Transformation of 3-hydroxy-steroids by Fusarium moniliforme 7alpha-hydroxylase.

Transformation of physiologically important 3-hydroxy-steroids by the DHEA-induced 7alpha-hydroxylase of F. moniliforme was investigated. Whereas DHEA was almost totally 7alpha-hydroxylated, PREG, EPIA and ESTR were only partially converted into their 7alpha-hydroxylated derivatives because hydroxylation at other undetermined positions as well as reduction of ketone at C17 or C20 into hydroxyl also occurred. Cholesterol was not transformed by the enzyme. Kinetic parameters of the 7alpha-hydroxylation for these substrates were determined and confirmed that DHEA was the best substrate of the 7alpha-hydroxylase. Inhibition studies of DHEA 7alpha-hydroxylation by the other 3-hydroxy-steroids were also carried out and proved that DHEA, PREG, EPIA and ESTR shared the same active site of the enzyme. Induction effects of these steroids were compared, and DHEA appeared to be the best inducer of the 7alpha-hydroxylase of F. moniliforme.     

Dehydroepiandrosterone 7alpha-hydroxylation in human tissues: possible interference with type 1 11beta-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7alpha-hydroxy-DHEA that is a substrate for 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which exists in the same tissues and carries out the inter-conversion of 7alpha- and 7beta-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11beta-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7alpha-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11beta-HSD1. Human skin was selected then and used to test its ability to produce 7alpha-hydroxy-DHEA, and to test the interference of 7alpha- and 7beta-hydroxy-DHEA and 7-oxo-DHEA with the 11beta-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11beta-HSD1 in the liver, skin and tonsils. DHEA was readily 7alpha-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11beta-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA was competitive with a Ki at 1.85+/-0.495 and 0.255+/-0.005 microM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a Ki at 1.13+/-0.15 microM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7alpha-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Transformation of 3-hydroxy-steroids by Fusarium moniliforme 7α-hydroxylase

Transformation of physiologically important 3-hydroxy-steroids by the DHEA-induced 7α-hydroxylase of F. moniliforme was investigated. Whereas DHEA was almost totally 7α-hydroxylated, PREG, EPIA and ESTR were only partially converted into their 7α-hydroxylated derivatives because hydroxylation at other undetermined positions as well as reduction of ketone at C17 or C20 into hydroxyl also occurred. Cholesterol was not transformed by the enzyme. Kinetic parameters of the 7α-hydroxylation for these substrates were determined and confirmed that DHEA was the best substrate of the 7α-hydroxylase. Inhibition studies of DHEA 7α-hydroxylation by the other 3-hydroxy-steroids were also carried out and proved that DHEA, PREG, EPIA and ESTR shared the same active site of the enzyme. Induction effects of these steroids were compared, and DHEA appeared to be the best inducer of the 7α-hydroxylase of F. moniliforme.     

Analysis of pregnenolone and dehydroepiandrosterone in rodent brain: cholesterol autoxidation is the key

Pregnenolone (PREG) and dehydroepiandrosterone (DHEA), and their respective sulfated forms PREGS and DHEAS, were among the first steroids to be identified in rodent brain. However, unreliable steroid isolation and solvolysis procedures resulted in errors, particularly in the case of brain steroid sulfates analyzed by radioimmunology or GC-MS of liberated free steroids. By using a solid-phase extraction recycling/elution procedure, allowing the strict separation of sulfated, free, and fatty acid esters of PREG and DHEA, PREGS and DHEAS, unlike free PREG, were not detected in rat and mouse brain and plasma. Conversely, considerable amounts of PREG and DHEA were released from unknown precursor(s) present in the lipoidal fraction, distinct from fatty acid ester conjugates. Chromatographic and mass spectrometric studies of the nature of the precursor(s) showed that autoxidation of brain cholesterol (CHOL) was responsible for the release of PREG and DHEA from the lipoidal fraction. When inappropriate protocols were used, CHOL was also the precursor of PREG and DHEA obtained from the fraction assumed to contain sulfated steroids. In contrast, free PREG was definitely confirmed as an endogenous steroid in rat brain. Our study shows that an early removal of CHOL from brain extracts coupled to well-validated extraction and fractionation procedures are prerequisites for reliable measurements of free and conjugated PREG and DHEA by GC-MS or other indirect methods.     

Neurosteroids: metabolism in human intestine microsomes

Both dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA) are substrate for cytochrome P450 species and enzymes that produce 7alpha- and 7beta-hydroxylated metabolites in the brain and other organs. In contrast to DHEA and EpiA, the 7-hydroxylated derivatives were shown to mediate neuroprotection, and 7beta-hydroxy-EpiA was the most potent. The suggested use of any of these steroids as drugs administered per os for neuroprotection requires the assessment of their metabolism in the human intestine and liver. To achieve this, we produced radio-labeled 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA that were used as substrates in incubations with human intestine microsomes supplemented with reduced or oxidized cofactors. Identity of the radio-labeled metabolites obtained was determined by gas chromatography/mass spectrometry after comparison with authentic steroid references. The proportions of metabolites produced resulted from their radioactivity contents. The only metabolite obtained with DHEA, EpiA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA substrates was its 17beta-reduced derivative, thus inferring the presence of 17beta-hydroxysteroid oxidoreductases in the human intestine microsomes. In addition to the 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA substrates, their 17beta-reduced metabolites were obtained with 7beta-hydroxy-EpiA and 7alpha-hydroxy-EpiA, respectively. The identity of the enzyme responsible for the 7alpha-hydroxy-EpiA/7beta-hydroxy-EpiA inter-conversion is unknown. The incubation conditions used produced these metabolites in low but significant yields that suggest their presence in the portal blood before access to the liver.     

Simultaneous determination of dehydroepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography--mass spectrometry

A highly sensitive and specific method has been developed for the simultaneous measurement of free (unconjugated) or sulfate-conjugated forms of dehydroepiandrosterone (DHEA), 7alpha-hydroxy-DHEA (7alpha-OH-DHEA), 7beta-hydroxy-DHEA (7beta-OH-DHEA), and 7-oxo-DHEA (7-oxo-DHEA) in human serum. This method is based upon a stable isotope-dilution technique by gas chromatography-selected-ion monitoring mass spectrometry. Free steroids were extracted from serum with an organic solvent and the sulfate-conjugated steroids remained in aqueous phase. Free steroids were purified by solid-phase extraction, while sulfate-conjugated steroids were hydrolyzed by sulfatase and deconjugated steroids were purified by solid-phase extractions. The extracts were treated with O-methylhydroxylamine hydrochloride and were subsequently dimethylisopropylsilylated. The resulting methyloxime-dimethylisopropylsilyl (MO-DMIPS) ether derivatives were quantified by gas chromatography-selected-ion monitoring mass spectrometry in a high-resolution mode. The detection limits of MO-DMIPS ether derivatives of DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA were 1.0, 0.5, 0.5 and 2.0pg, respectively. Coefficients of variation between samples ranged from 10.6 to 22.9% for free 7-oxygenated DHEA to less than 10% for DHEA and sulfate-conjugated 7-oxygenated DHEA. The concentrations of these steroids were measured in 18 sera samples from healthy volunteers (9 males and 9 females; aged 23-78 years). Free DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA levels ranged between 0.21-3.55, 0.001-0.194, 0.003-0.481, and 0.000-0.077ng/ml, respectively, and the sulfate-conjugated steroid levels of these metabolites ranged between 253-4681, 0.082-3.001, 0.008-0.903, and 0.107-0.803ng/ml, respectively. The free DHEA-related steroid concentrations were much lower than those previously measured by RIA and low-resolution GC-MS. The present method made it possible to determine simultaneously serum DHEA-related steroid levels with sufficient sensitivity and accuracy.     

Metabolism of neuroactive steroids in day-old chick brain

Metabolism of the neuroactive steroids pregnenolone (PREG), progesterone (PROG), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) was investigated in day-old chick brain following direct injection of the ³H-labelled compounds into the intermediate medial mesopallium and sampling at times known to be crucial for memory formation in this brain region. ³H-label from these steroids was cleared rapidly from the brain, decreasing to barely detectable levels within 5 h. Following extraction and fractionation, the ³H-labelled brain steroids were identified by TLC, coupled with acetylation and/or separation in different solvent systems. PREG and PROG were converted within 10 min mostly to 20β-dihydropregnenolone (20β-DHPREG) and 5β-dihydroprogesterone, respectively. There was no detectable metabolism of DHEA. Label from DHEAS persisted for longer (half-time 18.9 min) than the free steroid but with no detectable metabolism other than a small amount (4%) of desulphation to DHEA. Further investigation of chick brain steroid metabolism by incubation of subcellular fractions (1–3 h, 37°C) with PREG, PROG or DHEA plus NADPH led to the formation of the following compounds: 20β-DHPREG from PREG (particularly in cytosol); 5β-dihydroprogesterone and 3α,5β-tetrahydroprogesterone from PROG and no detectable metabolism of DHEA. Following incubation of the same brain fractions and labelled steroids with NAD⁺, there was no detectable metabolism of PREG or PROG but some conversion of DHEA to androstenedione, especially in the nuclear fraction. The results suggest direct actions of DHEA(S) on the early stages of memory formation in the chick and introduce the possibility that PREG may act indirectly via 20β-DHPREG.     

Antioxidant effects of dehydroepiandrosterone and 7alpha-hydroxy-dehydroepiandrosterone in the rat colon, intestine and liver

This study examined in healthy male Wistar rats the in vivo antioxidant effect of dehydroepiandrosterone (DHEA) and 7alpha-hydroxy-DHEA administered by intraperitoneal injections (50 mg/kg body weight) for 2 or 7 days. Markers of oxidative damage to lipids (thiobarbituric acid-reacting substances, TBARS) and to proteins (protein carbonyls) were assessed in colon, small intestine, and liver homogenates. DHEA and 7alpha-hydroxy-DHEA caused a decrease in body weight. DHEA treatment significantly increased liver, colon, and small intestine cell weights. After 7 days, DHEA exerted an antioxidant effect in all organs studied. In the colon, oxidative damage protection was accompanied by a goblet cell proliferation and increase in acidic mucus production. After 2 days, the antioxidant effect of 7alpha-hydroxy-DHEA was mainly observed in the liver. Nonprotein sulfhydryl groups (mostly glutathione levels) were altered by DHEA in the liver whereas they remained unchanged after 7alpha-hydroxy-DHEA treatment. The results indicate that in healthy animals, DHEA exerts a protective effect, particularly in the colon, by reducing the tissue susceptibility to oxidation of both lipids and proteins. This effect was not limited to a specific tissue, whereas the metabolite 7alpha-hydroxy-DHEA exerted its antioxidant effect towards the two markers of oxidative damage earlier than DHEA, and mainly in the liver.     

Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3- ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha- OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta- HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inter-conversion of 7alpha- and 7beta-hydroxy-dehydroepiandrosterone by the human 11beta-hydroxysteroid dehydrogenase type 1

The dehydroepiandrosterone (DHEA) 7alpha-hydroxylation in humans takes place in the liver, skin, and brain. These organs are targets for the glucocorticoid hormones where 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisone through its reduction into cortisol. The putative interference of 7alpha-hydroxy-DHEA with the 11beta-HSD1-catalyzed reduction of cortisone into cortisol has been confirmed in preliminary works with human liver tissue preparations of the enzyme demonstrating the transformation of 7alpha-hydroxy-DHEA into 7-oxo-DHEA and 7beta-hydroxy-DHEA. However, the large production of 7beta-hydroxy-DHEA could not be explained satisfactorily. Therefore our objective was to study the role in the metabolism of oxygenated DHEA by recombinant human 11beta-HSD1 expressed in yeast. The 7alpha- and 7beta-hydroxy-DHEA were each oxidized into 7-oxo-DHEA with quite dissimilar K(M) (70 and 9.5 microM, respectively) but at equivalent V(max). In contrast, the 11beta-HSD1-mediated reduction of 7-oxo-DHEA led to the production of both 7alpha- and 7beta-hydroxy-DHEA with equivalent K(M) (1.1 microM) but with a 7beta-hydroxy-DHEA production characterized by a significantly greater V(max). The 7alpha-hydroxy-DHEA produced by the cytochrome CYP7B1 in tissues may exert anti-glucocorticoid effects through interference with the 11beta-HSD1-mediated cortisone reduction.     

Dehydroepiandrosterone and its 7-hydroxylated metabolites do not interfere with the transactivation and cellular trafficking of the glucocorticoid receptor

The human brain is a target tissue for glucocorticoids (GC). Dehydroepiandrosterone (DHEA) is a neurosteroid produced in the brain where it is transformed into 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA. The antiglucocorticoid effects of both 7-hydroxylated metabolites have been investigated with evidence in mice that neither form of DHEA interfered with the binding of GC to its glucocorticoid receptor (GR), but contributed to a decreased nuclear uptake of the activated GR. Our objective was to use COS-7 cell culture to research DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA interferences with GR trafficking. These cells did not carry out the 7alpha-hydroxylation of DHEA and the oxidation of cortisol into cortisone. The cDNA of the human GR was inserted into pcDNA3 for a transient transfection of COS-7 cells. Human GR transactivation activity was measured from a luciferase-MMTV reporter gene. The transfected COS-7 cells were cultured using 10(-12) to 10(-5) M dexamethasone (DEX) or cortisol, which triggered the reporter expression. Treatment with 10(-12) to 10(-5) M DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA caused no change in the GC-induced GR transactivation. A reconstruction of the process associated EGFP to the human GR cDNA. Confocal microscopic examination of COS-7 cells transiently expressing the fusion protein EGFP-GR showed nuclear fluorescence 60 min after incubation with 10(-8) M DEX or cortisol. The addition of 10(-5) M DHEA, 7alpha-hydroxy-DHEA or 7beta-hydroxy-DHEA did not change its kinesis and intensity. These results contribute to the knowledge of DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA, in relation to antiglucocorticoid activity. We conclude that direct interference with GR trafficking can be discounted in the case of these hormones, therefore proposing new possibilities of investigation.     

The role of T cells in immunoglobulin class switching of specific antibody production system in vitro in humans

Only antibodies of the IgM class were produced in vitro by peripheral blood mononuclear cells stimulated with streptococcal carbohydrate. B cells of the peripheral blood mononuclear cells, however, synthesized both IgM and IgG class antibodies when combined with tonsillar T cells, suggesting that T cells inducing immunoglobulin class switching are present in the tonsils. Peripheral blood T cells also became capable of inducing B cells to produce IgG class antibodies when the T cells were incubated with antigen-pulsed macrophages. Surface IgM-positive, IgG-negative high-density B cells produced IgG antibodies for streptococcal carbohydrate in the presence of these T cells or tonsillar T cells. The culture supernatant solutions from these T cells or tonsillar T cells, however, failed to cause the B cells to produce IgG, indicating that class switching is not mediated by factors released from T cells. Lymphokines such as interleukin-2, human B cell growth factor, helper T cell factor, or interferon-gamma were also incapable of inducing IgG production. These results suggest that the cognate interaction between T cells and B cells is necessary for the immunoglobulin class switching.     

Dexamethasone-induced apoptosis of mouse thymocytes: prevention by native 7alpha-hydroxysteroids

Dehydroepiandrosterone (DHEA) has been shown to decrease the dexamethasone (DEX)-induced apoptosis of thymocytes and to be one of the native 3beta-hydroxysteroids extensively 7alpha-hydroxylated in thymus. This led us to question whether DHEA or 7alpha-hydroxy-DHEA is responsible for the decrease in DEX-induced apoptosis of thymocytes and whether this property is shared with other native 3beta-hydroxysteroids and their 7alpha-hydroxylated metabolites. Treatment of mice with DHEA or 7alpha-hydroxy-DHEA prior to DEX led to a smaller decrease in thymus weight than with DEX alone and to a disappearance of the DEX-induced changes in thymocyte phenotypes. Thymocyte apoptosis induced by DEX treatment was significantly lowered in DHEA- and 7alpha-hydroxy-DHEA-treated thymi, even after 18 h culture with additional 10-6 mol/L DEX. Extensive apoptosis of thymocytes cultured with 10-7 mol/L DEX was brought back to control levels when 10-5 mol/L 7alpha-hydroxy-DHEA or 10-5 mol/L 7alpha-hydroxy-epiandrosterone was added. After use of DHEA and epiandrosterone or pregnenolone, less significant and no significant changes were obtained, respectively. These findings imply that the 7alpha-hydroxylation of 3beta-hydroxysteroids may be a prerequisite for an exquisite regulation of the thymocyte-positive selection driven by the glucocorticoids produced in thymic epithelial cells.     

Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions

Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [(3)H]-labeled 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA substrates from 50 microM [1,2,6,7-(3)H]DHEA (specific radioactivity 60-80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography-mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7 alpha-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2h/2 ml, respectively; 82% conversion), while in the presence of 1mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), more 7-oxo-DHEA than 7 alpha-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed. When longer reaction times were used with NADPH and NADP(+), a mixture of 7 alpha-OH-DHEA, 7 beta-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.     

The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.     

Human peripheral blood mononuclear cells produce IgA anti-influenza virus antibody in a secondary in vitro antibody response

The function and immunoregulation of human IgA memory B cells producing anti-influenza virus antibody was analyzed in vitro in antigen-stimulated cultures. Peripheral blood mononuclear cells (PBMC) from seven of eight normal adult volunteers naturally immunized to influenza virus produced IgA anti-influenza virus antibody when stimulated in vitro with inactivated A/Aichi/68 [H3N2] influenza virus. This IgA antibody response was approximately one-eighth the IgG antibody response. PBMC from each of five patients with selective IgA deficiency failed to produce any measurable IgA antibody. When tonsillar mononuclear cells (TMC) were studied in a similar manner, a relatively higher IgA antibody response was obtained (one-third the IgG antibody) than with PBMC. Additional studies were undertaken to investigate the immunoregulation of this IgA antibody production and the relatively lower amount produced by PBMC than by TMC. Co-cultures of peripheral blood B cells with irradiated peripheral blood T cells (to possibly inactivate a radiosensitive IgA suppressor cell) did not result in a relative increase in IgA antibody production. Also, co-cultures of B cells with increasing numbers of T cells produced parallel increases of IgG and IgA antibody when plotted on a log scale with slopes of approximately 1, suggesting that a single helper T cell was limiting for both isotypes. Finally, pokeweed mitogen-stimulated co-cultures of peripheral blood and tonsillar B and T cells revealed that the B cell population, but not the T cell population, determined the amount of IgA anti-influenza virus antibody produced. Precursor frequency analyses of tonsillar and peripheral blood B cells in antigen-stimulated cultures confirmed that tonsils contained a higher precursor frequency of B cells for IgA anti-influenza virus antibody production (3.95/10(6) B cells) than did peripheral blood B cells (0.65/10(6) B cells). Thus, IgA memory cells are preferentially found in tonsillar tissue as compared with the peripheral blood, consistent with the role of the tonsils as a mucosal immune organ.     

Characterization of the structure-activity relationships of rat types I and II 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase by site-directed mutagenesis and expression in HeLa cells

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase (3 beta-HSD) catalyzes the conversion of delta 5 -3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus representing an essential step in the biosynthesis of all classes of hormonal steroids. We have recently characterized two types of cDNA clones encoding rat 3 beta-HSD proteins, the rat type I protein being much more active than type II. In order to characterize further the functional difference between these two 3 beta-HSD types, transient expression of type I and type II 3 beta-HSD cDNAs was performed in HeLa human cervical carcinoma cells. The present study demonstrates that the type I 3 beta-HSD protein has a relative specificity 64- and 46-fold higher than type II protein for pregnenolone (PREG) and dehydroepiandrosterone (DHEA) as substrates, respectively. The Km values of type I and type II enzymes were calculated at 0.74 and 14.3 microM, respectively, using PREG as substrate whereas the respective Km values were 0.68 and 12.9 microM when DHEA was used, thus showing that their different relative specificity results largely from a different affinity for substrates. Since the change of 4 amino acid residues in type II could prevent the formation of a putative membrane-spanning domain (MSD) predicted between amino acid residues 75 and 91, chimeric cDNAs containing either type I MSD in type II (II + MSD) or an absence of this MSD in type I (I-MSD) were constructed and transiently expressed. The addition of MSD intype II 3 beta-HSD markedly increased the affinity leading to Km values similar to those found in type I 3 beta-HSD, namely 0.36 and 0.40 microM for PREG and DHEA, respectively. II + MSD chimera thus encodes a protein having a relative specificity for PREG and DHEA of 58 and 73%, respectively, to that of native type I 3 beta-HSD. Moreover, removal of MSD in the type I protein (I-MSD chimera) decreased the relative specificity of type I 3 beta-HSD protein for PREG and DHEA to only 0.37 and 0.48%, with respective Km values of 11.7 and 11.0 microM, thus strongly indicating the functional importance of this putative MSD which is predicted in wild type rat type I as well as in macaque and human 3 beta-HSD proteins.     

Protection against dextran sodium sulfate-induced colitis by dehydroepiandrosterone and 7alpha-hydroxy-dehydroepiandrosterone in the rat

In this study the anti-oxidant effect of DHEA and 7alpha-hydroxy-DHEA against oxidative stress induced by colitis was investigated in vivo in rats. The two steroids were intraperitoneally injected once daily (50 mg/kg body weight) for 7 days before the induction of colitis that was effected by a daily treatment of 5% (w/v) dextran sodium sulfate (DSS) in drinking water for 7 days. This was quantified by the evidence of weight loss, rectal bleeding, increased wall thickness, and colon length. The inflammatory response was assessed by neutrophil infiltration after a histological examination and myeloperoxidase (MPO) activity measurement. Two markers of oxidative damage were measured in colon homogenates after the onset of DSS treatment: protein carbonyls and thiobarbituric acid-reacting substances. The colonic metabolism of corticosterone by 11beta-hydroxysteroid dehydrogenases types 1 and 2 (11beta-HSD) was investigated in control and treated animals. Results indicated that colitis caused a decrease in body weight and colon length. Severe lesions were observed in the colon with a reduced number of goblet cells which contained less mucins. The lesions were associated with increased MPO activity and oxidative damage. Colonic inflammation down and up regulated the 11beta-HSD2 and 11beta-HSD1, respectively. Treatments by DHEA and 7alpha-hydroxy-DHEA attenuated the inflammatory response when MPO activity decreased; but this did not increase the colonic oxidation of corticosterone into 11-dehydrocorticosterone. Both DHEA and 7alpha-hydroxy-DHEA exerted a significant anti-oxidant effect against oxidative stress induced by colitis through reducing the oxidative damage to proteins and lipids. This resulted in a moderate increase in the amount of colonic mucus. Both DHEA and 7alpha-hydroxy-DHEA may prove useful in the prevention or treatment of colitis.     

Formation of pregnenolone- and dehydroepiandrosterone-fatty acid esters by lecithin-cholesterol acyltransferase in human plasma high density lipoproteins

Pregnenolone- (PREG-), and dehydroepiandrosterone- (DHEA-) fatty acid esters (FA) are present in human plasma, where they are associated with lipoproteins. Because plasma has the ability to form PREG-FA and DHEA-FA in vitro from their unconjugated steroid counterparts, we postulated that the LCAT enzyme might be responsible for their formation. Here we show that lecithin-cholesterol acyltransferase (LCAT) has PREG and DHEA esterifying activities. First, VLDL, IDL, LDL, and HDL were isolated by the sequential ultracentrifugation micromethod from the plasma of fasting men and women and tested for their ability to form PREG-FA, DHEA-FA, and cholesteryl esters in vitro from their respective unconjugated counterparts. The results showed that the three steroids were esterified only in HDL subfractions. The rate of tritiated PREG esterification was clearly higher than that of tritiated cholesterol and DHEA, both in total plasma and isolated HDL, and no gender difference was observed. Second, human and guinea pig LCAT were purified and used in phosphatidylcholine-reconstituted vesicles containing human apoAI to show their ability to esterify tritiated cholesterol, PREG, and DHEA in the absence of unlabeled steroid. The amount of cholesteryl ester, PREG-FA, and DHEA-FA increased after incubation as a function of time and amount of purified LCAT, showing that PREG is preferentially acylated by LCAT compared to cholesterol and DHEA. The PREG and DHEA esterifying activities of LCAT were cofactor-dependent, as shown by the absence of acylation without apoAI. Finally, we determined by HPLC the fatty acid moiety of PREG-GA and DHEA-FA formed in human plasma and guinea pig and rat sera in vitro after incubation with unconjugated tritiated PREG and DHEA. We showed that the fatty acid moieties of newly formed tritiated PREG-FA and DHEA-FA were similar to that reported for cholesteryl esters in the plasma of the three species. We conclude that LCAT has a lecithin-steroid acyltransferase activity and that PREG is probably the preferential substrate of this enzyme. In addition, the fact that the differences in the fatty acid moieties of cholesteryl esters of human, guinea pig, and rat plasmas are also observed for PREG-FA and DHEA-FA suggests that the LCAT is the sole circulating enzyme that has PREG and DHEA esterifying activities.