthy subjects and patients with thyroid disease

Background Thyroglobulin (Tg) mRNA is expressed focally in thyroid tissue. In recent years, the Tg gene has been detected in other tissues, including lymphocytes, although the significance of its presence has not been elucidated yet. We measured Tg mRNA expression in the lymphocytes of healthy subjects and those with thyroid disease. Methods Analysis of the quantification of Tg mRNA from 20 healthy subjects and 47 subjects with thyroid disease was carried out by real-time PCR. Furthermore, in cultured lymphocytes we compared changes in Tg mRNA expression following stimulation with TSH. Results Tg mRNA was detected in the lymphocytes of all subjects. Tg mRNA in the lymphocyte sequence matched that derived from thyroid tissue, and mRNA levels were higher in subjects with thyroid disease than in healthy subjects. Following lymphocyte stimulation, Tg mRNA levels were observed to be increased 2.7-fold in Graves' disease and 1.6-fold in chronic thyroiditis compared to healthy subjects. Conclusions Tg mRNA in the lymphocytes was quantified by real-time PCR. The levels of Tg mRNA in the TSH-stimulated lymphocytes were noticeably increased in subjects with thyroid disease. These results suggest an interesting relationship between production of Tg antigen in peripheral blood and autoimmunity in thyroid disease.

A “possible” involvement of TNF-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis

Feline infectious peritonitis (FIP) cats show a decrease in peripheral blood lymphocyte counts, and a particularly marked decrease in T cells including CD4 + and CD8 + cells. In this study, we showed that lymphopenia observed in FIP cats was due to apoptosis, and that the ascitic fluid, plasma, and culture supernatant of peritoneal exudate cells (adherent cells with macrophage morphology, or PEC) from FIP cats readily induced apoptosis in specific pathogen-free cat peripheral blood mononuclear cells, particularly CD8 + cells. In addition, TNF-alpha released from macrophages and TNF-receptor (TNFR) 1 and TNFR2 mRNA expression in lymphocytes were closely involved in this apoptosis induction. In particular, in CD8 + cells cultured in the presence of the PEC culture supernatant, the expression levels of TNFR1 and TNFR2 mRNA were increased, indicating that CD8 + cells are more susceptible to apoptosis induction by TNF-alpha than other lymphocyte subsets, particularly B cells (CD21 + cells). The results of this study suggest that TNF-alpha, produced by virus-infected macrophages, is responsible for induction of apoptosis in uninfected T cells, primarily CD8 + T cells.

The Role of SPECs, Small Cdc42-binding Proteins, in F-actin Accumulation at the Immunological Synapse

SPEC1 and SPEC2 are structurally similar Cdc42-binding proteins of 79 and 84 amino acid residues, respectively. We investigated the role of SPEC2 in T cell function due to its high mRNA expression in lymphocytes. Western blot analysis revealed abundant SPEC2 protein in lymphocytes, which in glutathione S-transferase-capture experiments specifically interacted with only GTP-bound Cdc42. Immunofluorescence experiments revealed that the SPEC2 protein was diffusely localized in the cytoplasm and at the cell membrane in unstimulated Jurkat T cells and Raji B cells. Recruitment of SPEC2 within Jurkat T cells to the antigen-presenting cell interface occurred following incubation with staphylococcal enterotoxin E superantigen-loaded B cells and colocalized there with F-actin and Cdc42. T cell receptor (TCR) activation studies using anti-CD3 antibody-coated polystyrene beads showed that SPEC2 was recruited to the site of bead contact, which was not observed with anti-major histocompatibility complex antibody-coated beads. Accumulation of SPEC2 following TCR engagement occurred as early as 5 min, before obvious F-actin accumulation. Biochemical studies with Jurkat T cells demonstrated that N-terminal cysteine residues in SPEC2 were palmitoylated. Overexpression studies of the related SPEC1 showed that it also was recruited to the activated TCR. Mutational analysis revealed that localization of SPEC1 to the TCR required two N-terminal cysteine residues. Furthermore, a SPEC1 Cdc42 Rac-interacting binding mutant, containing an intact N terminus but defective in Cdc42 binding, completely blocked F-actin accumulation at the activated TCR. Taken together these results suggest that SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse.

Expression of Matrix Metalloproteinase-9 in Pleural Effusions of Tuberculosis and Lung Cancer

Background: Matrix metalloproteinase (MMP)-9 has been implicated in the development of pleural effusions. Objectives: The aim of this study was to assess the expression of MMP-9 in pleural effusions of tuberculosis, lung cancer and transudates. Methods: Ninety-one patients (37 tuberculous pleural effusions, 42 malignant pleural effusions of lung cancer and 12 transudates) were included. Concentrations of pleural fluid MMP-9 and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were determined by immunoassay. We also investigated the cellular localization of MMP-9 and TIMP-1 by reverse-transcriptase polymerase chain reaction on lymphocytes from pleural effusions and by immunohistochemical analysis of pleural tissues. Results: Pleural fluid MMP-9 levels, MMP-9/total protein and MMP-9/TIMP-1 ratios were significantly higher in tuberculous pleural effusions, whilst TIMP-1 levels were similar in the three groups. MMP-9 levels positively correlated with TIMP-1 and lactate dehydrogenase levels, and negatively with pH and glucose levels in pleural effusions. MMP-9 mRNA expression in lymphocytes tended to be higher in malignant pleural effusions of lung cancer than in the other groups without reaching statistical significance. The strongest immunoreactivity for MMP-9 was observed in epithelioid cells of tuberculous pleural tissues. Much lower levels of MMP-9 expression were found in tumor cells of pleural tissues. Conclusions: MMP-9 is increased in tuberculous pleural effusions compared with transudates and malignant pleural effusions of lung cancer and is produced predominantly by epithelioid cells in the granulomas of tuberculous pleural tissues.

Effects of Photoperiod During the Dry Period on Prolactin, Prolactin Receptor, and Milk Production of Dairy Cows

Cows exposed to short day photoperiod during the dry period produce significantly more milk in their subsequent lactation than cows exposed to long days. The mechanism(s) underlying this effect are unknown. Because concentrations of prolactin (PRL) in circulation are consistently affected by changes in photoperiod, we hypothesized that alterations in the prolactin axis and sensitivity of the mammary gland to prolactin signaling may mediate photoperiodic effects in dry cows. The objective of this study was to determine the effects of exposure to different lengths of daylight during the dry period on circulating PRL and PRL receptor (PRL-R) mRNA expression in lymphocytes and mammary tissue during the transition to lactation. Multiparous Holstein cows were dried off 62 d before calving and assigned to long day (16h light: 8h dark) or short day photoperiod (8h light: 16h dark). During the dry period, PRL and PRL-R mRNA were analyzed biweekly in plasma and lymphocytes, respectively. Expression of PRL-R mRNA was assessed in mammary biopsies during the dry and periparturient periods. Dry matter intake (DMI) was recorded through 21 d of lactation, and milk yield was recorded until 120 d in milk. Short day photoperiod was associated with reduced PRL, whereas milk yield and expression of PRL-R mRNA in lymphocytes and mammary tissue were increased. Cows on short days had higher DMI during the dry period but did not differ in DMI after parturition. These data support the concept that greater responsiveness and sensitivity to PRL during transition to lactation may be associated with an increase in subsequent milk yield.

Comparison of cytochrome P4502E1 (CYP2E1) activity and hepatic and lymphocyte mRNA expression in patients with chronic hepatitis C

The induction of cytochrome P4502E1 (CYP2E1) is believed to play a role in the development of fibrosis in hepatitis C patients. However, information about CYP2E1 activity in chronic hepatitis C patients is fragmentary and the relationship between CYP2E1 activity and mRNA expression is unknown in this disease. The purpose of this study was (a) to characterise CYP2E1 activity in those patients and (b) to analyse its relationship with CYP2E1 mRNA expression in the liver and in peripheral blood lymphocytes (PBLs), previously proposed as a surrogate to assess changes in CYP2E1 activity. Fourteen chronic hepatitis C patients were submitted to a routine transcutaneous liver biopsy. CYP2E1 activity was assessed by using chlorzoxazone (CZX) pharmacokinetic parameters and hepatic and PBLs CYP2E1 mRNA expression was measured by real-time RT-PCR. The mean oral clearance of CZX (CL T: 21.5 ± 10.1 L/h) was within the normal range and the chlorzoxazone metabolic ratio (CMR) at t = 2 h was closely related to other CZX pharmacokinetic parameters. None of the pharmacokinetic parameters did significantly correlate with CYP2E1 mRNA, neither in the liver nor in PBLs. Furthermore, there was no significant relationship between CYP2E1 mRNA levels in paired liver and PBL samples. Our data indicate that early stages of chronic hepatitis C are not associated with CYP2E1 induction. In this disease, the determination of the CMR at t = 2 h represents a reliable index to assess CYP2E1 activity. The measurement of CYP2E1 expression, at the mRNA level, in PBLs or in liver is not useful for that purpose.

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.

Lack of correlation between lymphocytic mrna expression and activity of CYP3A4 and CYP2E1 In-vivo in alcoholics and non-alcoholics

Lymphocyte mRNA levels may serve as a surrogate for activity of drug metabolizing enzymes. We conducted a study to examine the relationship between CYP2E1 and CYP3A4 lymphocyte mRNA levels and the clearances (CL) of oral chlorzoxazone (CHZ) and oral and IV midazolam (MDZ), respectively. 20 alcoholics (ALC) (> 140gm of alcohol/wk) and 20 non-alcoholics (N-ALC) (20 African-Americans 20 Caucasians) enrolled in the study. MDZ was given IV (0.05mg/kg over 30 min) and orally (5mg) on consecutive days. CHZ (500mg) was administered orally 8 hr after IV MDZ. mRNA was isolated from blood lymphocytes, CYP3A4 and 2E1 mRNA was quantitated using real time PCR. Serum CHZ and MDZ concentrations were determined by HPLC-UV or LC-MS. There was no significant difference (p>0.05) between ALC and N-ALC for CYP3A4 mRNA, MDZ CLORAL (149.4±87.8 vs 120.3±84.1 L/hr) and CLIV (36.9±12 vs 36.6±14.1 L/hr). No correlation was observed between lymphocytic CYP3A4 mRNA and CYP3A activity. CYP2E1 mRNA levels were not significantly different between ALC and N-ALC. In contrast, CHZ CLORAL was significantly greater (p<0.05) in ALC (31.5±11.9) compared to N-ALC (23.4±8.7) but no correlation was observed between CYP2E1 activity and mRNA. No significant race based differences were observed for either CYP mRNA or activity. In conclusion, lymphocyte CYP2E1 and CYP3A4 mRNA levels did not reflect hepatic CYP2E1 activity nor hepatic and intestinal CYP3A activity. Clinical Pharmacology & Therapeutics (2004) 75, P36–P36; doi: 10.1016/j.clpt.2003.11.137

Analysis of the immunological microenvironment at the tumor site in patients with non-small cell lung cancer.

Inactivation of tumor-infiltrating lymphocytes by immunomodulating cytokines shed by tumor cells into the tumor local microenvironment might be a potential escape strategy of various tumors from immune-immediate killing. Here, we provide an analysis of the cytokine profile at the tumor site in patients with non-small cell lung cancer (NSCLC). Using in situ hybridization (ISH), we determined the mRNA expression in lymphocytes and tumor cells for IL-2, INF-gamma, IL-12 (p40), IL-18, IL-4, IL-10, TGF-beta1, IL-1, IL-3, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF-alpha, and TGF-alpha in five fresh pleural effusion samples and 18 tumor tissue samples of patients with NSCLC. In pleural effusion as well as in tumor tissue of NSCLC patients, the mRNA expression of IL-4, IL-10, TGF-alpha, and TGF-beta1 was significantly higher than that of IL-2, IL-12, IL-18 and INF-gamma. In contrast, the analysis of tuberculosis pleural effusion samples revealed lower mRNA levels for all cytokines and did not show any significant difference among them. The predominant mRNA expression of type II and immunosuppressive cytokines in pleural effusion and tumor tissue of NSCLC patients mirrors an immunosuppressive state in the immunological microenvironment. The present study may, therefore, help to elucidate mechanisms of tumor escape and contribute to the development of an effective immunomodulatory treatment of NSCLC.

D3 dopamine receptor mRNA expression in lymphocytes: a peripheral marker for schizophrenia?

Identification of schizophrenia, a common neuropsychiatric disorder, is based on clinical examination. An easily measurable peripheral marker, which may enable a more rapid and more accurate diagnosis, is not available. A possible candidate is the D3 dopamine receptor on lymphocytes. The D3 receptor is investigated for its clinical significance as a marker for diagnosing schizophrenia. From eight schizophrenic patients and eight controls lymphocyte RNA was isolated. A semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out and the intensities of the specific D3 dopamine receptor bands of patients and controls were compared. No difference could be seen between the intensities of the bands from patients and controls. An aberrant D3 dopamine receptor mRNA expression in lymphocytes of schizophrenics could not be demonstrated. This might be caused by down-regulation of D3 receptor production by antipsychotic drug treatment. At present, the D3 receptor seems to have no diagnostic value in schizophrenia.

Comparison of Calcitonin Gene-Related Peptide Release from Rat Lymphocytes and Dorsal Root Ganglia Neurons

Calcitonin gene-related peptide (CGRP), a neuropeptide contained in primary sensory neurons, has been demonstrated to be synthesized and released by rat lymphocytes in our previous studies. In this study, the release properties and molecular characteristics of CGRP such as immunoreactivity (CGRP-LI) from lymphocytes were compared with those from dorsal root ganglia (DRG) neurons by using CGRP-specific RIA, reverse-phase HPLC, and RT–PCR. Con A and IL-2 could trigger CGRP-LI release from lymphocytes in a time-dependent manner. After 3 days stimulation with 4 μg/ml Con A, the level of CGRP-LI released by lymphocytes was increased from 77.4 ± 9.6 pg/108 cells to 191.1 ± 13.6 pg/108 cells and increased further to 374.5 ± 38.3 pg/108 cells after 5 days. Stimulation with 750 U/ml human IL-2 recombinant (rhIL-2) caused a significantly elevated CGRP-LI release from 75.4 ± 6.5 pg/108 cells to 266.2 ± 16.2 pg/108 cells after 3 days and to 469.1 ± 43.2 pg/108 cells after 5 days. Con A and IL-2 also augmented CGRP mRNA expression in lymphocytes. In the tested period (1–5 days), Con A and rhIL-2 had no stimulating effect on CGRP release from DRG neurons. In contrast, a high concentration of potassium and LPS could induce an acute release of CGRP from DRG neurons, but not from lymphocytes. Lymphocyte-released CGRP-LI was shown to coelute with synthetic rat CGRP (rCGRP) and DRG neuron-released CGRP by reverse-phase HPLC. In addition, to displace 125I-CGRP from CGRP antibody by lymphocyte-released CGRP-LI was similar to that by synthetic rCGRP. These data suggest that lymphocyte- and nerve-derived CGRP-LI are similar in terms of immunological characteristics, molecular size, and polarity. However, lymphocytes secrete CGRP-LI in response to different stimuli compared to nerve-derived CGRP.

The role of IL-2 in allograft rejection--a lesson learned from experimental work.

The role of IL-2 in allograft rejection--a lesson learned from experimental work.

Reduced sensitivity to glucocorticoid feedback and reduced glucocorticoid receptor mrna expression in the luteal phase of the menstrual cycle

We examined the effects of the menstrual cycle on hypothalamic-pituitary adrenal axis regulation in healthy women with no history of psychiatric illness by measuring plasma cortisol responses to a low-dose (0.25 mg) of dexamethasone (N = 23) and by measuring glucocorticoid receptor (type II) mRNA expression in lymphocytes using Northern blotting (N = 19). Both measures were performed in the early follicular and mid-luteal phases of the menstrual cycle. Dexamethasone suppression of plasma cortisol was greater in the follicular phase of the menstrual cycle compared to the mid-luteal phase (p < .01). In addition, type II glucocorticoid receptor mRNA expression in lymphocytes was 78% higher in the follicular phase compared to the mid-luteal phase (p < .02). These results indicate that glucocorticoid feedback regulation of the hypothalamic-pituitary-adrenal axis is reduced in the mid-luteal phase of the menstrual cycle. Reduced feedback regulation of central stress response systems may play a role in generation of common premenstrual symptoms of irritability and dysphoria.

Angiotensin Converting Enzyme Inhibition Corrects Na +/H + Exchanger Overactivity in Essential Hypertension

In this study, we investigated whether antihypertensive treatment with the angiotensin converting enzyme inhibitor quinapril modifies Na +/H + exchanger activity or NHE-1 (isoform of the exchanger) mRNA expression in lymphocytes from patients with essential hypertension. Thirty-three hypertensive patients and 27 normotensive subjects were studied. Maximal sodium-proton exchange activity was determined by acidifying cell pH and measuring the initial rate of the net sodium-dependent proton efflux driven by an outward proton gradient. The transcript level of NHE-1 was measured by reverse transcription-polymerase chain reaction in comparison with a constitutively expressed reference gene ( β-actin). With the 100% confidence (upper) limit of the normotensive population as a cutoff point, a subgroup of 11 hypertensive patients had an abnormally high lymphocyte Na +/H + exchange activity (group A). The activity of the exchanger was within the normal range in the remaining patients (group B). After 6 months of quinapril treatment the activity of the exchanger decreased to normal values ( P < .001) in patients from group A, but remained unchanged in patients from group B. The NHE-1 mRNA expression was not modified with treatment neither in patients from the group A, nor in patients from the group B. These results suggest that chronic angiotensin enzyme inhibition with quinapril abolishes Na +/H + exchange overactivity present in lymphocytes from a subgroup of hypertensive patients. This effect appears to be independent of changes in the expression of the mRNA encoding for the NHE-1 isoform of the exchanger.

Involvement of P-Glycoprotein in the Transmembrane Transport of Interleukin-2 (IL-2), IL-4, and Interferon-γ in Normal Human T Lymphocytes

The physiological role of the multidrug resistance P-glycoprotein (P-gp), which is expressed by normal human T lymphocytes, is still largely unknown. To investigate whether or not P-gp is involved in the transport of cytokines, peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA) in the absence or presence of P-gp inhibitors, and concentrations of cytokines (interleukin-2 [IL-2], IL-4, IL-6, interferon-gamma [IFN-gamma]) in the supernatants of these cultures were quantitated by enzyme-linked immunosorbent assay. P-gp inhibitors included verapamil (Ver), tamoxifen (Tmx), and the P-gp specific monoclonal antibody UIC2. Release of IL-2 was significantly suppressed by these inhibitors at concentrations that were also effective in blocking efflux of Rhodamine-123 from normal T lymphocytes. IL-2 mRNA expression in lymphocytes was not different between PHA control and the cultures with P-gp inhibitors. Ver and Tmx did not interfere with T-cell activation as determined by CD25 and CD69 expression. In a nonhematological model, the P-gp expressing HCT-8 adenocarcinoma cell line, exogenously added IL-2 was shown to exert an inhibitory effect on P-gp mediated Rhodamine-123 efflux. In addition, transepithelial transport of IL-2 by electrophysiologically tight and polarized HCT-8 monolayers was examined. A time-dependent flux of IL-2 across dense monolayers, which was partially inhibited by Ver, was observed. We also investigated whether or not P-gp inhibitors suppressed release of other cytokines produced by activated T cells (IL-4, IL-6, IFN-gamma). Release of IL-4 and IFN-gamma was significantly inhibited by Ver, Tmx, and UIC2; however, release of IL-6 remained unaffected. These data show P-gp mediated transmembrane flux of IL-2 in T lymphocytes and HCT-8 cells. We conclude that P-gp participates in the transport of cytokines (IL-2, IL-4, and IFN-gamma) in normal peripheral T lymphocytes.