Mononuclear Leukocytes Of Patients Research Articles

The State of Intracellular Molecular Regulators during the Reconvalescence of Community-Acquired Pneumonia under the Influence of Microwaves at 1 GHz

We evaluated the levels of protein p53, retinoblastoma protein (RB), β-catenin, SMAD2, protein kinases AKT and focal adhesion kinase (FAK), and transcription factor CREB using ELISA in mononuclear leukocytes of patients with community-acquired pneumonia on days 15–17 of the disease. The research results showed that the subclinical immune-inflammatory process was characterized by a higher content of β-catenin by 19.8%, CREB by 23.3%, RB protein by 14.7%, increased phosphorylation level of protein kinase FAK by 19.8%, АKТ1 at serine-473 by 65.6%, and RB by 13.7%. In the cells, there was a decrease in p53 of 15.7%, SMAD2 of 16.9%, and protein kinase АKТ1 of 31.2%. Three hours after 1-GHz microwave irradiation, MNCs displayed a statistically significant increase in their content of the p53 protein of 20.0‰, RB of 9.35‰, β-catenin of 10.9‰, protein kinase FAK of 10.0‰, and CREB of 8.55‰. A day after a single irradiation of the cultures of whole blood cells, in irradiated cells compared to nonirradiated control, we observed a statistically significant increase in the content of p53 of 24.2‰, β-catenin of 15.1‰, SMAD2 of 20.4‰, RB of 9.8‰, as well as a reduction of the initially elevated levels of protein kinase AKT1 of 25.1‰, its phosphorylated form at serine-473 of 16.5‰, and a decrease in the phosphorylated form of the transcription factor CREB at serine-133 of 4.1‰. The research results indicate that a low-intensity microwave frequency of 1 GHz can be considered as a factor in molecular immune rehabilitation in conditions of community-acquired pneumonia.

The state of membranes of mononuclear leukocytes in patients with tuberculosis of lungs.

The sampling of examined patients comprised 308 individuals with medication sensitive (or with absence of data about resistance) tuberculosis of lungs without decay of pulmonary tissue with purpose to detect characteristics of alteration of content and structural organization of monocytic membranes before treatment and also against the background of application of chemotherapy according 1 standard regimen. The data of clinical radiologic examination of patients, bacterioscopy, molecular genetic analysis and analysis of phospholipid spectrum of membranes of mononuclears of peripheral blood were used. To detect particular classes of phospholipids the thin-layer flow chromatography was applied. The following fractions of phospholipids were detected: aggregated lysophospholipids, sphingomyelin, phosphatidylinositol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine. To evaluate structurally functional condition of mononuclears of peripheral blood under tuberculosis the coefficients of activity of phospholipase and membrane permeability were applied. It is established that in case of tuberculosis infection an alteration of functional activity and restructuring of membranes of mononuclears of peripheral blood is observed expressed by increasing of content percentage of phosphatidylinositol and phosphatidylethanolamine and in decreasing of level of sphingomyelin, phosphatidylcholine and phosphatidylserine. In patients with tuberculosis prior to treatment decreasing of coefficient of membrane permeability. After application of chemotherapy coefficient of membrane permeability returned to standard. The anti-tuberculosis chemotherapy gives rise to activation of phospholipase A2 that shows membrane destructive effect on mononuclears of peripheral blood and results in disorganization of their membranes manifesting in cumulation of lysophospholipids at simultaneous decreasing of percentage content of phosphatidylserine and phosphatidylcholine. The toxic effect of anti-tuberculosis chemotherapy has no effect on number of monocytes in peripheral blood. At that, disorganization of membrane structures of these cells and possible suppression of their functional activity occur.

Insulin Receptor Substrate-1 Expression Is Increased in Circulating Leukocytes of Patients with Acute Coronary Syndrome

The mechanisms underlying the increased risk of cardiovascular disease associated with diabetes mellitus (DM) are not fully defined. Insulin resistance in human metabolic syndrome patients is associated with decreased expression of the insulin receptor substrate-2- (Irs2-) AKT2 axis in mononuclear leukocytes (MLs). Moreover, acute coronary syndrome (ACS) has been linked through genome-wide association studies to the 2q36-q37.3 locus, which contains the Irs1 gene. Here, we investigated the expression of insulin-signaling pathway genes in MLs from patients with DM, ACS, and ACS plus DM. Quantitative real-time PCR expression studies showed no differences in the mRNA levels of Irs2, Akt2, and Akt1 among all patients. However, Irs1 mRNA expression was significantly increased in patients with ACS—diabetics and nondiabetics—compared with diabetic patients without ACS (P < .02 and P < .005, resp.). The present study reveals for the first time an association between increased Irs1 mRNA levels in MLs of patients with ACS which is not related to DM.

Cytoprotective Mechanisms of the Lung in Sickle Cell Disease.

Abstract 1538Poster Board I-561Circulating plasma hemoglobin contributes to major vasculopathies including pulmonary hypertension in patients who have sickle cell disease (SCD). There is an emerging concept that such vasculopathies are relatively mild because of activation of several cytoprotective pathways in SCD. The biochemical profile of plasma and the transcriptome of peripheral blood cells in patients who have SCD offer indirect support for this concept. Indeed, heme oxygenase-1 (HO-1), an acute phase enzyme that degrades heme into intermediates and byproducts with vasculoprotective properties is markedly elevated in mononuclear leukocytes of patients who have SCD. Nonetheless, the scope of the cytoprotective mechanisms of the lung and other organs impacted directly by sickle vasculopathies remain poorly appreciated. We previously identified an array of cytoprotective enzymes in lung endothelial cells chronically exposed to non-toxic concentrations of hemin in vitro. In this study, we examined the expression of NAD(P)H oxidase and candidate cytoprotective enzymes in two models of transgenic mice with SCD, and examined HO-1 expression in sickle chronic lung disease. Although NAD(P)H oxidase catalyzes reactive oxygen species generation by heme and is responsible for increased adhesion of leukocytes to the endothelium in SCD mice, there was no elevation of any of its subunits (gp91Phox and p22Phox, p47Phox, p67Phox and p40 Phox) in sickle mice lungs compared to hemizygote control mice lungs. Quantitative RT-PCR analysis revealed unexpectedly no difference in HO-1 mRNA level in sickle and non-sickle control lungs. On the contrary, analysis of the same tissues showed significantly higher NAD(P)H quinone oxidoreductase-1 (NQO1) mRNA level in both Berkeley and Townes knock-in sickle mice compared to controls (p<0.001). Enhanced expression of NQO1 but not HO-1 in sickle mice lungs was confirmed at the protein level by western blot analysis and by immunohistochemistry. Finally, we studied cases of sickle chronic lung disease (n=17) and discovered that NQO1 is expressed in the pulmonary endothelium at significantly higher levels than in normal lung tissues (p<0.002). In agreement with the data in mice, we found no difference in HO-1 staining in sickle human lung and normal lung tissues. Our findings indicate that despite its established role in acute elimination of excess heme, HO-1 is not elevated in sickle lungs. This study highlights the importance of dissecting the cytoprotective phenotype of individual organs impacted by SCD towards a rationale approach to developing efficacious antioxidant therapy for this disorder. DisclosuresNo relevant conflicts of interest to declare.

Increased Expression of Cell–Cell Signaling Genes by Stimulated Mononuclear Leukocytes in Patients with Previous Atherothrombotic Stroke

Background/Aims: Inflammation plays an important role in atherosclerosis and stroke. Acute infections are recognized as trigger factors for ischemic stroke. Methods: In this whole genome expression profile study of 15 patients and 15 control subjects, we tested the hypothesis that patients with a history of atherothrombotic stroke show enhanced transcription of inflammatory genes in circulating leukocytes. RNA from unstimulated or lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) was analyzed with Affymetrix U133A GeneChips using a pooling design. Expression of single genes and functional groups of genes was analyzed by global statistical tests. Results: A total of 10,197 probe sets showed positive calls. After correction for multiple testing no single probe set revealed significant differences either without or with LPS stimulation. However, significant global expression differences were found upon LPS stimulation for the group of genes that are involved in cell–cell signaling. Conclusion: LPS stimulation of PBMCs, a condition mimicking bacterial infection, induces differential expression of a group of cell–cell signaling genes in patients with previous atherothrombotic stroke. This finding can be caused by genetic differences between both groups, but acquired risk factors, medication and technical factors may also have contributed to the result.

Codetection of Human Mineralocorticoid Receptor and Both Type 1 and 2 11-Hydroxysteroid Dehydrogenase Transcript Variants in Aldosterone Producing Adenoma Tissue, and in Mononuclear Leukocytes of Patients with Conn’s Syndrome

Codetection of Human Mineralocorticoid Receptor and Both Type 1 and 2 11-Hydroxysteroid Dehydrogenase Transcript Variants in Aldosterone Producing Adenoma Tissue, and in Mononuclear Leukocytes of Patients with Conn’s Syndrome

Myobacterium tuberculosis Induces Selective Up-Regulation of TLRs in the Mononuclear Leukocytes of Patients with Active Pulmonary Tuberculosis

Human and mouse studies indicate that TLRs are important in mycobacterial infections. We investigated TLR gene expression in fresh unstimulated blood and bronchoalveolar lavage from patients with pulmonary tuberculosis using a well-validated, real-time PCR. A human splice variant of TLR1, designated hsTLR1, was found in all donors tested. hsTLR1 mRNA lacks exon 2, which is a 77-bp region of the 5'-untranslated region, but contains the same coding sequence as TLR1. Compared with the matched controls, whole blood from patients had increased levels of mRNA encoding TLR2 (p = 0.0006), TLR1 (p = 0.004), hsTLR1 (p = 0.0003), TLR6 (p < 0.0001), and TLR4 (p = 0.0002). By contrast, expression of these TLRs was not increased in bronchoalveolar lavage. An increased level of hsTLR1 mRNA was found in both CD3- (p = 0.0078) and CD4+ cells (p = 0.028), resulting in an increased ratio of hsTLR1 mRNA to TLR1 and to TLR6 mRNA. An in vitro study in THP1 cells suggested that this relative increase in hsTLR1 might be attributable to a direct effect of mycobacterial components because it could be mimicked by mycobacterial preparations in the absence of IFN-gamma or T cells and by the TLR1/2 agonist Pam3CysK4. Half-life studies using blood from patients with pulmonary tuberculosis and THP1 cells exposed to Myobacterium tuberculosis in vitro showed p38 MAPK-independent stabilization of mRNAs encoding hsTLR1 and TLR1. We conclude that M. tuberculosis exerts direct effects on patterns of TLR expression, partly via changes in mRNA half-life. The significance of these changes in the pathogenesis of disease deserves further investigation.

Dynamics of beta-arrestin1 protein and mRNA levels elevation by antidepressants in mononuclear leukocytes of patients with depression

Background Beta-arrestins interfere in G protein-receptor interaction leading to desensitization of G protein-mediated receptor signaling. G protein-receptor signaling and its desensitization were previously implicated in the pathophysiology, diagnosis and treatment of mood disorders. The present study aims at evaluating alterations in beta-arrestin1 protein and mRNA levels in mononuclear leukocytes of untreated patients with major depression and the effects and time course of antidepressant treatments on these alterations. Methods Repeated beta-arrestin1 protein and mRNA measurements, through immunoblot analyses using monoclonal antibodies against beta-arrestin1 and reverse transcriptase polymerase chain reaction, respectively, were carried in mononuclear leukocytes of 18 patients with major depression and compared with 18 healthy subjects. Each patient was examined while untreated and after 1, 2, and 4 weeks of antidepressant treatment. Results Beta-arrestin1 protein and mRNA levels in mononuclear leukocytes of untreated patients with major depression were significantly lower than those of healthy subjects. The low beta-arrestin1 protein and mRNA levels were alleviated by antidepressant treatment. Normalization of beta-arrestin1 measures preceded, and thus predicted clinical improvement. Conclusions These findings support the implication of beta-arrestin1 in the pathophysiology of major depression and in the mechanism underlying antidepressant-induced receptor down-regulation and therapeutic effects. Beta-arrestin1 measurements in patients with depression may potentially serve for biochemical diagnostic purposes and for monitoring and predicting response to antidepressants.

β-Arrestin-1 Levels: Reduced in Leukocytes of Patients With Depression and Elevated by Antidepressants in Rat Brain

Beta-arrestins play a pivotal role in G protein-coupled receptor desensitization. beta-Arrestins interfere in G protein receptor interaction, thus leading to desensitization of G protein-mediated receptor signaling. G protein receptor signaling and its desensitization were previously implicated in the pathophysiology of mood disorders and in the mechanism of action of antidepressant and mood-stabilizing treatments. The present study aims at quantitatively evaluating beta-arrestin-1 levels in leukocytes of patients with major depression and the effect of antidepressants on beta-arrestin-1 levels in rat brain. Beta-arrestin-1 measurements were carried out in cortical, hippocampal, and striatal brain regions of rats chronically intragastrically treated with either imipramine, desipramine, or fluvoxamine. Similar measurements were conducted in mononuclear leukocytes of 36 untreated patients with major depression and 32 healthy volunteer subjects. Beta-arrestin-1 levels were evaluated through immunoblot analyses using monoclonal antibodies to beta-arrestin-1. Beta-arrestin-1 levels were significantly elevated by all three antidepressants in rat cortex and hippocampus, while in the striatum no alterations could be detected. This process became significant within 10 days and took 2-3 weeks to reach maximal increase. Mononuclear leukocytes of patients with depression showed significantly reduced immunoreactive quantities of beta-arrestin-1. The reduction in beta-arrestin-1 levels was significantly correlated with the severity of depressive symptoms. The findings in the rat study suggest beta-arrestin-1 elevation as a biochemical mechanism for antidepressant-induced receptor down-regulation. The findings in human subjects support the implication of beta-arrestin-1 in the pathophysiology of mood disorders. Beta-arrestin-1 measurements in patients with depression may potentially serve as a biochemical marker for depression.

Decreased nitric oxide production after intravenous immunoglobulin treatment in patients with Kawasaki disease

Objective: To assess nitric oxide (NO) production and different displays of NO synthase (NOS) isoforms in patients with Kawasaki disease (KD) before and after intravenous immunoglobulin (IVIG) treatment. Study design: Blood and urine samples were collected from patients with KD before IVIG treatment and 3 days afterward. Age-matched febrile patients with various diseases served as control patients. Plasma NOx (NO3- + NO2-) levels were measured as NO production. Real-time reverse transcriptase–polymerase chain reaction and immunohistochemical staining were used to detect differential NOS isoform expression in mononuclear leukocytes (MNCs). Results: Patients with KD (n = 40) had higher levels of NOx than the non-KD febrile control patients. The elevated NOx levels in patients with KD were significantly associated with the occurrence of coronary artery dilation (>3 mm). These elevated NOx levels significantly decreased after IVIG treatment (157.8 ± 15.9 μmol/L vs 57.0 ± 4.27 μmol/L, P <.01). However, excretion of urinary NOx levels was not changed after IVIG treatment. Inducible but not constitutive NOS mRNA and protein in MNCs were prominently expressed but significantly decreased after IVIG treatment. Conclusions: IVIG may suppress iNOS expression of MNCs in patients with KD, thus decreasing NO-mediated inflammatory responses and coronary artery dilation.( J Pediatr 2002;141:560-5)

Elevated dopamine receptor-coupled G s protein measures in mononuclear leukocytes of patients with schizophrenia

Heterotrimeric G proteins play a pivotal role in post-receptor information transduction and were previously implicated in the pathophysiology and treatment of mood disorders. Changes previously detected in G protein levels in post-mortem brain of patients with schizophrenia could reflect effects of antipsychotic medication. The present study aims at quantitatively and functionally evaluating receptor-coupled G proteins in mononuclear leukocytes obtained from 23 untreated patients with schizophrenia and 30 healthy subjects in an attempt to unravel a pattern of G protein measures in schizophrenia distinctive from patterns previously obtained in mood disorders. Dopamine-enhanced guanine nucleotide binding capacity to G s protein through D 1/D 5 receptor in mononuclear leukocytes of untreated patients with schizophrenia was significantly increased in comparison with healthy subjects, and positively correlated with both the total PANSS score and the positive subscale. β-Adrenergic and muscarinic receptor-coupled G protein functions, as well as G sα, G iα and Gβ immunoreactivities, were similar to healthy subjects. These findings, distinctive for schizophrenia, unrelated to drug treatment, and differential from previous findings in mania and depression, may potentially help to differentially diagnose, after the first psychotic episode, between the major psychoses: schizophrenia and manic-depressive illness.

Genetic alterations in patients with primary aldosteronism.

The syndrome of primary aldosteronism is characterized by hypertension with excessive production of aldosterone, potassium loss, and suppression of the renin-angiotensin system. The most common clinical subtypes of primary aldosteronism are aldosterone-producing adrenocortical adenoma (APA) and bilateral adrenal cortical hyperplasia (idiopathic hyperaldosteronism, or IHA). It has been reported that renin suppression and aldosterone levels are lower and hypokalemia milder in patients with IHA than in patients with APA. In the present study, we investigated the genetic analysis of aldosterone synthase gene, CYP11B2 in patients with primary aldosteronism and review the recent studies. The chimeric CYP11B1/CYP11B2 gene, which is a candidate gene for glucocorticoid-remediable hyperaldosteronism, was not found in either the DNA from aldosteronoma or in the genomic DNA from patients with APA or IHA. Mutations in the CYP21 or CYP11B1 gene were not present in patients with APA. No mutations in the coding region of the CYP11B2 gene were found in patients with IHA or APA. The level of CYP11B2 messenger RNA (mRNA) was much higher in the aldosteronoma portion than in nonadenomatous portion. The overexpression of CYP11B2 mRNA seen in the mononuclear leukocytes of patients with IHA suggests that unidentified aldosterone-stimulating factors or abnormalities of the CYP11B2 promoter region may cause the overproduction of aldosterone characteristic of IHA. The variants of the CYP11B2 gene may also contribute to dysregulation of aldosterone synthesis and lead to susceptibility to IHA.

The effects of seasons and light therapy on G protein levels in mononuclear leukocytes of patients with seasonal affective disorder.

Information-transducing heterotrimeric G proteins have been implicated previously in the mechanism of action of mood stabilizers and in the pathophysiology of mood disorders. Mononuclear leukocytes of patients with unipolar and bipolar depression have been characterized by reduced measures of the stimulatory and inhibitory G proteins. In this study, patients with seasonal affective disorder (SAD) were measured for mononuclear leukocyte G protein levels while depressed during the winter, following light therapy, and in remission during the summer. Twenty-six patients with SAD and 28 healthy subjects were assessed in the study. The immunoreactivities of Gs alpha, Gi alpha, and Gbeta subunit proteins were determined by Western blot analysis of mononuclear leukocyte membranes with selective polyclonal antibodies for the various G subunit proteins, followed by densitometric quantitation using an image analysis system. Untreated patients with SAD and winter, atypical-type depression showed significantly reduced mononuclear leukocyte immunoreactive levels of Gs alpha and Gi alpha proteins, similar to previous observations in patients with nonseasonal major depression. The reduced G protein levels were normalized with 2 weeks of light therapy. The same patients while in remission during the summer had G protein levels that were similar to those of healthy subjects. G protein-immunoreactive measures in patients with SAD are suggested as a state marker for winter depression, which is normalized by light treatment and during the summer. We speculate that light may exert its effects via normalization of transducin (Gt protein) levels, which are thought to be reduced in winter depression.

Dynamics of ECT Normalization of Low G Protein Function and Immunoreactivity in Mononuclear Leukocytes of Patients With Major Depression

Heterotrimeric G proteins were previously implicated in the biochemical mechanisms underlying the pathophysiology and treatment of mood disorders. Low function and immunoreactivity of G proteins were observed in patients with major depression. In the present study the authors evaluated the effects of ECT on the low measures of G proteins in patients with major depression. Repeated G protein measurements in mononuclear leukocytes of 10 patients with major depression were made. Each patient was examined while untreated and after successive sessions of ECT; 14 normal subjects were also studied. G protein function was evaluated through beta-adrenergic- and muscarinic-agonist-enhanced guanine nucleotide binding capacity, substantiated by quantitative measures of G proteins through immunoblot analyses using polyclonal antibodies against Gs alpha, Gi alpha, and G beta proteins. Mononuclear leukocytes of patients with depression showed immunoreactive levels of Gs alpha and Gi alpha that were significantly lower than those of normal subjects; the depressed patients also had markedly hypofunctional Gs and Gi. The low levels of G protein function and immunoreactivity were alleviated by ECT. Repeated measurements in the same patients after successive ECT sessions showed that the normalization of G protein measures preceded, and thus predicted, clinical improvement. The function and quantity of Gs and Gi proteins in patients given ECT were significantly correlated. These findings support the implication of G proteins in the pathophysiology and treatment of mood disorders. G protein measurements in patients with depression may potentially serve not only as a biochemical marker for affective state but also for biochemical prediction and evaluation of responses to ECT.

Differential G protein measures in mononuclear leukocytes of patients with bipolar mood disorder are state dependent

Quantitative and functional measurements of G proteins were undertaken in mononuclear leukocytes of bipolar disordered patients comparing bipolar depressed with manic patients groups in order to verify whether any alterations observed in G protein functional or immunoreactive measures in bipolar mood disorder are state- or trait-dependent characteristics. Compared with the control group of 30 subjects, isoproterenol- and carbamylcholine-enhanced Gpp(NH)p binding capacities were highly significantly increased in the group of 20 manic patients, while highly significantly reduced in the group of 11 bipolar depressed patients. While manic patients showed highly significant elevations in mononuclear leukocytes levels of G αs and G αi , evaluated through immunoblot analysis using specific polyclonal antibodies against the subunit proteins, mononuclear leukocytes of bipolar depressed patients show significant reductions in G αs and G αi immunoreactive levels. G β subunit levels were found to be similar in all three groups. The changes in G protein measures observed in mononuclear leukocytes of mood disordered patients thus represent state characteristics of the disorder.

Reduced Gs protein function and Gαs levels in leukocytes of patients with Parkinson's disease

Early events in signal information transduction beyond dopamine, beta-adrenergic, and muscarinic receptors, involving receptor-coupled G-protein function and G alpha subunit immunoreactive levels were measured in mononuclear leukocytes (MNLs) of 12 never-treated patients with Parkinson's disease in comparison with 10 age- and sex-matched healthy control subjects. Both beta-adrenergic and dopamine receptor-coupled Gs protein function as measured by cholera toxin-sensitive, isoproterenol- and dopamine-induced increases in Gpp(NH)p-binding capacity, in MNLs of patients with Parkinson's disease were found to be significantly reduced in comparison with those in the control group. Muscarinic receptor-coupled non-Gs (Gi or G(o)) protein function: pertussis toxin-sensitive, carbamylcholine-induced increase in Gpp(NH)p-binding capacity, was not found to be significantly different between patients with Parkinson's disease and control subjects. G protein alpha subunits were measured through immunoblotting analyses with specific polyclonal antibodies against G alpha s, G alpha i, and G alpha q subunits. MNL levels of the 45-kDa species of G alpha s were found to be significantly reduced in patients with Parkinson's disease in comparison with control subjects. Other non-Gs proteins (Gi, Gq) did not show any significant quantitative differences between patients with Parkinson's disease and control subjects. The reductions in G alpha s levels in MNLs of patients with Parkinson's disease may explain the beta-adrenergic and dopamine receptor-coupled Gs protein hypofunction detected in MNLs of these patients. As previous studies have failed to observe significant changes in receptor levels in MNLs of patients with Parkinson's disease, our findings of reduced dopaminergic and beta-adrenergic receptor-coupled Gs function and of G alpha s immunoreactive levels in MNLs of Parkinson's patients point to alterations distal to these receptors at the level of the signal-transducing Gs protein.

Reduced G protein functions and immunoreactive levels in mononuclear leukocytes of patients with depression.

Heterotrimeric G proteins play a pivotal role in postreceptor information transduction. These proteins were previously implicated in the biochemical mechanism underlying lithium action and in the pathophysiology of mood disorders. The present study sought to quantitatively and functionally evaluate G proteins in patients with major depression. G proteins were measured in mononuclear leukocytes of 37 untreated patients with major depression and 31 comparison subjects. Receptor-coupled G protein function was evaluated through beta-adrenergic and muscarinic-agonist-induced increases in guanine nucleotide binding capacity, which were substantiated by quantitative measures of G proteins through immunoblot analyses that used polyclonal antibodies against stimulatory (Gs alpha) and inhibitory (Gi alpha) G proteins. Mononuclear leukocytes of depressed patients showed significantly reduced immunoreactive quantities of Gs alpha and Gi alpha together with markedly hypofunctional Gs and Gi. The reductions in both function and quantity of Gs and Gi were significantly correlated with the severity of depressive symptoms. Moreover, simultaneous quantitative and functional measurements in a large number of patients showed significant correlations between the function and the quantity of mononuclear leukocyte Gs and Gi proteins: These findings lend further support to the implication of G proteins in the pathophysiology of mood disorders. G protein functional and quantitative measurements in mononuclear leukocytes of patients with mood disorders may potentially serve as a biochemical marker for the affective state of these patients.

Reduced β-adrenergic receptor-coupled Gs protein function and Gsα immunoreactivity in mononuclear leukocytes of patients with depression

beta-Adrenergic receptor-coupled Gs protein function was measured in 26 depressed patients through cholera toxin-sensitive, isoproterenol-induced increases in 3H-Gpp(NH)p binding capacity to mononuclear leukocytes (MNL). Highly significant reductions in receptor-coupled Gs protein function were observed in the depressed patients: 2.0 +/- 1.3% increases in guanine nucleotide-binding capacity, in comparison with the control group values of 28.3 +/- 6.9%. Similar reductions in Gs protein function were detected in both uni- and bipolar depressed patients. A significant negative correlation was found between receptor-coupled Gs protein measures and the severity of depression. Adding semiquantitative measures of MNL Gs alpha through immunoblot analysis by use of polyclonal antibodies against Gs alpha subunit, it was found that Gs alpha relative immunoreactivity was reduced from 100 +/- 2.0% in the control group of subjects to 75.9 +/- 2.3% in the depressed patients. We have previously described hyperfunctional Gs proteins in leukocytes of patients with mania. The present findings of reduced function of Gs in depressed patients suggests receptor-coupled Gs protein activity as a biochemical parameter indicatory of the affective state. Reduced receptor-coupled Gs protein function may reflect reduced levels of the beta-adrenergic receptor previously shown in leukocytes of depressed patients; however, our complementary immunoblot studies suggest a direct, postreceptor, quantitative, and functional reduction in Gs protein in MNL of depressed patients.

Gene Expression of Angiotensin II Receptor in Blood Cells of Cushing’s Syndrome

The relation between serum cortisol, plasma renin activity, angiotensin II (Ang II), or aldosterone levels and peripheral blood cell (mononuclear leukocytes and platelets) angiotensin II type 1A (AT1A) and 1B (AT1B) receptor mRNA levels was examined in both patients with Cushing's syndrome (seven patients with Cushing's syndrome due to unilateral adrenal cortical adenoma) and control subjects (seven normotensive patients with renal cell carcinoma). Blood was collected from each participant for estimation of plasma renin activity and plasma angiotensin II, aldosterone, and cortisol concentrations and for isolation of mononuclear leukocytes and platelets, which were then used to measure AT1A and AT1B receptor mRNA levels before and after adrenalectomy with the use of reverse transcription-polymerase chain reaction. In patients with Cushing's syndrome, both mononuclear leukocyte and platelet AT1A mRNA levels, which were elevated, were reduced after removal of the adrenal tumors, whereas AT1B receptor mRNA levels of both types of blood cells did not significantly change after adrenalectomy. In contrast, in control subjects, both AT1A and AT1B receptor mRNA levels did not significantly change after unilateral adrenalectomy and nephrectomy. In the adrenal tumors of patients with Cushing's syndrome, gene expression of AT1A receptor was decreased compared with that from adrenals of control subjects. AT1A receptors of the platelets were shown to be upregulated in a manner similar to those of mononuclear leukocytes in patients with Cushing's syndrome. These results suggest that cortisol excess is an important factor upregulating AT1A receptor mRNA levels in human blood cells.

Differential adaptation of glucocorticoid sensitivity of peripheral blood mononuclear leukocytes in patients with sepsis or septic shock.

In view of the immunosuppressive action of glucocorticoids (GCs), the activation of the hypothalamo-pituitary-adrenal axis in patients with sepsis or septic shock is paradoxical. At the same time, administration of GCs to these patients is not clearly beneficial. We investigated the role of GCs in severe illness by measuring the sensitivity of peripheral blood mononuclear leukocytes to GCs in a mitogen-stimulated lymphocyte proliferation assay. In addition, we studied the role of interleukin-2 and several other cytokines in this system. Cells from patients with sepsis or septic shock (n = 15) were more sensitive to the antiproliferative action of GCs than were cells from normal controls (IC50 6.7 +/- 2.1 nmol/L for patients vs. 19.5 +/- 2.5 nmol/L for controls; P < 0.01). This increased sensitivity of the peripheral mononuclear cells to dexamethasone during the period of sepsis normalized during the ensuing period of clinical recovery of these patients. Dexamethasone inhibited the production of interleukin-2 in the mitogen-stimulated cells. Addition of interleukin-2 antagonized the suppressive effects of dexamethasone in a dose-dependent manner, both in cells from controls and in cells from patients with sepsis. To a lesser extent, the combination of interleukin-1, interleukin-6, and tumor necrosis factor-alpha also counteracted the effects of dexamethasone. In conclusion, our results suggest that not only the activity of the hypothalamo-pituitary-adrenal axis but also the sensitivity to GCs is regulated during sepsis and septic shock. Generally there is an increased sensitivity to GCs, which might help to protect the organism as a whole through supportive effects on metabolism and vasculature. This hypersensitivity is counteracted, possibly at the site of inflammation, by high local concentrations of cytokines. This would enable an adequate local response of the immune system in the presence of elevated cortisol levels. In view of the increased sensitivity of peripheral leukocytes to GCs, treatment of these patients with high doses of GCs may not be beneficial or may even be harmful.