c-Fos-like immunoreactivity in the brainstem following gastric loads of various chemical solutions in rats.

The distribution of c-Fos-like immunoreactivity (c-FLI) in the lower brainstem especially in the area postrema (AP), nucleus of the tractus solitarius (NTS) and parabrachial nucleus (PBN) was examined following gastric loads of various chemical solutions in rats. An aliquot of 7.5 ml of each stimulus was intragastrically infused, and c-FLI was detected. The most remarkable c-FLI was induced by LiCl, lactose and ethanol which are known to be effective unconditioned stimuli in conditioned taste aversions. Polycose and disaccharides such as sucrose and maltose induced more c-FLI than monosaccharides such as glucose, fructose and galactose. Relatively low levels of c-FLI were observed for other sweeteners such as saccharin, glycine and alanine, and other basic taste stimuli such as NaCl, HCl, quinine and umami substances. Each stimulus induced a similar proportion of c-FLI among the subnuclei of the NTS, but not in the PBN, where chemicals effective in inducing conditioned taste aversions elicited stronger c-FLI in the external lateral subnucleus, and those in inducing conditioned taste preferences such as Polycose and glucose elicited stronger c-FLI in the dorsal lateral subnucleus. Vagotomy reduced c-FLI to about 50% for LiCl stimulation and to about 30% for sucrose stimulation, suggesting that LiCl has a larger proportion of extravagal inputs than sucrose.

Comparison of c-fos-like immunoreactivity in the brainstem following intraoral and intragastric infusions of chemical solutions in rats.

To examine whether the activation of brainstem neurons during ingestion is due to orosensory afferents or post-ingestive factors, neuronal activation in response to intraoral and intragastric infusions of taste stimuli was compared in the area postrema (AP), nucleus tractus solitarius (NTS) and parabrachial nucleus (PBN) by the c-fos immunohistochemical method. An aliquot (7.5 ml) of 0.5 M sucrose, 5 mM sodium saccharin, 1 mM quinine hydrochloride and distilled water was delivered into the oral cavity or the stomach in each rat, which had been deprived of water and food overnight. Water induced little c-Fos-like immunoreactivity (c-FLI), but both intraoral and intragastric infusions of sucrose, but not non-caloric saccharin, induced strong c-FLI in the AP, caudal NTS and the external lateral subnucleus of the rostral PBN, suggesting that these areas receive general visceral inputs. Other areas in the NTS and PBN may receive gustatory inputs since more dominant c-FLI was detected by intraoral rather than intragastric infusions of the stimuli. Functional segregation of neurons reflecting qualitative and hedonic aspects of sweeteners (sucrose and saccharin) and bitter-tasting substance (quinine) was suggested in the PBN, but less evident in the NTS. These results indicate that c-fos induction in brainstem neurons during ingestion reflects gustatory inputs and postingestional factors depending on the kind of food ingested.

Establishment of T-helper type 2 clone-induced eosinophilia in mouse conjunctiva.

Eosinophil infiltration is one of the aspects of the inflammatory response to an allergen. The aim of this study was to establish T-helper type-2 (Th2)-induced conjunctival eosinophilia in mice to evaluate the effect of anti-allergic drugs. Th2 clone, D10.G4.1. (D10) with its specific antigen, conalbumin was co-injected into conjunctival tissue in mice. At 3-6 hr after D10 injection, eosinophil and neutrophil recruitment into conjunctival tissue was observed. Cellular infiltration into conjunctival tissue reached a maximum level between 24-48 hr after D10 injection. The number of eosinophils which infiltrated after 24 hr was dose dependent on T cell injection titer from 1x10(4), 1x10(5)to 1x10(6)cells site-1. The ratio of eosinophils to neutrophils at 24 hr after D10 cell (1x10(5)cells site-1) injection was about 9 to 1. The eosinophil infiltration into conjunctival tissue was significantly reduced by the intraperitoneal administration of anti-IL-5 monoclonal antibody (100 micrograms animal-1). Anti-IL-4 monoclonal antibody (500 micrograms animal-1) partially inhibited eosinophilia. The combined inhibitory effect of anti-IL-4 and anti-IL-5 monoclonal antibodies was larger than the inhibitory effect of anti-IL-5 monoclonal antibody alone. In conclusion, a Th2-induced mouse conjunctival eosinophilia model was established in which D10 activation results in IL-4 and IL-5 release. These cytokines elicit eosiniophil infiltration. This response shows that the model will be effective for screening candidates of anti-allergic drugs.

[Rapid identification of Bilophila wadsworthia].

Biophila wadsworthia is a recently recognized nonspore-forming anaerobic gram-negative rod and is reported to be associated with various infections such as gangrenous perforated appendicitis, peritonitis, osteomyelitis and bacteremia. Although the isolation of B. wadsworthia seems to be facilitated by using Bacteroides bile esculin (BBE) agar, the reliable scheme of identification of this species has been published. This study was conducted to find simple, rapid, and reliable measures for identification of B. wadsworthia. A total of 32 B. wadsworthia-suspected clinical isolates as well as B. wadsworthia WAL 7959 and Desulfomonas pigra DSM 749, which is the species that shows bacteriological characteristics similar to B. wadsworthia, were used. Through the study of various biochemical and enzymatic tests and culture on selective media, it is indicated that a nonspore-forming, gram-negative anaerobic rod that is nonmotile, forms "black-eyed" colonies on BBE agar, and demonstrates strongly positive catalase test, positive acid phosphatase test, and no-growth on Bacterides medium or to be susceptible to colistin (10 micrograms disk) can be identified as B. wadsworthia.

16 beta-Methyl-17 alpha,21-diesterified glucocorticoids as partial agonists of glucocorticoid in rat endotoxin-induced inflammation.

OBJECTIVE AND DESIGN: We investigated the anti-inflammatory effects of 16 beta-methyl-17 alpha,21-diesterified glucocorticoids which are well known as potent topical glucocorticoids in man on endotoxin-induced uveitis (EIU) in rats. MATERIAL: Female Lewis rats were used. TREATMENT: Glucocorticoids were instilled (0.01%-1.0%) or subcutaneously injected (0.1-10 mg/kg) to rats. METHODS: To elicit EIU, LPS (500 micrograms/kg) was injected into the footpad of rats. Twelve hours after LPS injection, cell number in aqueous humor was counted by flow cytometry. Endotoxin-induced in vivo tumor necrosis factor-alpha (TNF-alpha) production was also examined. RESULTS: 16 beta-methyl-17 alpha,21-diesterified glucocorticoids showed no effects or some enhancement of cell infiltration into the aqueous humor in EIU by topical instillation. Systemic injection of these glucocorticoids showed only weak inhibition of cell infiltration and TNF-alpha production. On the other hand, betamethasone phosphate strongly inhibited the cell infiltration and TNF-alpha production. Combined systemic injection of 16 beta-methyl-17 alpha,21-diesterified glucocorticoids and betamethasone phosphate reduced the inhibitory effects of the latter. CONCLUSIONS: These results suggest that 16 beta-methyl-17 alpha,21-diesterified glucocorticoids might act as partial agonists of glucocorticoid in rats.

The effects of betamethasone derivatives on endotoxin-induced uveitis in rats.

The anti-inflammatory effects of betamethasone and its derivatives (betamethasone 21-phosphate, betamethasone 21-acetate, betamethasone 17-valerate, clobetasol 17-propionate and betamethasone dipropionate) on endotoxin-induced uveitis (EIU) in rats was investigated. Among the compounds examined, betamethasone, betamethasone 21-phosphate, betamethasone 21-acetate and clobetasol 17-propionate strongly inhibited cell infiltration into the aqueous humor in the EIU by instillation into the eye in a dose-dependent manner (0.01-1.0%) and by systemic administration (1 mg kg-1). On the other hand, betamethasone 17-valerate and betamethasone dipropionate showed only weak inhibitory effects or enhancement of cell infiltration by instillation into the eye (0.01-1.0%) and by systemic administration (1 mg kg-1). Combined systemic administration of betamethasone dipropionate and betamethasone reduced the inhibitory effect of betamethasone on cell infiltration and gene expression of IL-1 beta. In an in vitro interleukin-8 (IL-8) release assay, betamethasone showed stronger inhibition of the IL-8 release from rat peritoneal exudate cells (PEC) than betamethasone dipropionate, and simultaneous addition of betamethasone dipropionate with betamethasone reduced the inhibitory effect of the latter. These results suggest that the betamethasone derivative betamethasone dipropionate might behave as an anti-glucocorticoid in rats.

The effects of betamethasone derivatives on endotoxin-induced uveitis in guinea pigs.

OBJECTIVE AND DESIGN: We reported previously that the betamethasone derivative betamethasone dipropionate behaves as an anti-glucocorticoid in rat endotoxin-induced uveitis (EIU). In the present study, we produced EIU in guinea pigs and investigated the effects of betamethasone dipropionate on the EIU. MATERIAL: Male Hartley guinea pigs were used. TREATMENT: Glucocorticoids were instilled into the eye. METHOD: To elicit EIU, lipopolysaccharide (LPS) was injected into the anterior chamber of the eye. Cell numbers in the aqueous humor after LPS injection were determined by flow cytometry. Prostaglandin E2 (PGE2) production after LPS injection into the anterior chamber was also examined. RESULTS: Intracameral injection of LPS (1 microgram/eye) induced cell infiltration into the anterior chamber and PGE2 production. Betamethasone dipropionate inhibited cell infiltration and PGE2 production more strongly than betamethasone. These results suggest that betamethasone dipropionate is a potent glucocorticoid in guinea pigs. CONCLUSIONS: Structure-activity relationships of glucocorticoids in the guinea pig EIL model may differ from those in the rat EIU model.

Effects of bucillamine and antigen-presenting cells in experimental autoimmune uveitis in rats.

Bucillamine, an anti-rheumatic drug, was compared with cyclosporine (CYA) in its effects on the antigen-presentation activity of antigen-presenting cells (APCs) and the antigen-specific proliferation of T cells from S-antigen-immunized rats. In vitro assay showed that bucillamine did not affect the proliferative response of T cells, but suppressed the antigen-presenting activity of APCs, such as macrophages and retinal pigment epithelial cells. On the other hand, CYA suppressed both T cell proliferation and the antigen-presenting activity of macrophages. However, the doses of CYA required to produce significant suppression of antigen presentation were higher than those needed to inhibit T cell proliferation. Daily systemic administration of bucillamine for 14 days after immunizing rats with S-antigen suppressed the intensity of experimental autoimmune uveitis (EAU) and the antigen-presenting activity of macrophages in treated rats, but not the antigen-specific proliferation of the T cells. EAU intensity was completely suppressed by CYA for 14 days post-immunization, and antigen-specific proliferation of T cells was suppressed, but the antigen-presenting activity of macrophages was not affected. These results suggested that the suppressive effects of bucillamine on the antigen-presenting activity of APCs contributed to its suppressive effects on EAU; whereas, the suppressive effects of CYA on EAU resulted principally from its suppression of the T-cell function.

Cloning and sequencing of human complementary DNA for the phosphoribosylpyrophosphate synthetase-associated protein 39.

A human cDNA encoding a human homologue of the rat phosphoribosylpyrophosphate synthetase-associated protein of 39 kDa was isolated. The deduced protein contains 356 amino acids and has calculated molecular mass of 38561. The amino acid sequence is 98% identical to that of the rat. The corresponding mRNA is present in all human tissues examined.

Promoter region of the rat phosphoribosylpyrophosphate synthetase-associated protein 39.

The 5' region of the 39-kDa rat phosphoribosylpyrophosphate synthetase-associated protein (PAP39) gene was isolated and sequenced. The promoter region of the rat PAP39 is GC-rich and contains potential binding sites for regulatory factors. Its promoter activity was demonstrated by transfection of the promoter region in fusion with the chloramphenicol acetyltransferase gene into rat pheochromocytoma PC12 cell.

A canine case of profound granulomatosis due to Paecillomyces fungus.

A 5-year-old dog showed remarkable edematous swelling of the left hock with lameness and local cellulitis, and paecillomyces fungus was isolated from ulcerative lesion of the hock joint and mediastinum. At autopsy severe effusive pleuritis was shown and numerous necrotizing and granulomatous lesions with fungal elements were seen in the liver, pancreas, kidney and mediastinal lymph nodes.

Promoter region of the mouse cyclin-dependent kinase 5-encoding gene.

While cyclin-dependent kinases, such as CDC2 and CDK2, are key regulators of cell-cycle progression, cyclin-dependent kinase 5 (CDK5) is highly expressed in mature neurons with no evident cell-cycle regulation. The 5'-region of the mouse CDK5 gene was isolated and sequenced. The isolated clone included exons 1 through 7. The 5'-flanking region has a high G+C content. There is no TATA box around the transcriptional start points (tsp), as determined by primer extension analysis. One CCAAT box, one AP-1-binding site, two AP-2-binding sites, and one cAMP-responsive element are located upstream from the tsp. Promoter/cat fusion assays showed that the 5.8-kb fragment of this 5'-flanking sequence possessed the promoter activities expressing cat in rat PC12 pheochromocytoma cells. The effect of deletions of the promoter suggested the presence of two negative control elements located from -2.9 kb to -546 bp, and from -212 to -155 upstream from the 5' end of the tsp. Two positive elements from bp -300 to -212 and from -155 to -41 were also detected. In the element from bp -300 to -212, there was a putative NF-IL6-binding sequence. Thus, the CDK5 promoter region contains multiple positive and negative cis-acting regulatory elements, an arrangement which suggests that the regulation of transcription of CDK5 is under complex control.

Effects of bucillamine, a new anti-rheumatic drug, on the experimental autoimmune uveitis in rats.

The in vitro and in vivo effects of a new immunomodulating agent, bucillamine, on experimental autoimmune uveitis (EAU) was studied in the rat. The capacity of S-antigen-sensitized lymphocytes to proliferate in response to the antigen or to produce antigen-specific antibodies was significantly suppressed by bucillamine in culture in a dose-dependent manner. The inhibitory effect of bucillamine was significantly enhanced by adding cyclosporin A (CYA) in the culture. The in vivo effects of bucillamine alone or in combination with CYA were further examined in Lewis rats immunized with S-antigen. All untreated rats developed severe EAU 17 days after S-antigen immunization, while rats treated with either bucillamine (200 mg kg-1 day-1) or CYA (2 mg kg-1 day-1) demonstrated milder symptoms of EAU. A combination therapy with bucillamine (20 or 200 mg kg-1 day-1) and CYA (2 mg kg-1 day-1) exhibited much more significant suppression of EAU induction. Although the in vivo treatment with bucillamine or CYA had no effects on the T-cell populations of spleen cells, the combination therapy significantly decreased the CD4+ T-cell population. As for the immune responses to S-antigen in drug-treated rats, bucillamine suppressed the lymphocyte proliferation to S-antigen, which was further suppressed by combination therapy with CYA. The serum antibody levels specific to S-antigen were not affected by tested dose of bucillamine.

Bacterial flora detected in the uterine endometrial cavity of normal puerperae on the puerperal first day and on the puerperal fifth day after incidental use of cefpodoxime proxetil.

In Japan, oral antimicrobial agents are prophylactically used with oxytocics after normal delivery to prevent puerperal infections. The present study was designed to investigate bacterial floras in the endometrial cavity immediately after normal delivery and the effect of prophylactic use of anti-microbial agents on those floras. Sixty-six puerperae who underwent uneventful courses of pregnancy and delivery were subjected for this study. Intrauterine contents were collected on the first day and the fifth day of the puerperium and submitted to microbiological examinations. Cefpodoxime proxetil (CPDX-PR) was orally given to the puerperae for prophylaxis for 5 days after the initial sampling. On the puerperal first day, a total of 98 strains (71 strains of aerobic bacteria, 27 strains of anaerobic bacteria) was detected in the uteri of the 66 subjects. The incidences of aerobic Gram-positive cocci, aerobic Gram-negative bacilli and anaerobic bacteria were 59.2%, 12.2%, 27.6% of the 98 strains, respectively. On the puerperal fifth day, a total of 82 strains (51 strains of aerobic bacteria and 31 strains of anaerobic bacteria) were detected in the uteri of the 66 subjects. The incidences of aerobic Gram-positive cocci, aerobic Gram-negative bacilli and anaerobic bacteria were 52.5%, 8.6% and 37.7% of 82 strains, respectively.

Effects of topical FK506 on experimental allergic conjunctivitis.

In vitro studies of FK506 demonstrated that the agent inhibited the T-cell receptormediated signal transduction that results in the transcription of interleukin 2. Recent in vitro studies have demonstrated an identical spectrum of activities of FK506 on IgE receptor-mediated signal transduction that results in exocytosis of secretory granules from basophils and mast cells. The effects of topical FK506 on allergic conjunctivitis were therefore studied in the rat. Passive anaphylaxis was induced in Wistar rats by injecting anti-ovalbumin IgE at the conjunctiva followed by intravenous injection of antigen (ovalbumin) and Evans blue. Topical FK506 (0.03-0.1%) significantly suppressed the passive anaphylaxis in the rat conjunctiva when given at 15 and five minutes, or six, four and two hours before the antigen challenge. The efficacy was more intense than that of 0.1% betamethasone or 2% disodium cromoglycate. These data thus suggest that topical FK506 might be beneficial for the therapy of severe allergic diseases in the eye.

[Incidence of Mobiluncus spp. from the patients with clinical bacterial vaginosis].

Aerobic and anaerobic cultures as well as a Gram stain and wet mount preparations were made of vaginal swabs in twenty patients with clinical bacterial vaginosis. Mobiluncus spp. were detected in 7 cases (35%). Cultures appeared to indicate that mixed abnormal flora between aerobic and anaerobic bacteria are found in bacterial vaginosis, and that Mobiluncus spp. may play a role in bacterial vaginosis.

A comparison of the opacifying effects of carteolol.HCl and 8-hydroxycarteolol.HCl in the isolated porcine cornea.

Using an in vitro method the authors have investigated whether 8-hydroxycarteolol.HCl(8-OH carteolol) and 8-OH carteolol with benzalkonium chloride affected intact isolated porcine corneas to the same or to a different degree as carteolol.HCl (carteolol) or carteolol with benzalkonium chloride. These ophthalmically used drugs were applied as solutions of varying concentrations to the epithelial surface only, to the endothelial surface only, or to both surfaces of porcine corneas. The resultant opacities were determined using an opacitometer. In general, 8-OH carteolol and 8-OH carteolol with benzalkonium chloride caused less opacity to develop than carteolol and carteolol with benzalkonium chloride. This suggests that 8-OH carteolol may be a safer drug than carteolol for ophthalmic use. It is very interesting to note that compounds with two nitrogens, e.g., 8-OH carteolol, caused greater opacity in the intact cornea when applied to the endothelial surface than when applied to both the epithelial and endothelial surfaces; however, compounds with none or one nitrogen caused greater opacity in the intact cornea when applied to both surfaces than when applied to the endothelial surface.