Low-dose 17-ß-estradiol cream for vaginal atrophy in a cohort without prolapse: Serum levels and vaginal response including tissue biomarkers associated with tissue remodeling.

OBJECTIVES: Describe the effect of 50 mcg vaginal 17-ß-estradiol (E2) cream on vaginal maturation, serum estrogen levels, atrophic symptoms, and biomarkers of oxidative stress and tissue remodeling in postmenopausal women without prolapse. METHODS: Seventeen women, 65 years or older, applied intravaginal E2 cream nightly for eight weeks, then twice weekly for eight weeks. Vaginal biopsies, serial blood draws, and atrophic symptoms were obtained at baseline, eight, and sixteen weeks. Changes in atrophic symptoms, vaginal maturation indices (VMI), and serum E2 were measured. Immunohistochemical staining characterized levels of transforming growth factor-beta (TGF-ß), nuclear factor kappa B (NFKB), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and thrombospondin (TSP). RESULTS: Serum E2 levels (pg/ml) were unchanged from baseline (mean (SD)) 7.7 (3.3) to eight 9.7 (5.7) and sixteen 8.7 (5.8) (p=0.24) weeks. VMI (mean (SD)) improved from baseline 34.2 (18.3) to eight 56.7 (13.1) and sixteen 54.5 (11.3) (p<0.001) weeks with no difference between eight and sixteen weeks. Vaginal dryness (p=0.03) and itching (p=0.02) improved. Tissue biomarker levels did not change (TGF-ß p=0.35, NFKB p=0.74, eNOS p=0.80, iNOS p=0.24, TSP p=0.80). DISCUSSION: Vaginal E2 improved atrophic symptoms and VMI without elevating serum E2. Tissue remodeling biomarkers did not change.

Development of monoclonal antibodies against human CYP11B1 and CYP11B2.

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.

Differential gene expression in the adrenals of normal and anencephalic fetuses and studies focused on the Fras-1-related extracellular matrix protein (FREM2) gene.

PRECIS: Many genes are differentially expressed in normal compared to anencephalic human fetal adrenals (HFAs), especially the Fras-1-related extracellular matrix protein (FREM2) gene. FREM2 expression appears to be regulated by adrenocorticotrophic hormone (ACTH). CONTEXT: The expression profiles of genes responsible for cortical growth and zonation in the HFA gland are poorly characterized. The neural tube disorder anencephaly is associated with fetal adrenal hypoplasia with a large size reduction of the fetal zone of the HFA. OBJECTIVE: To determine gene expression profile differences in the adrenals of anencephalic compared to normal HFAs to identify genes that may play important roles in adrenal development. DESIGN AND METHODS: Fresh tissues were obtained at the time of autopsy from normal and anencephalic human fetuses delivered at mid-gestation. The following techniques were used: cell culture, messenger RNA (mRNA) extraction, microarray analysis, complementary DNA (cDNA) synthesis, quantitative real-time reverse transcriptase polymerase chain reaction (QT-PCR). RESULTS: We identified over 40 genes expressed at levels 4-fold or greater in the normal versus anencephalic HFAs and that 28 genes were expressed at increased levels in the anencephalic HFA. The expression of FREM2 at approximately 40-fold greater levels in the normal HFA compared to the HFA of anencephalic fetuses was confirmed by QT-PCR. Expression of FREM2 in the kidney was not significantly different between normal and anencephalic fetuses. In cultured HFA cells, ACTH treatment for 48 hours increased the expression of FREM2 and a gene responsive to ACTH, CYP17, but not tyrosine hydroxylase. CONCLUSIONS: Abnormal expression of many genes may be involved in the adrenal hypoplasia seen in anencephaly. FREM2 appears to be regulated by ACTH and is the most differentially expressed gene, which may be important in the development and function of the HFA, particularly the fetal zone of the HFA.

ACTH is a potent regulator of gene expression in human adrenal cells.

The adrenal glands are the primary source of minerocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it has been established that ACTH can stimulate steroidogenesis, the effects of ACTH on overall gene expression in human adrenal cells have not been established. In this study, we defined the effects of chronic ACTH treatment on global gene expression in primary cultures of both adult adrenal (AA) and fetal adrenal (FA) cells. Microarray analysis indicated that 48 h of ACTH treatment caused 30 AA genes and 84 FA genes to increase by greater than fourfold, with 20 genes common in both cell cultures. Among these genes were six encoding enzymes involved in steroid biosynthesis, the ACTH receptor and its accessory protein, melanocortin 2 receptor accessory protein (ACTH receptor accessory protein). Real-time quantitative PCR confirmed the eight most upregulated and one downregulated common genes between two cell types. These data provide a group of ACTH-regulated genes including many that have not been previously studied with regard to adrenal function. These genes represent candidates for regulation of adrenal differentiation and steroid hormone biosynthesis.

Interlaboratory comparison study of serum total testosterone [corrected] measurements performed by mass spectrometry methods.

BACKGROUND: Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays. METHODS: Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis. RESULTS: The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range. CONCLUSIONS: The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.

Antenatal betamethasone depresses maternal and fetal aldosterone levels.

Antenatal glucocorticoids are used to mature lung function in fetuses at risk for preterm delivery, but they also suppress cortisol synthesis in both pregnant women and their fetuses. We recently discovered in pregnant rabbits that even though exogenous betamethasone is not a mineralocorticoid, it also suppresses production of aldosterone. Lower aldosterone levels were linked to reduced P450 side chain cleavage(P450scc) messenger RNA levels in the rabbit maternal and fetal adrenal cortex. To establish whether this occurs in humans, we assayed aldosterone levels in women and newborns treated with antenatal betamethasone for preterm labor. In mothers treated with betamethasone, maternal cortisol depression after 48 hours was accompanied by aldosterone depression. Both pregnant women and their newborns treated with betamethasone showed depressed aldosterone levels in a 1- to 3-day period after the first betamethasone dose. We conclude that suppression of aldosterone biosynthesis is a side effect of antenatal steroids that has been largely overlooked, but may be clinically relevant at a time when the newborn is learning to control plasma electrolytes and blood volume.

Relation of glucose control in diabetic pregnancy to fetal cholesterol homeostasis.

OBJECTIVE: To determine the impact of maternal diabetic glucose control on fetal cholesterol homeostasis. METHODS: Singleton pregnancies of 150 women complicated by gestational (n = 90) and pre-gestational (n = 60) diabetes were evaluated. Those with insulin-requiring diabetes had fasting blood glucose levels determined daily during the last 4 weeks of pregnancy and weekly fasting glucose values were obtained in those with A1 (diet-controlled) gestational diabetes. Umbilical cord venous serum was collected at delivery and total cholesterol levels were determined. RESULTS: Among the 150 women, 69 had A1 gestational diabetes and 81 were insulin-requiring. Fasting glucose levels were inversely correlated (p < 0.001) to gestational age, regardless of diabetes classification. Umbilical serum cholesterol levels were inversely related (p < 0.002) to gestational age and weight, but did not vary according to diabetic classification. Among term infants (n = 110), umbilical serum cholesterol levels were inversely related to maternal fasting glucose levels at 4 weeks (p = 0.006) and 2 weeks (p = 0.006) before delivery. CONCLUSION: Maternal hyperglycemia is associated with decreased plasma fetal cholesterol levels in term infants.

Marshmallows used as saliva stimulant do not affect cortisol concentrations: finally a palatable alternative for toddler saliva collection.

Two studies were conducted to validate marshmallows as a saliva stimulant for use with toddlers. First, cortisol concentrations from 14 subjects (ages 6-46 years) were compared using three saliva collection methods: (1) plain cotton dental roll, (2) dental roll with one mini-marshmallow, and (3) expectorating into a collection tube using no cotton or stimulant. EIA was used for analyses. There were no significant differences among cortisol concentrations. Second, saliva collection compliance rate was compared for 21-month-olds (n = 51) using either flavored drink crystal- (compliance rate = 16.7%) or marshmallow-flavored (compliance rate = 60%) dental rolls for saliva collection (chi(2) (1) = 4.02, p = .045). These studies indicate that marshmallow is a viable option for saliva stimulation to determine toddler cortisol concentrations using EIA.

The regulation of adrenocorticotrophic hormone receptor by corticotropin-releasing hormone in human fetal adrenal definitive/transitional zone cells.

As gestation progresses, human fetal adrenals (HFA) initiate the production of cortisol, which increases placental corticotropin-releasing hormone (CRH) biosynthesis. While adrenocorticotrophic hormone (ACTH) is important for the onset of cortisol production, the late gestational surge in cortisol production occurs despite falling ACTH levels in the fetal circulation. The authors determine if CRH directly regulates the expression of the ACTH receptor (ACTHR) in HFA definitive/transitional zone (DZ/TZ) cells. DZ/TZ cells isolated from midgestation HFA were cultured before treatment with 0.01 nM to 100 nM CRH or ACTH. Cortisol was measured by radioimmunoassay. Real-time reverse-transcriptase polymerase chain reaction was used to measure ACTHR mRNA. Whole-cell ACTH binding studies were performed using I(125) (Tyr-23) ACTH. CRH produced a dose-dependent rise in cortisol production and caused a time-dependent increase in ACTHR mRNA levels between 12 and 24 hours. As little as 0.1 nM CRH induced ACTHR transcript by 12-fold at 24 hours. Together with ACTH 0.01 nM, 0.03 or 0.1 nM CRH increased ACTHR expression more than ACTH alone. Binding assays demonstrated a 3.5-fold increase in ACTHR protein at 48 hours with combined CRH and ACTH treatment. Physiologic levels of CRH seen in the late-gestation fetus stimulate DZ/TZ ACTHR expression. Since placental CRH production increases strikingly near the end of gestation, the authors suggest that CRH-induced ACTH receptor expression may increase TZ responsiveness to circulating ACTH and contribute to the late gestational rise in cortisol secretion by the HFA, participating in an endocrine cascade that is involved in fetal organ maturation and potentially in the timing of human parturition.

Corticotropin-releasing hormone (CRH) and urocortin act through type 1 CRH receptors to stimulate dehydroepiandrosterone sulfate production in human fetal adrenal cells.

CONTEXT: Near term, the human fetal adrenal increases the production of cortisol and dehydroepiandrosterone sulfate (DHEAS). DHEAS, which acts as substrate for placental estrogen production, induces key changes involved in parturition. OBJECTIVE: The objective of this study was to determine quantitatively the effect of CRH on mRNA levels of enzymes needed for DHEAS production (steroidogenic acute regulatory protein, CYP11A, CYP17, and SULT2A1), to determine the CRH receptor (CRH-R) subtype(s) responsible for CRH action, and to determine the effect of CRH on CRH-R mRNA expression in human adrenal fetal zone (FZ) cells. DESIGN: Human adrenal FZ cells were treated with CRH, ACTH, urocortin (Unc), and CRH antagonists, and RNA was analyzed by microarray and real-time RT-PCR. SETTING: This study was performed at an academic research laboratory. MAIN OUTCOME MEASURE: The main outcome measure was the expression of steroidogenic enzymes and CRH-R. RESULTS: Microarray analysis of human FZ cells treated for 24 h with CRH or ACTH showed increased mRNA expression levels of the genes needed for DHEAS production. Real-time RT-PCR analysis confirmed these data. Induction was lost in the presence of CRH-R1 antagonists, but not CRH-R2 antagonists. Stimulation was reproduced by Unc. The CRH-R1alpha mRNA splice variant was the only type 1 receptor isoform expressed in the fetal adrenal, and treatment with CRH up-regulates its mRNA levels. CONCLUSIONS: CRH, Unc, and ACTH stimulate all elements of the DHEAS synthetic pathway and activate CRH-R1 as well. The resulting increased DHEAS levels can be used for placental estrogen synthesis and contribute to the process leading to parturition in humans.

Effects of aging on cytochrome b5 expression in the human adrenal gland.

CONTEXT: Aging in humans is characterized by a selective decline in circulating levels of adrenal androgens. The results of in vivo studies are suggestive of reduced adrenal 17,20-lyase activity in aging men and women. OBJECTIVE: We sought to determine whether there are changes in the distribution and/or expression of cytochrome B5 (CytB5), an accessory protein important in the regulation of 17,20-lyase activity, in the adrenals of aging humans. DESIGN: Comparison between younger and older adrenal glands. SETTING: The study was conducted in a University Center. PATIENTS OR OTHER PARTICIPANTS: Adrenal glands obtained at autopsy after sudden death as a result of trauma from 46 young (age 20-40 yr) and 26 older (age 50-91 yr) humans were obtained and fixed within 24 h postmortem. INTERVENTIONS: Paraffin sections were stained with hematoxylin and eosin and also were subjected to immunohistochemical staining for CytB5. All sections were quantitatively evaluated using an image capture and analysis program and qualitatively evaluated with respect to staining intensity. MAIN OUTCOME MEASURES: To determine whether there are any changes in CytB5 distribution in the adult human adrenal cortex during the aging process using qualitative and quantitative analysis with respect to age, gender, race, and postmortem interval. RESULTS: CytB5 immunoreactivity was found in cells that corresponded to those of the zona reticularis. The percentage of the adrenal cortex immunoreactive for CytB5 decreased with aging (38.6 +/- 7.6% for young and 30.1 +/- 5.9% for older, mean +/- sd; P < 0.0001) as did the percentage of adrenocortical tissue comprising the zona reticularis (36.8 +/- 10.8% for young and 27.2 +/- 5.9% for older; P < 0.001). However, there was no apparent change in the staining intensity of CytB5 among those cells that were immunopositive for this factor with aging. CONCLUSIONS: There appears to be a reduction in the proportion of the adrenal cortex that expresses CytB5 with aging, and this likely corresponds to a shrinkage of the zona reticularis. The mechanism and cause for this cortical regression are unknown.

Corticotropin-releasing hormone directly stimulates cortisol and the cortisol biosynthetic pathway in human fetal adrenal cells.

Near term the human fetal adrenals (HFAs) initiate production of cortisol, which promotes organ maturation and acts to increase placental CRH biosynthesis. The objective of the present study was to determine whether CRH directly stimulates both cortisol production and expression of the steroidogenic enzymes in HFA-definitive zone cells. CRH stimulated the production of cortisol in a time- and dose-dependent manner, with an effective concentration of as low as 0.01 nm. In real-time RT-PCR experiments, CRH treatment increased the mRNA levels of steroidogenic acute regulatory protein and each of the enzymes needed to produce cortisol. CRH induced 3beta-hydroxysteroid dehydrogenase type II (HSD3B2) by 34-fold, 21-hydroxylase (CYP21) by 55-fold, and 11beta-hydroxylase by 41-fold. Induction of steroidogenic acute regulatory protein, cholesterol side chain cleavage (CYP11A), and 17alpha-hydroxylase (CYP17) mRNA by CRH was 6-, 4-, and 6-fold, respectively. We also demonstrated that submaximal concentrations of CRH (30 pm) and ACTH (30 pm) that are seen in fetal circulation were additive on cortisol biosynthesis and 3beta-hydroxysteroid dehydrogenase type II mRNA induction. We suggest that CRH may play an important role in the late gestational rise in cortisol secretion from the HFAs, which may serve to augment placental CRH production and therefore participate in the endocrine cascade that is involved in fetal organ maturation and potentially in the timing of human parturition.

Biopsychological markers of distress in informal caregivers.

BACKGROUND: Thirty caregiving wives participated in a study of caregiving distress and negative mood (depressive symptoms) by making diary entries on stressful caregiving situations and collecting saliva samples 4 times a day. At the end of the 7-day study period, caregivers' salivary cortisol production was compared with their diary entries and correlated with pencil and paper self-report scores of caregiver distress and depressive symptoms. FINDINGS: Despite the inability to control a number of factors thought to confound cortisol production (exercise, smoking, alcohol ingestion, and prescription medications), there was a statistically significant difference between No Caregiving and Caregiving cortisol, F(1,739) = 7.67, P = 0.006, with cortisol production higher when caregiving events occurred. However, efforts to code specific types of caregiving situations (e.g., 1 = indirect care; 4 = AD problem behavior care) did not further differentiate cortisol production. Although caregivers' self-reports for the same 7-day period indicated they were depressed, pencil-and-paper measures of distress and negative affect were not significantly correlated with cortisol production. CONCLUSIONS AND RECOMMENDATIONS: The finding that this caregiving group was significantly stressed by caregiving, as evidenced by increased cortisol production during caregiving episodes, verifies the importance of further exploration of specific caregiving situations as contributory factors in caregiver health and well-being. In that saliva is a relatively economical and comparatively noninvasive biological data source for community-based stress studies, methodological limitations of the study are identified and 5 recommendations are made for future biological marker studies of caregiver distress in community-based settings.

Colocalization of P450c17 and cytochrome b5 in androgen-synthesizing tissues of the human.

Androgens are an integral part of human physiology. The de novo production of androgens is generally limited to the adrenal cortex and the gonads. Androgen synthesis by these steroidogenic tissues requires the bifunctional enzyme cytochrome P450c17, which catalyzes both 17 hydroxylase and 17,20 lyase activities. 17,20-lyase activity is relevant to the regulation of androgen production, and is allosterically modulated through the action of an accessory protein, cytochrome b5 (CytB5). Our objective was to determine the cellular localization of P450c17 and CytB5 in androgen-synthesizing tissues of the human. Immunohistochemical analyses of P450c17 and CytB5 were performed on fetal and adult human adrenals, ovaries, and testes. In the fetal adrenal, CytB5 and P450c17 were both found in the cells of the fetal zone, but not in the neocortex. In the adult adrenal, the zona fasciculata was immunoreactive for P450c17 only, whereas the zona reticularis was immunopositive for both P450c17 and CytB5. In the adult gonads, P450c17 and CytB5 were colocalized in the Leydig cells of the testis, theca interna cells of the follicle, theca lutein cells, and isolated cell clusters in the ovarian stroma. Whereas P450c17 and CytB5 were colocalized in the Leydig cells of the fetal testes, there was no immunostaining for either in the midgestational fetal ovary. Our findings of colocalization of CytB5 and P450c17 are strongly supportive of the view that CytB5 plays an important role in the regulation of the androgen biosynthetic pathway in the fetal and adult human.

Is reduced cell size the mechanism for shrinkage of the adrenal zona reticularis in aging?

In aging humans, corticosteroid production is preserved, or even increased, but there is an unexplained reduction in adrenal androgen secretion that likely has significant health implications. Preliminary analyses on adrenocortical morphology have revealed an age-associated reduction in the thickness of the zona reticularis (ZR), the cortical zone responsible for the majority of DHEA/DHEA sulfate production in the adult human, but no change in the overall thickness of the adrenal cortex. The ZR width could decrease in aging due to loss of ZR cells and/or to shrinkage of ZR cells. In the current study, we investigated whether there was a relation between thickness of the zona reticularis in young and old humans and the cell density in this zone. Paraffin-embedded sections of the adrenal cortex of 10 young (21-35 yr old) and 10 old (54-89 yr old) adults who had died suddenly as the result of trauma were stained with hematoxylin and eosin. These specimens were chosen from a larger cohort of samples for having a broad ZR in the young group and a narrower ZR in the older group. After determining the overall cortical thickness and the width of the ZR by use of computerized image analysis software, we counted the number of adrenocortical cells in two random high power fields of the ZR of each specimen. The ZR width of the older group (57 +/- 7 arbitrary units, Mean +/- SE) was significantly reduced compared to that of the young group (124 +/- 21), P < 0.001. On the other hand, the overall cortical width in the old group (232 +/- 17) was similar to that of the young adults (249 +/- 38). In the old group, the ZR comprised 24.7 +/- 3% of the total cortical width, whereas it was 50 +/- 2% of the cortical width in the young adrenals, P < 0.001. The cell density (cell number/60 x high power field) of the ZR of old adults (83 +/- 9) was similar to that of the young group (87 +/- 5). In summary, although the width of the ZR regresses with aging, cell size in this zone is preserved. Therefore, loss of trophic support for ZR cells would not appear to be the explanation for zonal shrinkage in aging. Rather, it is likely that aging effects may be due to increased cell loss in the ZR or else reduced rates of differentiation/migration of cells into this cortical zone.

Adrenal androgens and aging.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) are the principal C19 steroids produced by the human adrenals. Their plasma levels decline to less than 20% of their maximal value during aging. Because these steroids appear to play a role in the maintenance of immunity, musculoskeletal integrity, and cardiovascular health, age-associated declines in adrenal androgen production may contribute to decreased immune function, osteoporosis, and atherosclerosis. Production of DHEA and DHEAS has been localized to the zona reticularis (ZR) of the adrenal cortex and can be modulated by intra-adrenal or extra-adrenal modulators. Extra-adrenal modulators include corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), insulin, and transforming growth factor beta (TGF-beta). Intra-adrenal regulators include enzymes and proteins involved in the steroidogenic pathway, specifically 17,20 lyase activity and DHEA sulfotransferase (DST). The natural histories of the emergence of adrenal androgen production and the ontogeny of the ZR appear to correlate closely. In addition, aging results in a decline in adrenal androgen production, and our data suggest a parallel diminution in the area represented by the ZR. This decline in the ZR may result from apoptosis, cellular and humoral immunity, or a reduction in the replicative capacity of the cells of the ZR.

Adrenal androgens and the immune system.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are the main adrenal androgens (AAs) produced in humans. Production of these steroids, like that of cortisol, is under the control of hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH. Other factors, however, appear to be involved in AA secretion because there are many instances in which their circulating levels do not change in parallel to those of cortisol. Apart from physiological alterations associated with fetal adrenal regression, adrenarche and aging, the main instances of divergence in AA production compared with those of corticosteroids occur when immune function is activated or is aberrant. Relative reductions in DHEA and DHEAS have been noted in subjects with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), human immunodeficiency virus (HIV) and autoimmune deficiency syndrome (AIDS), sepsis, and trauma. In some instances, differences in the AA responses have been linked to a clinical course. The mechanisms for impairments in AA production in the absence of suppressed corticoid secretion are unclear but may involve circulating cytokines or locally released mediators from immune system cells in the adrenal gland. There also is evidence that DHEA and DHEAS play a role in immune competence, displaying biological effects opposite to those of corticosteroids.

Macrophage migration inhibitory factor is a constitutively expressed cytokine in the human adrenal gland.

Macrophage migration inhibitory factor (MIF) has been found to be widely expressed in many cell types throughout the body and appears to play several physiologic roles. We sought to determine the expression and cellular distribution of MIF in human fetal (HFA) and adult (HAA) adrenals. A single band of approximately 0.8 kb was revealed by northern blot hybridization using cDNA probes for MIF in total RNA extracts of both HFA and HAA tissues. Immunohistochemical analysis showed strong immunostaining for MIF in the broad fetal zone of the HFA and in both the zona glomerulosa and zona reticularis of HAA. The cells of the zona fasciculata of the HAA and of the neocortex of the HFA were only minimally, if at all, immunopositive for MIF and medullary elements were consistently negative for MIF. These results are indicative of local production of MIF in adrenocortical cells during intrauterine development and also in adulthood. The role of MIF in cortical cells of the human adrenal gland remains to be determined.