Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation.

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares.

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.

Differential expression of steroidogenic enzymes by the primary corpora lutea of pregnant mares during equine chorionic gonadotrophin secretion.

At the onset of equine chorionic gonadotrophin (eCG) secretion, eCG stimulates luteal androgen and oestrogen production. Although eCG concentrations increase exponentially from day 37 to day 60 of gestation and eCG is detectable in maternal serum until about day 120-150 of gestation, luteal androgen and oestrogen production peaks between 5 and 10 days after initial exposure to eCG and then decreases gradually. It is not clear how eCG regulates luteal androgen and oestrogen production. In the present study, the steady-state mRNA expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450(17alpha)) and cytochrome P450 aromatase (P450arom) in primary corpora lutea before, during and after eCG secretion was determined by northern blotting. Expression of 3beta-HSD was similar at all the stages examined. Cytochrome P450(17alpha) expression increased at the onset of eCG secretion, decreased between days 42 and 46 of gestation and was constant for the remaining period of eCG secretion. Cytochrome P450arom expression was highest before and after eCG secretion and lowest during periods of peak eCG secretion. The differential expression of P45017alpha and P450arom indicates that production of luteal androgen and oestrogen is regulated by P450(17alpha), activity. The effect of eCG on luteal steroidogenic enzyme mRNA expression appears to be stage-specific.

Equine chorionic gonadotropin regulates luteal steroidogenesis in pregnant mares.

The onset of eCG secretion in pregnant mares coincides with an increase in luteal steroid production and a relative shift toward androgen and estrogen synthesis. However, a cause-effect relationship between eCG and the shift in luteal steroidogenesis has not been demonstrated. In this study, we have investigated the effect of eCG on steroid production by the corpus luteum (CL) during equine pregnancy. All mares were supplemented with 44 mg altrenogest (a progestogen) per day on Days 18-50. Increasing doses of eCG were administered on Days 26-28, before the onset of endogenous eCG secretion, to four mares with and four mares without a functional CL (prostaglandin F2alpha administered on Day 18). Four mares with a functional CL received no exogenous eCG. In eCG-treated mares without a functional CL, progestin, androstenedione, and estrogen concentrations did not significantly increase after exogenous eCG administration or endogenous eCG secretion. In eCG-treated mares with a functional CL, progestin and estrogen production increased significantly after exogenous eCG administration and endogenous eCG secretion, whereas androstenedione concentrations tended to increase following exogenous eCG and increased significantly following endogenous eCG secretion. In mares with a functional CL that did not receive exogenous eCG, progestin and estrogen concentrations increased and androstenedione concentrations tended to increase only after the onset of endogenous eCG secretion. These data demonstrate that the increase in luteal steroidogenesis that coincides with the onset of eCG secretion is induced by eCG and results in an increase in luteal androgen and estrogen synthesis. Our findings support the hypothesis that eCG has a luteotropic action in pregnant mares.

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase, cytochrome P450 17 alpha-hydroxylase/17,20-lyase and cytochrome P450 aromatase in the equine corpus luteum of dioestrus and early pregnancy.

The onset of equine chorionic gonadotrophin (eCG) secretion in pregnant mares is associated with an increase in luteal androgen and oestrogen production. The luteal cell type(s) responsible for the increased production of androgens and oestrogens has not been identified in the equine corpus luteum. In this study, we examined the pattern of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450(17 alpha)) and cytochrome P450 aromatase (P450arom) by immunohistochemistry in equine luteal tissue collected during dioestrus (days 7-10; n = 4) and early pregnancy, before (days 29-35; n = 4) and after (days 39-45; n = 4) the onset of endogenous eCG secretion. All luteal cells expressed 3 beta-HSD, P450(17 alpha) and P450arom. The distribution of 3 beta-HSD, P450(17 alpha) and P450arom did not differ with stage of the reproductive cycle. The intensity of immunohistochemical staining for 3 beta-HSD did not appear to differ with reproductive stage. In contrast, the intensity of immunostaining for P450(17 alpha) increased after the onset of eCG secretion. The intensity of immunostaining for P450arom increased during pregnancy before the onset of eCG secretion and diminished after the onset of eCG secretion to the intensity seen in dioestrous corpora lutea. This finding suggests that androgen and oestrogen production is not compartmentalized within the equine corpus luteum. Both large and small luteal cells express the steroidogenic enzymes necessary for oestrogen production, and the intensity of immunostaining for P450(17 alpha) and P450arom appears to be stage-specific.

Differential transcription of steroidogenic enzymes in the equine primary corpus luteum during diestrus and early pregnancy.

In pregnant mares, eCG stimulates luteal androgen and estrogen production, increasing plasma concentrations 2- to 3-fold. To study how these changes are regulated, we examined the expression of mRNA for the steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450 17 alpha), and cytochrome P450 aromatase (P450arom) in equine primary corpora lutea using Northern blot analyses. Three equine specific cDNAs were generated by reverse transcriptase polymerase chain reaction. When compared to human, bovine, and rat sequences, the nucleotide identities were 82%, 84%, and 76%, respectively, for 3 beta-HSD cDNA (843 base pairs [bp]); 79%, 80% and 66% for P450(17) alpha cDNA (541 bp); and 80%, 83% and 75% for P450arom cDNA (289 bp). The P450(17) alpha cDNA sequence demonstrated 99.6% nucleotide identity with the previously published sequence for equine testicular P450(17) alpha. Luteal tissue samples were collected at three times: diestrus (Days 8-10), early pregnancy before the onset of eCG secretion (Days 29-35), and early pregnancy after the onset of eCG secretion (Days 42-45). Although no significant changes were observed in 3 beta-HSD expression, P450(17) alpha and P450arom demonstrated stage-specific transcriptional regulation. Steady-state levels of P450(17) alpha mRNA were similar during diestrus and early pregnancy before the onset of eCG secretion but increased significantly after the onset of eCG secretion. Cytochrome P450arom mRNA levels decreased significantly after the onset of eCG secretion. Steady-state levels of P450arom mRNA were highest in luteal tissue collected during pregnancy before the onset of eCG secretion and intermediate during diestrus. Secretion of eCG appears to increase luteal estrogen synthesis by a transcriptional up-regulation of P450(17) alpha expression. These data suggest that availability of aromatizable androgens may be rate-limiting in luteal estrogen synthesis before the onset of eCG secretion.

Carcinogenicity of CoCrMo (F-75) implants in the rat.

A long-term study was conducted in rats to assess the contribution of the surface area of CoCrMo devices to carcinogenesis. Groups consisting of 104 rats each (52 male, 52 female) were either implanted with metal cylinders fixed on the left, lateral femur (groups 1-3) or injected with a suspension of metal microspheres in the dorsal subcutis (group 4). Group 1 (control) received solid Ti6Al4V cylinders [surface area to body weight (SA/BW) ratio measuring 1.35 times that of human total hip prosthesis (HTHP)]. Group 2 was implanted with solid CoCrMo (SA/BW ratio: identical to implants of group 1). Group 3 received sintered-porous CoCrMo devices (SA/BW ratio: 30 x HTHP). Group 4 was injected with a suspension of CoCrMO microspheres (SA/BW ratio: 135 x HTHP). Implant-associated tumors (IATs) were observed in 23, 14, 3, and 15 rats of groups 1, 2, 3, and 4, respectively. Within groups 1 and 2, 34 IATs were associated with loose implants, three with undetermined implant fixation status, and none with fixed implants. A significantly increased accumulation of chronic inflammatory tissues around loose rather than fixed implants suggested a foreign-body reaction as the primary mechanism of carcinogenesis. A secondary role in carcinogenesis was ascribed to the increased CoCrMo implant SA/BW ratios as indicated by a 14.6% IAT incidence in group 4 versus 3% in group 3. These results support the notion that early intervention in the removal of loose metal devices is warranted to mitigate against foreign body-induced carcinogenesis, at least in this animal model.

Aerosols, cloud microphysics, and fractional cloudiness.

Increases in aerosol concentrations over the oceans may increase the amount of low-level cloudiness through a reduction in drizzle-a process that regulates the liquid-water content and the energetics of shallow marine clouds. The resulting increase in the global albedo would be in addition to the increase due to enhancement in reflectivity associated with a decrease in droplet size and would contribute to a cooling of the earth's surface.