Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

Mechanism of down regulation of luteinizing hormone receptors and steroidogenesis in corpora lutea

The mechanism of ‘down regulation’ of luteinizing hormone receptors was investigated in pseudopregnant rats using a modified radioimmunoassay capable of measuring endogenous tissue-bound hormone. Treatment of pseudopregnant animals with a desensitizing dose (desensitization treatment) of human chorionic gonadotropin resulted in a decrease in receptor concentration. This decrease was prevented if the animals were treated prior to the desensitization treatment with indomethacin, an inhibitor of prostaglandin biosynthesis, suggesting a role for prostaglandins in down regulation. The desensitization treatment resulted in a time-dependent decrease in subsequent responsiveness of the tissue to luteinizing hormone. Basal progesterone production rate was also decreased following desensitization. Total tissue cholesterol was found to be decreased following desensitization treatment, without any change in the ratio of free to esterified cholesterol. Mitochondrial cholesterol was significantly reduced and pregnenolone production by the mitochondria of desensitized corpora lutea was also markedly reduced. However, when cholesterol was added to the mitochondria of desensitized corpora lutea, pregnenolone production was increased, reaching values almost equal to that shown by the control mitochondria. These results show that decrease in the responsiveness following desensitization treatment is due to, besides receptor loss, decrease in tissue cholesterol, in particular mitochondrial cholesterol. The cholesterol side chain cleavage activity, although low, appears to be functionally intact; the low activity could be attributed to low levels of mitochondrial cholesterol.     

Antinociceptive action of FK506 in mice

Immunophilins are abundantly present in the brain as compared to the immune system. Immunophilin-binding agents like FK506 are known to inactivate neuronal nitric oxide synthase (nNOS) by inhibiting calcineurin and decrease the production of nitric oxide. Nitric oxide is involved in the mediation of nociception at the spinal level. In the present study, the effect of FK506 on the tail flick response in mice and the possible involvement of NO-L-arginine pathway in this paradigm was evaluated. FK506 (0.5, 1 and 3 mg/kg, ip) produced a significant antinociception in the tail flick test. Nitric oxide synthase (NOS) inhibitor L-NAME significantly and dose dependently (10-40 mg/kg, ip) potentiated the FK506 (0.5 mg/kg)-induced antinociception. On the other hand, NOS substrate L-arginine (100, 200 and 400 mg/kg) inhibited the FK506-induced antinociception in a dose-dependent manner. Concomitant administration of L-NAME (20 and 40 mg/kg) with L-arginine (200 mg/kg) blocked the inhibition exerted by L-arginine on the FK506-induced antinociception. Thus, it was concluded that NO- L-arginine pathway may be involved in the FK506-induced antinociception in tail flick test.     

Modulation of NSAID-induced antinociceptive and anti-inflammatory effects by alpha2-adrenoceptor agonists with gastroprotective effects

Tizanidine, an alpha2-adrenergic receptor agonist with myospasmolytic action, is indicated for the treatment of back pain either as monotherapy or in combination with nonsteridal anti-inflammatory drugs (NSAIDs). Tizanidine (0.25-1.0 mg/kg) significantly produced analgesic and anti-inflammatory effect in acetic acid induced writhing in mice and carrageenan-induced paw edema in rats, respectively. The effects were comparable with clonidine (0.25 and 0.50 mg/kg), another alpha2-agonist. Yohimbine (1 mg/kg), alpha2-adrenergic antagonist reversed the effect of tizanidine. Tizanidine (0.25 mg/kg) and clonidine (0.25 mg/kg) significantly potentiated the antinociceptive and anti-inflammatory effect of NSAIDs (nimesulide, meloxicam and naproxen). Tizanidine (1 mg/kg) did not alter basal pH, acidity (free and total) of gastric content and did not produce any mucosal injury in fasted rats. Tizanidine (1 mg/kg) significantly reduced meloxicam (UD50 3.21 mg/kg), nimesulide (UD50 24.52 mg/kg) and naproxen (UD50 14.10 mg/kg)-induced ulcerogenic effect (ulcer index, pH and free/total acidity). It is expected that tizanidine exerted gastrotprotection through stimulation of gastric and central alpha2-adrenergic receptors. Present investigation suggested that tizanidine not only enhance the analgesic and anti-inflammatory effect of NSAIDs but also improved gatstrointestinal tolerability of NSAIDs through modulation of central alpha-2-receptors. From this study, it can be speculated that tizanidine and NSAID combination therapy would prove to be a novel approach to treat nociceptive/inflammatory conditions with improved gastric tolerability of NSAIDs.     

Effect of nimesulide on acetic acid- and leukotriene-induced inflammatory bowel disease in rats

Inflammatory bowel disease (IBD) is a relapsing inflammation of intestine, which is mediated by release of inflammatory mediators. Both cyclo-oxygenase product prostaglandin (PGE2) and lipo-oxygenase product leukotriene (LTB4), may contribute to the pathogenesis of the inflammatory response. Nimesulide, a preferential COX-2 inhibitor was evaluated for its efficacy against experimental colitis in two different models (acetic acid- and LTB4-induced IBD) in rats. Inflammatory response was induced by intrarectal single administration of acetic acid or LTB4. Nimesulide (9 and 18 mg/kg, p.o.) significantly prevented development of inflammatory changes, decreased myeloperoxidase (MPO) activity, and also restored the altered contractility response of the isolated colon segment to KCl. The results suggested the involvement of both cyclo-oxygenase (COX) and lipo-oxygenase-mediated proinflammatory agents in colonic inflammatory process associated with IBD. Further, this study suggests that such therapeutic interventions may be of value in the treatment of IBD.     

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.     

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions.     

Effect of licofelone against NSAIDs-induced gastrointestinal ulceration and inflammation

The present study was aimed to evaluate the effect of licofelone, a dual inhibitor of cycloxygenase1/2-5-lipoxygenase against indomethacin-induced gastric damage in rats and mice in order to assess the role of leukotrienes if any, in non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrointestinal inflammation. Acute pretreatment with licofelone reversed the indomethacin-induced gastric ulceration, neutrophil adhesion in mesentery venules, neutrophil count in blood, lipid peroxides and vascularity in the stomachs of mice and rats. Further, chronic pretreatment of licofelone also prevented indomethacin-induced gastric morphological changes and cellular infiltration in mesentery venules. Moreover, acute administration of indomethacin elevated leukotriene B4 levels in gastric mucosa, which was reversed by pretreatment with licofelone The results suggest that licofelone offered gastroprotection against NSAIDs-induced gastropathy through its effect on leukotrienes and by inhibiting extravasation of neutrophils.     

Collection devices for cervicovaginal cytology: a comparison

OBJECTIVE: To compare cervicovaginal smears obtained by a cotton-tipped swab with those obtained by cervix brush and modified Ayre spatula. STUDY DESIGN: A combined cervicovaginal smear was collected from 100 women using 3 different collection devices: cotton-tipped swab, cervix brush, and modified Ayre spatula. In each patient a set of 3 smears was collected by the same cytotechnologist using all 3 devices in random order. Smears were evaluated using parameters mandatory for an optimal smear: evenly dispersed, well-preserved, adequate cells from the transformation zone. The cost and availability of the collection devices were also considered. RESULTS: The swab was the most effective device in obtaining thin, evenly spread, adequate, well-preserved smears as against the cervix brush and Ayre spatula. The pickup of abnormal cells was similar with the cotton-tipped swab and cervix brush, while the Ayre spatula failed to yield high grade squamous intraepithelial lesions, atypical glandular cells of undetermined significance and adenocarcinoma cells. The cotton-tipped swab proved to be the most cost effective. CONCLUSION: A properly prepared cotton-tipped swab is an inexpensive, readily available, nontraumatic collection device that yields smear of optimal quality.