Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity-2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy-diagnosed HTx rejection (cellular and/or antibody-mediated), serum sST2 was elevated compared to rejection-free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor-α and IL-1ß as upstream activators. In total, our data indicate that alloimmune-associated pro-inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

Sequential histologic changes in the healing process in small bowel allografts treated for acute cellular rejection.

INTRODUCTION: Acute cellular rejection (ACR) is a common complication seen in small intestinal transplant patients. Once diagnosed, follow-up endoscopy/biopsies may be performed to assess for response to therapy and the pathologist is often asked to determine whether the findings are compatible with treated/resolving or ongoing ACR. To this end, the sequence of resolution of ACR's changes in biopsies is important. METHODS: We retrospectively reviewed the clinical histories and hematoxylin and eosin-stained slides from 16 cases of ACR patients who underwent isolated small bowel or combined liver/small bowel/pancreas transplants. Selected cases were new diagnoses of mild ACR with prior negative biopsies in the preceding 2 months, treatment for rejection based on the ACR diagnosis, and biopsies in the following 4 weeks diagnosed as "treated ACR" or "normal." The presence of ACR diagnostic features (epithelial injury, lamina propria [LP] inflammation with resident cell population, and crypt apoptotic body [AB] quantification) were evaluated. A series of 15 age-matched screening intestinal allograft biopsies were used as controls. RESULTS: After treatment, epithelial injury as manifested by mucin depletion resolved by 2 week. LP inflammation was significantly reduced by 1 week after therapy, with a marked decrease in activated lymphocytes and eosinophils, and completely returned to control levels by week 3. Apoptosis fell below the diagnostic threshold for rejection (<6 AB/10 crypts) by week 2 and was equivalent to control biopsies at week 3. CONCLUSION: Knowledge of the sequence of the resolution of the histologic features of ACR after treatment may provide useful information to pathologists evaluating follow-up biopsies. These data show that both LP inflammation and crypt epithelial injury are reduced by 1 week after anti-rejection therapy and their persistence may signify ongoing rejection.

Positive and negative selection of antigen-specific B cells in transgenic mice expressing variant forms of the V(H)1 (T15) heavy chain.

Four variant forms of the V1 (T15-H chain) gene are synthesized in mice. Each V1 variant pairs with a distinct L chain to produce a binding site having specificity for phosphocholine (PC). Transgenic mice expressing variant forms of the V1 gene were analyzed to elucidate the factors driving B cell selection into the peripheral repertoire. In all four lines of H chain transgenic mice analyzed, transgene expression caused complete allelic exclusion of endogenous H chains in the bone marrow (BM), whereas most splenic B cells expressed endogenous H chains. The number of sIgM(+) BM B cells and their sIg receptor number was reduced compared to that of normal transgene-negative controls, suggesting that B cells expressing transgene-encoded H chains were being negatively selected in the BM. Mice expressing autoreactive forms of the V1 transgene with lower affinity for PC (M603H and M167H) exhibit positive selection of PC-specific B cells into the spleen, whereas mice expressing the higher affinity T15H variant exhibited elevated PC-specific B cells in the peritoneal cavity but few V(H)1(+) splenic B cells. These data suggest that the higher affinity T15-id(+) B cells preferentially survive in the peritoneal cavity. When these H chain transgenes were crossed into the mu MT knockout mouse in which surface expression of endogenous H chains is blocked, the percent of splenic V(H)1(+) PC-specific B cells increased up to 5-fold and T15-id(+) B cells were detectable in the spleen of T15H mice. This implies that T15-id(+) PC-specific B cells can be selected into the periphery, but they compete poorly with follicular B cells expressing endogenous Ig.

Highly reduced protection against Streptococcus pneumoniae after deletion of a single heavy chain gene in mouse.

Phosphocholine (PC) is the immunodominant epitope found on the surface of Streptococcus pneumoniae (SPn). T15-idiotype Abs, whose heavy (H) chain variable region is encoded by the V1 gene, are dominant in the anti-PC response in adult mice and protect mice from lethal pneumococcal infection. The ability of anti-PC Abs using H chains other than the V1 H chain to protect against pneumococcal infection remains controversial. We generated V1(-/-) knockout mice to determine whether protective anti-PC Abs could be produced in the absence of the V1 gene. No anti-PC Abs were produced in V1(-/-) mice immunized with avirulent SPn; however, PC-BSA binding Abs were induced after immunization with PC-keyhole limpet hemocyanin but at significantly lower levels than those in wild-type mice. These Abs provided poor protection against virulent SPn; thus, <25% of V1(-/-) mice survived challenge with 10(4) bacteria as compared with 100% survival of V1(+/+) mice. The anti-PC Abs in V1(-/-) mice were heteroclitic, binding to nitrophenyl-PC better than to PC. None of nine hybridomas produced from V1(-/-) mice provided passive protection. However, the V1(-/-) mice produced normal amounts of Ab to SPn proteins that can partially protect mice against SPn. These data indicate that the V1 gene is critical for the production of anti-PC Abs providing optimum protection against infection with SPn, and the V1(-/-) mice could be useful in unmasking epitopes other than the immunodominant PC epitope on SPn capable of providing cross protection.

Autoreactive B cells escape clonal deletion by expressing multiple antigen receptors.

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2-/-) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2-/- knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.

Sequence changes at the V-D junction of the VH1 heavy chain of anti-phosphocholine antibodies alter binding to and protection against Streptococcus pneumoniae.

X-linked immune deficient (Xid) mice fail to produce anti-phosphocholine (PC) antibodies even after immunization with Streptococcus pneumoniae. Consequently, Xid mice are extremely susceptible to infection with S. pneumoniae, PC-specific B cells appear to undergo clonal deletion in Xid mice; however, a new thymus-dependent form of PC, 6-(O-phosphocholine)hydroxyhexanoate (EPC), can rescue PC-specific B cells from the bone marrow presumably by providing T cell help before clonal deletion. Analysis of PC-specific IgG hybridomas from Xid mice revealed utilization of several V-D junctional variants of the VH1 gene segment rearranged to different D and JH gene segments. The majority of Xid anti-PC antibodies exhibit an Asp-->Gly95H replacement at the V-D junction. These Gly95H VH1 variants associate with kappa 1C L chains to produce anti-PC antibodies that: (1) have low relative affinity for PC, (ii) are heteroclitic for nitrophenylphosphocholine and (iii) fall to bind to or provide protection against S. pneumoniae. Single prototypic V-D variants of the T15 idiotype (Asp95H), M603 idiotype (Asn95H) and M167 idiotype (Asp95H-Ala96H) were also induced in Xid mice. The M603-like and M167-like antibodies bound to and protected against S. pneumoniae even though they exhibited Kas for PC which were lower than T15 idiotype+ antibodies. These data demonstrate that small changes in the V-D junctional sequence of the T15 (VH1) heavy chain alter L chain usage and the structure of the PC binding site so that the PC expressed on S. pneumoniae is no longer recognized.

Antiproliferative activity in vitro and in vivo of the spicamycin analogue KRN5500 with altered glycoprotein expression in vitro.

The spicamycin analogue KRN5500 (NSC 650426; SPA) is derived from Streptomyces alanosinicus. The unique structure contains a purine, an aminoheptose sugar, glycine, and a tetradecadiene fatty acid. SPA potently inhibits the growth of certain human tumor cell lines in vitro (IC50 for growth <100 nM) and displays marked activity in vivo in Colo 205 colon carcinoma xenografts. Selective inhibition of labeled precursor incorporation was not evident at 1 or 4 h of exposure to the drug, but at 8 h, [3H] leucine incorporation was inhibited by approximately 40% at or below the IC50 for cell growth. Because of the structural similarity of SPA to inhibitors of glycoprotein processing, we examined the effect of SPA on indicators of glycoprotein synthesis and processing in HL60TB promyelocytic leukemia and Colo 205 colon carcinoma cells. Brief periods of exposure ( approximately 30 min) to SPA at the IC50 for growth increased incorporation of [3H]mannose. When examined by Western blotting after prolonged (40-48 h) incubation with lectins that target mannose-containing carbohydrates, Galanthus nivalis agglutinin and concanavalin A, a qualitative change in the pattern of mannose-containing glycoproteins was observed in HL60TB cells. Significant changes in the pattern of surface glycoprotein expression in intact cells were demonstrated by flow cytometry using fluorescence-labeled lectins. An increase in the number of cells binding G. nivalis agglutinin (indicating terminal mannose) was noted, but a decrease in the amount of lectin bound per cell was noted for wheat germ agglutinin (detecting sialic acid and terminal beta-N-acetyl glucosamine residues). Electron microscopy revealed loss of microvilli, and the Golgi apparatus appeared inflated. Our findings, therefore, raise the possibility that cells exposed to SPA have altered glycoprotein processing after exposure to low concentrations of drug, prior to the occurrence of overt cytotoxicity. These effects are consistent with a prominent early effect of SPA on the enzymatic machinery or organelles important for proper glycoprotein processing and emphasize the novelty of this agent's likely mechanism of action.

bcl-2 alters the antigen-driven selection of B cells in mukappa but not in mu-only Xid transgenic mice.

A point mutation in the pleckstrin homology domain of the mouse Bruton's tyrosine kinase (btk) gene results in an X-linked immune defect, Xid, characterized by immunologic unresponsiveness to polymeric carbohydrate Ags. In Xid mice, B cells specific for phosphocholine (PC) do not develop in peripheral lymphoid tissues because they either fail to be positively selected from the marrow or they are clonally deleted via an Ag-driven, receptor-mediated process. Overexpression of the bcl-2 gene allows PC-specific B cells to survive and mature in Xid mukappa anti-PC transgenic mice, but PC-specific B cells are not rescued by bcl-2 in Xid mu-only transgenic mice. The failure of bcl-2 to rescue PC-specific B cells, in mu-only transgenic mice suggests that either it does not correct the btk defect in the Ag-driven selection process that occurs in pre-B cells and/or in very immature B cells or that a btk-dependent proliferative phase is required for the selection and amplification of the PC-specific B cells in mu-only transgenic mice. The rescue of PC-specific B cells in mukappa transgenic mice indicates that bcl-2 can alter receptor-mediated B cell selection at late stages in B cell development. The rescued PC-specific B cells in Xid male mice do not exhibit an altered proliferation profile in response to B cell-stimulating agents compared with B cells from unmanipulated Xid mice; thus, they fail to respond to soluble anti-mu, or PC-dextran, but they proliferate in response to PC, anti-mu, or anti-id conjugated to Sepharose.

Efflux of newly synthesized cholesterol and biosynthetic sterol intermediates from cells. Dependence on acceptor type and on enrichment of cells with cholesterol.

Previous studies suggest that during sterol synthesis in cells, cholesterol and precusor sterols are transported to the plasma membrane and that this transport is stimulated by the binding of high density lipoprotein (HDL) to its putative cell surface receptor, leading to enhanced sterol efflux. Little is known about the identities of synthesized sterols subject to efflux or whether efflux of cholesterol and precursor sterols are stimulated equally by HDL. To address these issues, cells were incubated with [3H]acetate or [3H]mevalonate and sterol acceptors, and then the labeled sterols in cells and efflux media were analyzed by high pressure liquid chromatography methods that resolved cholesterol and precursor sterols. In non-hepatic cells (Chinese hamster ovary (CHO), fibroblasts, and smooth muscle), cholesterol and multiple precursor sterols accumulated. In CHO cells, the major products were cholesterol and desmosterol, which together constituted 50% of labeled nonsaponifiable lipids. When media contained human HDL3 (1 mg of protein/ml), the molar efflux of synthesized desmosterol was four times that of cholesterol, and the 8-h efflux of these sterols, each normalized to its own production, averaged 48 and 16%, respectively. When media contained egg phosphatidylcholine vesicles (1 mg/ml), the efflux of these sterols averaged 18 and 2.4%, respectively. Thus, with both acceptors, desmosterol was the major synthesized sterol released from cells, and its efflux was substantially greater than that of synthesized cholesterol. High relative efflux of desmosterol (or a desmosterol-like sterol) occurred in all cell types and in both cholesterol-enriched and unenriched cells. These results demonstrated qualitatively similar efflux of synthesized sterols in the presence of HDL3 and phospholipid vesicles, arguing against an absolute requirement for acceptors that interact with the HDL receptor. To probe for possible quantitative differences in the capabilities of these two acceptors, the ratios of (efflux to HDL3)/(efflux to phosphatidylcholine vesicles) were calculated for synthesized cholesterol and desmosterol, plasma membrane cholesterol, and lysosomal cholesterol. In comparison to plasma membrane cholesterol, there was little or no HDL selectivity for lysosomal cholesterol or synthesized desmosterol, whereas there was a 2-3-fold selectivity for synthesized cholesterol, suggesting that the ability of HDL to enhance the efflux of synthesized sterols is a modest quantitative effect and confined to cholesterol.

Cholesterol quantitation by GLC: artifactual formation of short-chain steryl esters.

A simple and rapid method for the quantitation of total cholesterol in lipid extracts using gas-liquid chromatography is presented here as a modification of an earlier saponification procedure (Ishikawa, T. T., J. MacGee, J. A. Morrison, and C. J. Glueck. 1974. Quantitative analysis of cholesterol in 5 to 20 microliters of plasma. J. Lipid Res. 15: 286-291). Using the original method, as well as a slightly modified version, we found a systematic loss of cholesterol measured as total cholesterol that was attributable to the formation of a byproduct during the procedure. Depending on the nature of the solvent mixture used for extraction after saponification, different byproducts were produced that had longer retention times than cholesterol. The byproducts were identified as cholesteryl butyrate (produced when methyl butyrate was included in the solvent mix) and cholesteryl propionate (with ethyl propionate in the solvent mix) by comparison to authentic standards using gas chromatography-mass spectroscopy. Using mixtures of cholesterol standards, we compared several solvents in lieu of the solvent mixture used in the original extraction procedure to identify those that eliminate the formation of the byproducts. Our optimized microsaponification procedure uses a single solvent, tetrachloroethylene, to extract lipids after the saponification reaction, and improves the accuracy of the cholesterol determination.

Substrate-based inhibitors of lanosterol 14 alpha-methyl demethylase: II. Time-dependent enzyme inactivation by selected oxylanosterol analogs.

Selected 15-, 32-, and 15,32-substituted lanosterol analogs are shown here to display time-dependent inactivation and lanosterol 14 alpha-methyl demethylase. These molecules are competitive with respect to substrate and require NADPH and O2 in order to display time dependence, thus supporting the premise that they are mechanism-based inactivators. Structural features required for lanosterol demethylation by the lanosterol demethylase such as nuclear double bond location and availability of an abstractable 15 alpha-proton are also essential elements for time-dependent inactivation. 32-(S)-Vinyllanost-8-en-3 beta,32-diol is a potent time-dependent inactivator (Kinact/Ki = 0.36 min-1 microM-1), while the 32-(R)-vinyllanost-8-en-3 beta,32-diol functions solely as a competitive demethylase inhibitor. These results support the premise that stereoselective oxidation occurs during lanosterol demethylation and that the 32-pro-S proton is abstracted during the demethylation reaction.

Substrate-based inhibitors of lanosterol 14 alpha-methyl demethylase: I. Assessment of inhibitor structure-activity relationship and cholesterol biosynthesis inhibition properties.

A series of 15-, 32-, and 15,32-substituted lanost-8-en-3 beta-ols is described which function as inhibitors of cholesterol biosynthesis. These agents inhibit lanosterol 14 alpha-methyl demethylase activity as well as suppress HMG-CoA reduction activity in cultured cells. Several of these agents are extremely potent as both demethylase inhibitors and reductase suppressors, while others are more selective in their activities. Selected regio double bond isomers show preference for demethylase inhibition with the following order: delta 8 > delta 7 > delta 6 = unsaturated sterols. Comparisons also show that 4,4-dimethyl sterols are always more potent demethylase inhibitors and reductase suppressors than their 4,4-bisnomethyl counterparts. However, evaluation of an extensive oxylanosterol series leads us to conclude that demethylase inhibition and reductase suppression are not parallel in the same molecule. In addition, the oxylanosterols, but not the oxycholesterols, are able to disrupt coordinate regulation of HMG-CoA reductase from the LDL receptor. Thus, oxylanosterol treatment at levels which suppress reductase activity enhances LDL receptor activity. These results demonstrate that compounds can be made which (1) are selective reductase suppressors enabling dissection of the dual inhibitor nature of these compounds and (2) maximize reductase suppression and LDL receptor induction without demethylase inhibition which could lead to novel agents for serum cholesterol lowering.

A novel phosphocholine antigen protects both normal and X-linked immune deficient mice against Streptococcus pneumoniae. Comparison of the 6-O-phosphocholine hydroxyhexanoate-conjugate with other phosphocholine-containing vaccines.

A novel form of phosphocholine (PC), p-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate (EPC) coupled to keyhole limpet hemocyanin (KLH) has been compared with unencapsulated, avirulent Streptococcus pneumoniae (R36a) and with the traditional thymus-dependent form of PC, diazophenylphosphocholine (DPPC)-conjugated KLH for its vaccine potential against virulent S. pneumoniae. Immunization with any of these three PC-containing Ags protects normal mice against a lethal challenge with 10(4) S. pneumoniae, whereas only EPC-KLH provides total protection to Xid mice. DPPC-KLH and unencapsulated S. pneumoniae confer less than 40% protection in Xid mice. Passive transfer of a PC-specific hybridoma Ab made from EPC-KLH-immunized Xid mice also provided protection against lethal challenge with S. pneumoniae. Protective anti-PC Ab were capable of binding to the surface of virulent bacteria, whereas anti-PC Ab incapable of binding to the bacterial surface failed to protect. Furthermore, serum Ab from EPC-KLH immunized and protected mice bound to S. pneumoniae, whereas secondary Abs from DPPC-KLH- or R36a-immunized mice failed to bind to the bacteria. EPC-KLH is potentially a vaccine candidate for pneumococcal prophylaxis in settings of immune compromise.

Induction of phosphocholine-specific antibodies in X-linked immune deficient mice: in vivo protection against a Streptococcus pneumoniae challenge.

X-linked immune deficient (XID) mice are susceptible to infection with Streptococcus pneumoniae because they fail to mount an immune response to the immunodominant phosphocholine (PC) epitope on the bacterial cell wall. It is difficult to induce PC-specific antibodies in XID mice because PC-specific B cells expressing the T15-, M167- and M603 idiotype (Id), which provide protection against S. pneumoniae, are deleted in these mice via an antigen-specific, receptor-mediated process. In addition, the standard PC hapten, p-diazophenylphosphocholine (DPPC), induces high affinity phenylphosphocholine (PPC)-specific antibodies in XID mice, which are not protective against S. pneumoniae. We have used a novel PC hapten, p-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate (EPC), to induce PC-specific antibodies in XID mice. The immune response to EPC-keyhole limpet hemacyanin (KLH) is dominated by IgG1, VH1+, T15-Id-, PC-inhibitable antibodies. A small IgM anti-PC response having a consistent T15-Id+ component is also induced in XID mice, whereas normal mice produce a large IgM response dominated by T15-Id+ antibodies. The immune response to EPC-KLH remains predominantly PC-inhibitable even after multiple immunizations, while the response to DPPC-KLH becomes dominated by PPC-specific antibodies. C.CBA/N mice immunized twice with EPC-KLH are protected against 10(4) S. pneumoniae while as few as 10 bacteria are 100% lethal for the unimmunized controls. The ability of EPC-protein to induce a long-lived, PC-specific response should make this hapten a potential TD vaccine candidate for S. pneumoniae.

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by 15 alpha-fluorolanost-7-en-3 beta-ol. A mechanism-based inhibitor of cholesterol biosynthesis.

The chemical synthesis and metabolic characteristics of the lanosterol analogue, 15 alpha-fluorolanost-7-en-3 beta-ol, are described. The 15 alpha-fluorosterol is shown to be a competitive inhibitor of the lanosterol 14 alpha-methyl demethylase (Ki = 315 microM), as well as substrate for the demethylase enzyme. Metabolic studies show that the 15 alpha-fluorosterol is converted to the corresponding 15 alpha-fluoro-3 beta-hydroxylanost-7-en-32-aldehyde by hepatic microsomal lanosterol 14 alpha-methyl demethylase but that further metabolic conversion to cholesterol biosynthetic intermediates is blocked by virtue of the 15 alpha-fluoro substitution. When cultured cells are treated with the fluorinated lanosterol analogue, a decrease in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and immunoreactive protein was observed. However, when the lanosterol 14 alpha-methyl demethylase-deficient mutant cell line, AR45, is treated with the fluorosterol, no effect upon HMG-CoA reductase is observed. Thus, metabolic conversion of the sterol to its 32-carboxaldehyde analogue by the lanosterol 14 alpha-methyl demethylase is required for HMG-CoA reductase suppressor activity. Measurement of HMG-CoA reductase mRNA levels in 15 alpha-fluorosterol-treated Chinese hamster ovary (CHO) cells reveals that mRNA levels are not decreased by the sterol as would be expected for a sterol regulator of HMG-CoA reductase activity. The decrease in HMG-CoA reductase protein is due to inhibition of enzyme synthesis, suggesting that the 15 alpha-fluorosterol reduces the translational efficiency of the reductase mRNA. Measurements of the half-life of HMG-CoA reductase show that, in contrast to other oxysterols, the 15 alpha-fluorolanostenol does not increase the rate of degradation of the enzyme. Collectively, these data support the premise that oxylanosterols regulate HMG-CoA reductase expression through a post-transcriptional process which may be distinct from other previously described sterol regulatory mechanisms.

Selection of antigen-specific, idiotype-positive B cells in transgenic mice expressing a rearranged M167-mu heavy chain gene.

Flow cytometric analysis of antigen-specific, idiotype-positive (id+), B cell development in transgenic mice expressing a rearranged M167-mu gene shows that large numbers of phosphocholine (PC)-specific, M167-id+ B cells develop in the spleen and bone marrow of these mice. Random rearrangement of endogenous V kappa genes, in the absence of a subsequent receptor-driven selection, should give rise to equal numbers of T15- and M167-id+ B cells. The observed 100-500-fold amplification of M167-id+ B cells expressing an endogenous encoded V kappa 24]kappa 5 light chain in association with the M167 VH1-id transgene product appears to be an antigen driven, receptor-mediated process, since no amplification of non-PC-binding M167 VH1/V kappa 22, T15-id+ B cells occurs in these mu-only transgenic mice. The selection and amplification of antigen-specific, M167-id+ B cells requires surface expression of the mu transgene product; thus, no enhancement of M167-id+ B cells occurs in the M167 mu delta mem-transgenic mice, which cannot insert the mu transgene product into the B cell membrane. Surprisingly, no selection of PC-specific B cells occurs in M167-kappa-transgenic mice although large numbers of B cells expressing a crossreactive M167-id are present in the spleen and bone marrow of these mice. The failure to develop detectable numbers of M167-id+, PC-specific B cells in M167-kappa-transgenic mice may be due to a very low frequency of M167-VH-region formation during endogenous rearrangement of VH1 to D-JH segments. The somatic generation of the M167 version of a rearranged VH1 gene may occur in less than one of every 10(5) bone marrow B cells, and a 500-fold amplification of this M167-Id+ B cell would not be detectable by flow cytometry even though the anti-PC antibody produced by these B cells is detectable in the serum of M167-kappa-transgenic mice after immunization with PC.

Lanosterol 14 alpha-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediate.

Conditions have been identified which permit metabolic formation of the third oxidized intermediate in the lanosterol 14 alpha-methyl demethylase reaction cascade. Metabolism of either the immediate precursor substrate 3 beta-hydroxylanost-8-en-32-al or lanost-8-ene-3 beta,32-diol under mixed function oxidase conditions affords formation of the intermediate. It must be emphasized that the intermediate can only be detected if saponification procedures are omitted during sterol isolation. Comparative chemical and biochemical studies of the isolated metabolite with 3 beta,15 alpha-dihydroxylanost-8-en-32-al reveal that the metabolite is not the 15 alpha-hydroxylanosterol aldehyde, a putative demethylase intermediate. The metabolite is efficiently converted to the demethylated delta 8,14-diene sterol in the absence of molecular oxygen or NADPH, thus supporting its identity as the final oxidized intermediate in the lanosterol 14 alpha-methyl demethylase cascade. 1H NMR analysis shows a proton resonance at 7.86 ppm consistent with a formyloxy proton. Mass spectral and infrared analysis of the metabolite clearly establish oxygen insertion into the immediate precursor substrate, 3 beta-hydroxylanost-8-en-32-al. Collectively, the biochemical and chemical characteristics of the metabolite support a structural assignment for the metabolite as 14 alpha-formyloxy-lanost-8-en-3 beta-ol.

Generation of regulatory oxysterols: 26-hydroxylation of cholesterol by ovarian mitochondria.

De novo synthesis of cholesterol and low-density lipoprotein (LDL) receptor levels are suppressed in the presence of cholesterol. Recent evidence suggests that a cholesterol metabolite (possibly a hydroxysterol), not cholesterol per se, is the effector that inhibits transcription of genes encoding enzymes involved in sterol synthesis and LDL receptors. We found that 26-hydroxycholesterol inhibits human ovarian cell sterol synthesis, and that luteinized human granulosa cells contain 26-hydroxylase messenger RNA (mRNA). We proceeded to characterize the enzyme generating 26-hydroxycholesterol in the rat ovary. Mitochondria derived from ovaries of PMSG-human CG (hCG) primed immature rats (day 3 post-hCG) metabolized [3H] cholesterol into [3H]26-hydroxycholesterol in the presence of nicotinamide adenine dinucleotide phosphate and aminoglutethimide (100 micrograms/ml), added to inhibit metabolism of sterols by the cholesterol side-chain cleavage system. The identity of the product was confirmed by chromatography in several systems; recrystallization to constant specific activity and mass spectrometry. Negligible 26-hydroxylase activity was detected in other ovarian subcellular fractions. 26-Hydroxycholesterol formation progressed at a linear rate for up to 40 min and was linearly related to mitochondrial protein added to the incubation mixture. 26-Hydroxylase was markedly stimulated (5-fold) by calcium (0.2 mM). Maximal rates of 26-hydroxycholesterol formation observed were 1 pmol/min.mg protein. This activity is substantially lower than cholesterol side-chain cleavage measured in the absence of aminoglutethimide. Ketoconazole (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner. Pregnenolone (1-1000 microM) and progesterone (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner, with appreciable inhibitory effects in the 1-10 microM range. We suggest that 26-hydroxycholesterol is an intracrine regulator that controls cellular sterol metabolism. Formation of 26-hydroxcholesterol in ovarian cells may be regulated by steroidogenic activity in such a way as to ensure availability of steroid hormone precursors. When steroidogenesis is active, 26-hydroxylase is inhibited by products of the side-chain cleavage system, allowing increased de novo sterol synthesis and LDL uptake. With reduced steroidogenic activity and less demand for cholesterol, 26-hydroxylase is not blocked, permitting formation of 26-hydroxycholesterol with attendant reduction in sterol synthesis and LDL receptor gene expression.

Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme.

Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism.

A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity.

A Chinese hamster ovary cell mutant, AR45, was selected for amphotericin B resistance after treatment with the mutagen ethyl methanesulfonate. The mutant is a cholesterol auxotroph with a deficiency in cholesterol biosynthesis. Whole cell experiments demonstrate that the mutant accumulates the C30 sterols, lanosterol and dihydrolanosterol, under culture conditions which promote active sterol biosynthesis. Metabolic studies show that the C29 sterol demethylation product of lanosterol, but not lanosterol itself, is actively converted to end product cholesterol by whole cells as well as by microsomal preparations derived from the mutant. Detectable amounts of several cytochromes can be observed spectrally in the AR45 demonstrating that it is not a general heme-deficient mutant. Collectively, these results characterize the AR45 mutant cells as being lanosterol 14 alpha-methyl demethylase-deficient. The cell line should prove useful in studying regulation of the demethylase enzyme and the putative endogenous regulatory oxysterol. It should also be a useful tool in the molecular cloning and elucidation of genetic properties of the demethylase.