Levels of sex steroid hormones and their receptors in women with preeclampsia.

BACKGROUND: Pregnant women have high serum concentrations of sex steroid hormones, which are major regulators of paracrine and autocrine responses for many maternal and placental functions. The main purpose of this study was to compare patients with preeclampsia and patients with uncomplicated pregnancies in terms of serum steroid hormones (estradiol [E2], progesterone [P4], dehydroepiandrosterone sulfate [DHEAS], and testosterone [T]) throughout pregnancy and the levels of cord blood and placental steroid receptors during the third trimester. METHODS: Quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry were used to determine the levels of steroid hormones in the serum and cord blood and the placental levels of estrogen receptor-α (ERα), ERß, androgen receptor (AR), and progesterone receptor (PR). RESULTS: There were 45 women in the uncomplicated pregnancy group and 30 women in the preeclampsia group. Serum levels of T were greater and serum levels of E2 were reduced in the preeclampsia group, but the two groups had similar levels of P4 and DHEAS during the third trimester. Cord blood had a decreased level of DHEAS in the preeclampsia group, but the two groups had similar levels of P4, E2, and T. The two groups had similar placental mRNA levels of ERα, ERß, AR, and PR, but the preeclampsia group had a higher level of ERß protein and a lower level of ERα protein. Immunohistochemistry indicated that the preeclampsia group had a greater level of ERß in the nucleus and cytoplasm of syncytiotrophoblasts and stromal cells. CONCLUSIONS: Women with preeclampsia had lower levels of steroid hormones, estrogen, and ERα but higher levels of T and ERß. These molecules may have roles in the pathogenesis of preeclampsia.

Resilient Phenotype in Chronic Mild Stress Paradigm Is Associated with Altered Expression Levels of miR-18a-5p and Serotonin 5-HT1a Receptor in Dorsal Part of the Hippocampus.

Disturbed serotonergic signaling in the hippocampus observed in many individuals vulnerable to stress has been suggested as one of the primary factors contributing to the development of depression. However, little is known about the physiology of the brain in the resilient phenotype. Resilient subjects maintain a positive mood and psychological balance despite being under the stress influence. In our study, we generated stress-vulnerable and resilient rats by using a chronic mild stress (CMS) paradigm. Using different molecular approaches, we revealed that resilient animals exhibited a significantly decreased expression level of miR-18a-5p and, in the same time, an elevated level of 5-HT1AR in dorsal, but not ventral, part of the hippocampus. Described biochemical changes were not observed in animals behaviorally vulnerable to stress. Further, in vitro analysis showed that miR-18a-5p may be a negative epigenetic regulator of 5-HT1AR since the treatment of adult hippocampal neurons with miR-18a-5p mimic significantly lowered the expression level of mRNA encoding 5-HT1AR. Moreover, bioinformatic analysis of potential target genes expressed in the hippocampus and being regulated by miR-18a-5p showed that this microRNA may regulate biological processes, such as axonogenesis, which are important in the functioning of the hippocampus in both rats and humans. All these molecular features may contribute to serotonergic homeostatic balance at the level of serotonin turnover observed in hippocampi of resilient but not stress-vulnerable rats. Delineation of further molecular and biochemical markers underlying resilience to stress may contribute to the development of new antidepressant strategies which will restore resilient phenotype in depressed patients.

Altered NR4A Subfamily Gene Expression Level in Peripheral Blood of Parkinson's and Alzheimer's Disease Patients.

Parkinson's disease (PD) is a neurodegenerative pathology characterized by the degeneration of midbrain dopamine neurons, whose development and maintenance in brain is related to the transcription factor NR4A2 (also called Nurr1). Notably, NR4A2 is a neuroprotective agent with anti-inflammatory role in microglia and astrocytes. Furthermore, mutations in NR4A2 gene are associated to the familial form of PD, and its gene expression level is down-regulated in blood obtained from PD patients. NR4A2 belongs to the NR4A subfamily consisting of three members: NR4A1, NR4A2, and NR4A3. The NR4A subfamily shares high degree of homology in their molecular structure and cooperates in a spectrum of functions ranging from central nervous system to immune control during physiological and pathological conditions. Considering the close functional link between the member of NR4A subfamily, we performed a gene expression analysis of NR4A1, NR4A2, and NR4A3 in peripheral blood obtained from PD patients and healthy controls (HC). Then, in order to evaluate possible involvement of the NR4A subfamily in other neurodegenerative processes, we carried out the same analysis on blood obtained from Alzheimer's disease (AD) patients. A correlation between clinical features and gene expression was also evaluated. We found a marked down-regulated gene expression of the NR4A subfamily obtained from PD patients, but only a NR4A1 decrease in AD patients compared to HC. This study reports that the entire NR4A subfamily and not only NR4A2 could be systemically involved in PD suggesting that the study of these factors could be a promising approach to develop PD therapy.

Expression of oxysterol binding protein isoforms in opisthorchiasis-associated cholangiocarcinoma: a potential molecular marker for tumor metastasis.

Oxysterols are oxygenated derivatives of cholesterol generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by inflammation-associated non-enzymatic reactions. Oxysterol binding proteins (OSBPs) are cytosolic high affinity receptors for oxysterols. We previously found that OSBPL-8 is upregulated in liver fluke (Opisthorchis viverrini)-induced hamster cholangiocarcinoma (CCA). Our aims were to determine the expression patterns of OSBP isoforms in human CCA tissues and to evaluate whether OSBPs could be used as molecular markers for the identification of blood-borne CCA metastasis. Expression levels of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 in CCA tissues were detected using qRT-PCR and immunohistochemistry. Expression of OSBPs at mRNA level in the blood of CCA patients was also investigated. We confirmed increased expression of OSBPL-8 in O. viverrini -induced hamster CCA tissues. Moreover, increased expression of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 was seen in human CCA tissues. Notably, a significant increased level of OSBPL-7 mRNA was observed in tumor compared to non-tumor liver tissue. Immunohistochemistry supported the mRNA results, in that OSBPL-7 protein was over-expressed in cancer cells and hepatocytes but not in normal biliary cells and surrounding inflammatory cells. Interestingly, in our preliminary results, significantly higher levels of OSBP2 and OSBPL-7 mRNA were seen in blood samples from CCA patients than in healthy controls. These results suggest that OSBP2 and OSBPL-7 might serve as molecular markers for the identification of CCA metastasis in the bloodstream.

Exposure and effective dose biomarkers for perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) in infertile subjects: preliminary results of the PREVIENI project.

Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) have been used as surfactants in various industry and consumer products. PFOS/PFOA are very persistent in the environment and bioaccumulate in humans. They are potential reproductive and developmental toxicants and are considered to be emerging endocrine disrupters (EDs). The Italian project PREVIENI, funded by the Italian Environment Ministry, aims to link environment and human health through the investigation of selected endocrine disrupters (EDs) exposure and associated biomarkers related to human infertility conditions. In the early PREVIENI phase, PFOS and PFOA were determined in 53 couples affected by an infertility status, enrolled in a metropolitan area, according to established inclusion criteria and informed consensus. Nuclear receptors related to chemical compounds interactions were selected as biomarkers of effect and their gene expression modulations were analyzed in human peripheral blood mononuclear cell (PBMC). Among couples, subjects not presenting infertility factors (IF--) were separated from affected subjects (IF++). Most IF-- serum samples showed PFOS and PFOA concentrations overlapping the limit of detection (LOD) of 0.5 ng/g wet weight (ww). A substantial percentage of IF++ serum samples showed PFOS concentrations >20-fold the LOD, i.e. from 3 to 50 ng/g ww. In male (50%, n=26) and from 3 to 144 ng/g ww in female (37%, n=30) samples. PFOA values were below the LOD levels in 90% of the total samples. Peroxisome proliferator-activated receptor-gamma (PPARγ) and aryl hydrocarbon receptor (AhR) showed a low level of expression in PBMC of both IF++ and IF-- groups. Whereas alpha and beta estrogen receptors (ERα and ERß), androgen receptor (AR), and pregnane X receptor (PXR) were all upregulated in IF++ of both sexes with respect to IF-- group. Our preliminary results related to the metropolitan area indicate that subjects affected by infertility factors tend to have both higher PFOS levels and higher gene expression of specific nuclear receptors.

Corticosterone and cortisol binding sites in plasma, immune organs and brain of developing zebra finches: intracellular and membrane-associated receptors.

Glucocorticoids (GCs) affect the development of both the immune and nervous systems. To do so, GCs bind to intracellular receptors, mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). In addition, GCs bind to membrane-associated corticosteroid receptors (mCR). Two well-known GCs are corticosterone and cortisol. Whereas corticosterone is the primary GC in zebra finch plasma, cortisol is the primary GC in zebra finch lymphoid organs and is also present in the brain and plasma during development. Here, we characterized binding sites for corticosterone and cortisol in plasma, liver, lymphoid organs, and brain of developing zebra finches. In tissues, we examined both intracellular and membrane-associated binding sites. For intracellular receptors, there were MR-like sites and GR-like sites, which differentially bound corticosterone and cortisol in a tissue-specific manner. For mCR, we found little evidence for membrane-associated receptors in immune organs, but this could be due to the small size of immune organs. Interestingly, cortisol, but not corticosterone, showed a low amount of specific binding to bursa of Fabricius membranes. For neural membranes, corticosterone bound to one site with low affinity but a relatively high B(max), and in contrast, cortisol bound to one site with high affinity but a lower B(max). Our results indicate that intracellular and membrane-associated receptors differentially bind corticosterone and cortisol suggesting that corticosterone and cortisol might have different roles in immune and nervous system development.

[Radiotherapy for and prognosis of breast cancer patients with local-regional recurrence after mastectomy].

BACKGROUND AND OBJECTIVE: Controversies remain regarding the therapeutic principle for and prognosis of breast cancer patients with isolated local-regional recurrence after mastectomy. This study was to evaluate the role of radiotherapy in treating these patients and to investigate the prognosis. METHODS: Clinical data of 255 breast cancer patients with chest-wall and/or regional lymph node recurrence as first failure after mastectomy from 1990 to 2005 were analyzed. All patients received radiotherapy for recurrence. RESULTS: The median follow-up time was 45 months (9 months-15.5 years). The median disease-free interval (DFI) was 22 months (2-260 months); it was 37 months in patients with positive hormonal receptor and 17 months in those with unknown or negative receptor. The 2-, 5-, and 8-year overall survival (OS) rates were 86.4%, 56.5%, and 35.0%, respectively. The median survival time was 79 months. The 2-, 5-and, 8-year local control rates were 6.1%, 36.3%, and 27.6%, respectively. Univariate prognostic analysis showed that DFI, site and number of recurrence, receptor status, short-term therapeutic response, initial T status and axillary involvement significantly affected the survival (all P<0.05); multivariate analysis showed that DFI, receptor status, site and number of recurrence were independent prognostic factors. Prognostic index was established to classify the patients. The 2-, 5-and 8-year OS rates were 100%, 91.6%, and 56.4% in the favorable prognosis group, 88.1%, 59.1%, and 36.8% in the medium prognosis group, 68.0%, 8.5%, and 0 in the poor prognosis group (P<0.001). CONCLUSION: Radiotherapy is effective for breast cancer patients with isolated local-regional recurrence after mastectomy. Prognostic index could be applied to predict the prognosis.

Pharmacological characterization of intracellular, membrane, and plasma binding sites for corticosterone in house sparrows.

The diversity and specificity of glucocorticoid effects are dependent on cell-specific receptor mechanisms. Three known corticosteroid receptors mediate tissue effects of glucocorticoids in vertebrates: two intracellular receptors that act primarily as ligand-activated transcription factors, and a membrane-associated receptor. The intracellular receptor sub-types have been well characterized in mammals, however relatively little is known about them across non-mammalian vertebrates. The membrane-associated receptors are poorly characterized in most vertebrate taxa. To explore the basis for glucocorticoid action in birds, we pharmacologically characterized the three putative corticosteroid receptors in the brain, as well as a plasma corticosterone binding globulin, in the house sparrow (Passer domesticus). We found that house sparrow brain cytosol contained two distinguishable binding sites for corticosterone. A high affinity, mineralocorticoid-like receptor had subnanomolar affinity for corticosterone (K(d) approximately 0.2 nM). However, this 'MR-like' high-affinity receptor did not bind RU28318 or canrenoic acid, two compounds that bind mammalian MR with high affinity. A lower-affinity, glucocorticoid-like receptor in brain cytosol bound corticosterone with an average K(d)=5.61 nM. This GR-like receptor showed subnanomolar affinity for RU 486. MR- and GR-like receptors were found in equal numbers in whole brain assays (average B(max)=69 and 62 fmol/mg protein, respectively). House sparrow brain membranes contain a single binding site specific for glucocorticoids, with characteristics consistent with a steroid/receptor interaction. Corticosterone affinity for this putative membrane receptor was approximately 24 nM, with apparent B(max)=177 fmol/mg protein. House sparrow plasma contained a single binding site for [(3)H]corticosterone. Specific binding to plasma sites was inhibited by glucocorticoids, progesterone, and testosterone. Testosterone binding to this corticosteroid binding globulin is noteworthy as sex steroid-specific binding globulins have not been identified in birds. Taken together, these data extend our ability to evaluate the comparative actions of glucocorticoids, increase our understanding of mechanisms behind the tissue specificity of glucocorticoid action, and offer insight into the evolution of glucocorticoid action in vertebrates.

The differential expression of corticosteroid receptor isoforms in corticosteroid-resistant and -sensitive patients with rheumatoid arthritis.

OBJECTIVE: A proportion of patients with rheumatoid arthritis (RA) fail to respond adequately to corticosteroid (CS) therapy. Using an in vitro CS sensitivity bioassay, we have subdivided RA patients into steroid-sensitive (SS) and -resistant (SR) subgroups and this correlates with clinical responses to CS therapy. CSs exert their effects via the CS receptor (CR), which exists as two main isoforms, CRalpha and CRbeta. CRbeta can function as a negative inhibitor of CRalpha. We have hypothesized that steroid resistance in RA patients is due in part to a relative over-expression of the CRbeta. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from SS and SR RA patients. CRalpha and CRbeta mRNA expression was determined by quantitative real time polymerase chain reaction (qRT-PCR). The ratio of CRbeta/CRalpha mRNA expression was determined. CRalpha and CRbeta protein expression by PBMCs was analysed by flow cytometry. RESULTS: qRT-PCR analysis showed a trend towards higher expression of both CRbeta and basal CRbeta/CRalpha ratio in SR RA patients. Stimulation of PBMCs in vitro with concanavalin-A induced a significantly higher CRbeta mRNA expression, and CRbeta/CRalpha ratio in SR RA patients compared with SS patients, which was not inhibited by hydrocortisone. Flow cytometry showed that the percentage of PBMCs staining for CRbeta protein was significantly lower in the SS RA group (SS 43.3 +/- 14.8% vs SR 88.6 +/- 8.6%; P < 0.0010). The mean intensity of fluorescence CRbeta staining was higher in the SR RA patients (P < 0.001). CONCLUSION: We show for the first time that CRbeta is over-expressed in SR RA patients and that hydrocortisone fails to inhibit concanavalin-A stimulated increase in CRbeta mRNA in SR RA patients. This mechanism may contribute in part to the CS hyporesponsiveness seen in some RA patients.

Alpha-isoform of Ca2+/calmodulin-dependent kinase II autophosphorylation is required for memory consolidation-specific transcription.

Autophosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II switches the kinase into an autonomous activity mode. This molecular switch is important for hippocampal long-term memory formation, which requires de novo gene transcription and protein synthesis. Here, we have studied whether auto-phosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II is required for gene transcription induced in the hippocampus by contextual fear conditioning. We have shown that upregulation of a nonassociative transcript, the serum and glucocorticoid-induced kinase-1 messenger RNA, is normal in alpha-isoform of Ca2+/calmodulin-dependent kinase II autophosphorylation-deficient mutant mice, whereas upregulation of an associative transcript, the nerve growth factor-inducible gene B messenger RNA, is impaired. Thus, we suggest that autophosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II is a biochemical switch that regulates association-specific consolidation processes.

NR4A orphan nuclear receptor family in peripheral blood eosinophils from patients with atopic dermatitis and apoptotic eosinophils in vitro.

To identify novel genes related to the clinical signs of atopic dermatitis (AD), differentially expressed genes were sought in peripheral blood eosinophils from both AD patients and healthy volunteers. RNA was prepared from eosinophils, expression of various genes was monitored using the Affymetrix GeneChip, and expression was quantified by real-time RT-PCR. Two genes, Nur77 and NOR1, members of NR4A orphan nuclear receptor family, were expressed at a significantly higher level in AD patients than in healthy volunteers. Expression of another gene in the NR4A receptor family, Nurr1, was also higher in AD patients than in healthy volunteers. When peripheral blood leukocytes from healthy volunteers were fractionated, NOR1 expression was highest in eosinophils, but expression of Nur77 and Nurr1 genes was not eosinophil-specific. Extremely intense apoptosis was induced in both eosinophils and an eosinophil cell line, AML14.3D10, by treatment with antibody (Ab) to both CD30 and Fas. Rapid expression of the genes for the NR4A receptor family was observed with anti-CD30 Ab treatment but not with anti-Fas Ab. The NR4A orphan nuclear receptor family gene expression and the subsequent eosinophil apoptosis were downregulated by the MAPK inhibitor, U0126. These results suggest that the expression of the NR4A receptor family genes through CD30 signaling may regulate eosinophil apoptosis in allergic conditions such as AD.

MUC3 and MCA serum levels and steroid receptor content in breast cancer.

Mucins are an important class of complex glycoproteins expressed by many epithelial cells and their malignant counterparts. The aim of this study was to determine the serum levels of MUC3 and mucin-like carcinoma-associated antigen (MCA) in patients with primary breast cancer and to analyze the possible relationships between these two mucins and the steroid receptor status. The preoperative basal serum levels of MUC3 (ELISA assay with monoclonal antibody 1143/B7) and MCA (EIA assay with anti-MCA mouse monoclonal antibody b-12) were determined in 44 patients with breast cancer while estrogen receptor (ER) and progesterone receptor (PgR) levels were measured by the dextran-coated charcoal method in the cytosol of neoplastic tissue. MUC3 was expressed in 43/44 serum samples while high MCA serum levels were found in 16/44 only; the mean values of both markers did not correlate with menopausal status, tumor size, nodal involvement or ER. The only significant difference observed was a lower median value of MCA in patients with small tumors (T1-T2). No statistically significant correlation between MUC3 and MCA, MUC3 and ER or MCA and ER was observed; a statistically significant direct correlation between MUC3 and PgR+ status and a statistically significant inverse correlation between MCA and PgR+ were observed. Our results suggest that further investigations are necessary to establish whether progesterone can modulate MUC3 and MCA expression in breast cancer.

[Plasma oxysterols and vitamin E concentrations and lipid profile in morbidly obese women]. / Stezenie oksycholesteroli i witaminy E a profil lipidowy osocza u kobiet ze skrajna otyloscia.

Morbid obesity (BMI > or = 40 kg/m2) is accompanied by lipid disturbances which may be involved in the increased incidence of atherosclerosis, arterial hypertension and non-insulin-dependent diabetes mellitus. The aim of the study was to assess concentrations of total cholesterol (TC), HDL-cholesterol, LDL-cholesterol, triglycerides (TG), products of cholesterol peroxidation--oxysterols, and the major lipophilic antioxidant--vitamin E, in morbidly obese women without coexisting diseases. The study was performed in 11 morbidly obese women (BMI 42.21 +/- 2.21 kg/m2) and 11 healthy volunteers (BMI 23.0 +/- 2.31 kg/m2). Obese women demonstrated higher concentrations of TG (2.03 +/- 0.78 vs. 0.99 +/- 0.37 mmol/l; p < 0.05), 7-ketocholesterol (7-K) (89.85 +/- 63.03 vs. 41.90 +/- 17.33 ng/ml; p < 0.05) and 7-hydroxycholesterol (7-OH) (456.04 +/- 199.22 vs. 132.37 +/- 53.96 ng/ml; p < 0.05), and lower HDL-cholesterol level (0.74 +/- 0.10 vs. 1.30 +/- +/- 0.17 mmol/l; p < 0.05) compared to the control group, while there were no significant differences between the two groups in concentrations of TC, LDL-cholesterol and vitamin E. Plasma vitamin E/(TC + TG) ratio was lower in obese women (6.42 +/- 2.61 vs. 10.76 +/- 4.57 mumol/mmol; p < 0.05). Tocoferols concentration correlated positively with TG (r = 0.45; p < 0.05) and negatively with 7-OH (r = -0.44; p < 0.05) levels. Moreover, concentration of 7-K correlated positively with the level of HDL (r = 0.54; p < 0.05). In conclusion, despite normal TC and LDL-cholesterol concentrations, there are disturbances in cholesterol peroxidation processes, with the rise in oxysterol levels and the decrease in vitamin E concentration in lipoproteins, which may be involved in the increased incidence of cardiovascular diseases in morbidly obese women.

Identification of a gene encoding a human oxysterol-binding protein-homologue: a potential general molecular marker for blood dissemination of solid tumors.

This study describes a new potential role in human cancer for a gene, HLM, isolated by differential display, that bears homology to an oxysterol-binding protein. A significant association between increased expression of HLM with metastatic disease was found. HLM mRNA levels were increased in circulating tumor cells in patients' peripheral blood and in primary human epithelial cells expressing the human papillomavirus16 E6 and E7 proteins. HLM mRNA was not detected in most normal human tissues, including peripheral blood and lymph node. These findings indicate that HLM may function as a potential marker for tumor dissemination.

[Axillary lymph node dissection in clinically occult breast cancer]. / Le curage ganglionnaire axillaire dans les cancers infracliniques du sein.

The study concerns 265 patients with axillary lymph node dissection for non-palpable breast cancer. The mammographically detected breast tumors were: 36 ductal carcinomas in situ (DCIS), 23 microinvasive carcinomas, 206 invasive carcinomas of which 179 were invasive ductal cancers (IDC), 25 invasive lobular cancers (ILC) and 2 mucinous invasive carcinomas. The histologic size of the invasive component was < or = 5 mm in 38 cases, 6-10 mm in 84 cases, 11-15 mm in 53 cases, 16-20 mm in 16 cases, > 20 mm in 15 cases. Axillary dissection was performed immediately during the initial surgical procedure in 209 patients (79%) or secondarily in 56 (21%) according to the results of intraoperative examination of surgical specimens on frozen sections. Axillary lymph node involvement was not found in DCIS, microinvasive carcinomas or invasive carcinomas < or = 5 mm in size. Among all 206 invasive breast carcinomas, lymph node involvement was found in 7.8% (16/206) of cases. There were 9/84 (10.7%) in tumors > 10 mm, 7/122 (5.8%) in tumors < or = 10 mm. Thus, it is concluded that lymph node involvement is unlikely to be found in patients with non palpable breast cancers, specially those with carcinoma in situ, microinvasive breast tumors and invasive breast cancer with less than 5 mm maximum diameter size. Axillary dissection may be avoided in these patients. However, the use of new prognostic factors of lymph node involvement may help in the definition of patient group.

[Differential diagnostic value of receptors of 1,25-dihydroxyvitamin D3 (calcitriol) determination in lymphocytes of children with rickets and rickets-like diseases]. / Differentsial'no--diagnosticheskoe znachenie opredeleniia retseptorov 1,25-digidroksivitamina D3 (kal'tsitriola) v limfotsitakh u detei pri rakhite i rakhitopodobnykh zabolevaniiakh.

The authors provide the results of studying 1,25-dihydroxyvitamin D3 (calcitriol) in lymphocytes of children with rickets and rickets-like diseases. It is proposed that the character of their expression under the influence of vitamin D may be used with differential diagnostic purposes in view.

1,25-Dihydroxyvitamin D3 receptors in lymphocytes from patients with rheumatoid arthritis.

It has been previously found that activation of human lymphocytes in vitro causes the expression of receptors for the hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and that 1,25(OH)2D3 has immunoregulatory properties including the ability to inhibit interleukin-2, to suppress lymphocyte proliferation, and to inhibit antibody production. In the present study we found that 13 of a group of 17 (76%) seropositive patients with rheumatoid arthritis had lymphocytes that possessed 1,25(OH)2D3 receptors (without activation in vitro) compared with only three of 17 (18%) normal individuals. The biochemical characteristics of the 1,25(OH)2D3 receptor, including affinity, sedimentation coefficient, and DNA-binding properties in the rheumatoid arthritis lymphocytes were indistinguishable from those established for this receptor in the classic target tissue of the hormone. This finding raises the possibility that 1,25(OH)2D3, acting through its receptor, might play a previously unsuspected role on lymphocytes of patients with rheumatoid arthritis.

Decreased number of steroid receptors of circulating lymphocytes in Crohn's disease and ulcerative colitis.

The number of steroid receptors of circulating lymphocytes was determined in 13 patients with inflammatory bowel disease and in controls. Marked reduction of the number of receptors was observed both in Crohn's disease and in ulcerative colitis; no receptors were detected by radioactive hormone binding assay in 4 cases.

Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with end-organ resistance to 1,25-dihydroxyvitamin D.

Lectin-induced DNA synthesis by peripheral mononuclear cells from 17 normal donors was inhibited (40-60%) by 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) at physiological concentrations (10(-10)-10(-9) M). The lymphocytes acquire specific receptors for 1,25(OH)2D3 upon activation by the lectins. This process precedes the inhibitory effect of 1,25(OH)2D3. We studied lymphocytes from six patients from four different kindreds with the syndrome of hereditary end-organ resistance to 1,25(OH)2D (the so-called vitamin D-dependent rickets type II). In five patients (three kindreds) peripheral blood mononuclear cells did not acquire receptors for 1,25(OH)2D3 upon phytohemagglutinin-induced activation. Moreover, in contrast to normal lymphocytes, the mitogenic stimulation of these patients' lymphocytes by phytohemagglutinin and concanavalin A was not inhibited by 1,25(OH)2D3. Activated lymphocytes of the sixth patient from a fourth kindred exhibited normal binding of [3H]1,25(OH)2D3 but the hormone failed to inhibit the mitogenic stimulation. A similar pattern of the vitamin D effector system was previously observed in fibroblasts cultured from skin biopsies of the same group of patients. The conclusions from these findings are: (a) the inhibition of mitogenic stimulation by 1,25(OH)2D3 is mediated by specific functional receptors to the hormone; and (b) the receptors for 1,25(OH)2D3 in mononuclear cells are probably controlled genetically by the same mechanisms as the effector system in well-characterized target organs of the hormone, such as intestine and kidney.

Estimation of 1,25 dihydroxyvitamin D by cytoreceptor and competitive protein binding assays without high pressure liquid chromatography.

Using two different cultured rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8) we have investigated the recently described cytoreceptor assay for 1,25-dihydroxyvitamin D (1,25-(OH)2D). The assay method is relatively simple and sensitive to 2.4 fmole per tube. Using either cell line, assay of serum samples, whose only preparation consisted of extraction and purification on a disposable diatomaceous earth column, produced variable values for serum 1,25-(OH)2D. Additional purification, using a disposable silicic acid minicolumn to remove other vitamin D metabolites resulted in consistent values and additional HPLC resulted in no further decrease in the values obtained. Our results show that a single two stage non-HPLC column can purify serum samples for assay in the cytoreceptor assay. The method is also applicable to the competitive protein binding assay employing calf thymus cytosol and the correlation between values obtained by both methods is highly significant. It is a sensitive, simple, and accurate method with technical advantages which allow greater sample throughput than other 1,25-(OH)2D assays.