Storage stability of serum formulations containing ofloxacin for autologous serum eardrop therapy.

OBJECTIVE: The storage stability of serum formulations containing ofloxacin for autologous serum eardrop therapy was evaluated for microbiological quality and component stability. METHODS: Sterile serum formulations were prepared by mixing human serum and ofloxacin otic solution (1:1, v/v). To simulate eardrop contamination with external ear surface substances, prepared serum formulations were contaminated with a cotton swab that was rubbed sufficiently on the human external ear. Formulations were stored at 4 °C or room temperature in the dark. Colony forming units (CFUs), ofloxacin, and basic fibroblast growth factor (bFGF) concentrations in the stored serum formulations were determined. RESULTS: The growth of microorganisms derived from the external ear was not detected in serum formulations after storage for 14 days, regardless of temperature. However, microbial growth was detected in serum formulations stored without ofloxacin, indicating that this is necessary for storage. In addition, concentrations of ofloxacin and bFGF did not decrease over 14 days, indicating that ofloxacin and bFGF in serum formulations are stable for this time period. CONCLUSION: The present study indicates that the efficacy and safety of serum formulations used as a therapy for perforated eardrums are stable and safe for at least 14 days.

Expression and role of SNAT3 in the placenta.

Glutamine is the most versatile amino acid and its plasma concentration is the highest of all amino acid. Many transporters are therefore involved in glutamine uptake or efflux. Glutamine is actively released from the placenta into fetal circulation. In this study, we examined the alteration of transporters that transport glutamine into fetal circulation as gestation progresses. High expression levels of system A and y(+)L were found in the rat placenta in the late period of pregnancy and the expression levels of these transporters increased as gestation progressed (p<0.05). On the other hand, the expression of SNAT3, the system N transporter, was detected in the early period of pregnancy and its expression level decreased as gestation progressed (p<0.05). SNAT3 was also found to be expressed in isolated human primary cytotrophoblast cells and its expression level was decreased by their differentiation into syncytiotrophoblast cells (p<0.05). Since this regulation is closely related to glutamine synthetase expression, SNAT3 may play a key role in providing glutamine corresponding to glutamine synthetase function in the early period of gestation. This is the first report on the expression of SNAT3 in the placenta in the early stage of pregnancy.

Functional and molecular analysis of D-serine transport in retinal Müller cells.

D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.

New anti-malarial flavonol glycoside from Hydrangeae Dulcis Folium.

Bioassay-guided fractionation of the MeOH extract of Hydrangeae Dulcis Folium resulted in isolation of a new flavonol glycoside and two known congeners as anti-malarial principles. These flavonol glycosides showed characteristic proliferation inhibition of Plasmodium falciparum at significantly low concentration without showing any cytotoxicity. In addition, several naturally occurring flavonol glycosides were also shown to exert similar anti-malarial behavior.

Localization and age-related changes in cytochrome P450 expression in APA hamster livers.

To establish the baseline data, age-related changes and the regional expression of the hepatic P450 isozymes in Syrian hamsters of the APA strain at 3, 6, 12, 18 months old were examined by immunological techniques. Immunohistochemical analysis of liver serial sections revealed that the midzonal and perivenous regions (zones 2 and 3, respectively) were stained with the anti-rat CYP1A1/2, 2B1/2 and 2E1 antibodies. These three antibodies most intensely stained the hepatocytes around the central vein. An anti-rat CYP3A2 staining section had a staining pattern with equally intense reactions in zones 2 and 3. On the other hand, CYP2C6, 2C11 and 4A1 were distributed diffusely throughout the hepatic acinus. There was no age-related difference in the expression pattern of any of the P450 isozymes examined. Total P450 content had a peak at 6 months of age and decreased to 60% of that level thereafter. Western-blot analysis revealed that the peak expressions of the isozymes detected with anti-rat CYP1A1/2, 2C6, 2E1 and 3A2 antibodies were observed in 6-month-old hamsters and declined in older ones. The CYP2B and 2C11 content reached the maximum at the age of 6 months and maintained almost the same level thereafter. The CYP4A level did not change from 3 to 6 months, and then declined to about 40% of the younger level at 12 and 18 months of age. These results suggest that the hepatic P450 isozymes of APA hamsters have region-specific expressions and most isozymes have their peaks of expression at 6 months of age, which differs from the patterns for rat P450.

Aortic atheromatous lesions developed in APA hamsters with streptozotocin induced diabetes: a new animal model for diabetic atherosclerosis. 1. Histopathological studies.

To develop an adequate animal model for atherosclerosis in large vessels of patients with diabetes, i.e. diabetic macroangiopathy, we induced diabetes in APA hamsters with a single injection of streptozotocin (SZ) and examined the aorta histopathologically and immunohistochemically. As a result, hyperglycemia and hyperlipidemia were continuously observed for 26 weeks after the SZ injection (WAI) in APA hamsters. Fatty streaks characterized by a subendothelial accumulation of many foam cells were observed, limited to the aortic arches as early as 6 WAI. In addition to larger fatty streaks developing with the duration of diabetes, fibrous plaques and plaques containing calcium deposits or cholesterol clefts developed at 26 WAI. These lesions are generally similar to the atheromatous lesions developed in humans. Moreover, depositions of apolipoprotein E and advanced glycation end-products immunohistochemically detected in the lesions were very similar to those found in humans. The diabetic APA hamster is therefore considered to be a useful model for studying the formation of atheromatous lesions in diabetic patients.

APA hamster model for diabetic atherosclerosis. 2. Analysis of lipids and lipoproteins.

Syrian hamsters of the APA strain (APA hamsters) have recently been shown to have atheromatous lesions in the aortic arches under diabetic condition induced by a single injection of streptozotocin (SZ). In that model, fatty streaks, which are the initial lesions of atherogenesis, develop by 6 weeks after the injection (WAI). In this study, we evaluated plasma lipid concentrations and lipoprotein profiles in diabetic APA hamsters at 6 WAI to reveal the early stage of atherogenesis clinicopathologically. As a result, by biochemical analysis, hyperglycemic APA hamsters showed signs of hypercholesterolemia and hypertriglyceridemia. Low-density lipoprotein (LDL) cholesterol significantly increased, but high-density lipoprotein (HDL) cholesterol significantly decreased. Agarose gel electrophoresis showed an obvious increase in the fractions of chylomicron, LDL and abnormal lipoprotein. Plasma LDL in diabetic animals was in a state more susceptible to oxidization. In addition, a significant increase in glycated LDL was also found in the diabetic animals by enzyme linked immunosorbent assay (ELISA). Moreover, lipid peroxidation product (4-hydroxynonenal (4 HNE))-adducted proteins and advanced glycation end-products (AGE) were immunohistochemically detected in the foam cells of the fatty streaks. These results revealed that diabetic APA hamsters had hyperlipidemia characterized by increases in chylomicron, LDL and abnormal lipoprotein, and suggested that oxidized LDL and/or glycated LDL might be actively uptaken by macrophages and play an important role in the initial stage of atherogenesis.

Jejunal pouch interposition with an antiperistaltic conduit as a pyloric ring substitute after standard distal gastrectomy: a comparison with the use of an isoperistaltic conduit.

BACKGROUND/AIMS: We performed jejunal pouch interposition with a short antiperistaltic conduit as a pylorus substitute after gastrectomy for gastric cancer and compared the outcome with an isoperistaltic conduit. METHODOLOGY: After a standard distal gastrectomy and lymph node dissection, a 15-cm-long pouch was formed using 3 linear staples (Endo-GIA) and interposed between the residual stomach and duodenum. The distal jejunal limb was made into a 3-cm-long isoperistaltic conduit in the isoperistaltic group (n = 17), and the proximal jejunal limb was made into a 3-cm-long antiperistaltic conduit in the antiperistaltic group (n = 8). Postoperatively, the patients were interviewed periodically to document any complaints. A dual-phase, dual-isotope radionuclide gastro-pouch-emptying study was performed 1 and 6 months after surgery. RESULTS: None of the patients developed postoperative complications and showed discomforts of dumping, stasis or reflux esophagitis. The dietary volume and body weight of patients gradually increased in both groups after 6 months. The combined radioisotope retention rate for the pouch and residual stomach was 31% for liquid food and 35% for solid food in the isoperistaltic group after 120 min, and 41% and 57%, respectively, in the antiperistaltic group. The pattern and emptying rate for solid food in the antiperistaltic group were more similar to those in healthy individuals than in the isoperistaltic group. CONCLUSIONS: The gastro-pouch-emptying test in the antiperistaltic group demonstrated acceptable emptying for a pyloric ring substitute. A reasonably good quality of life has been obtained for patients having an antiperistaltic jejunal conduit.

Pulmonary lesions in guinea pigs experimentally infected with Actinobacillus pleuropneumoniae (A.p.) serovar 1.

Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 x 10(6-8) colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.

Inhibition of polyposis in the small intestine of BALB/c mice by intestinal bacteria.

The incidence of polyposis and the number of polyps per mouse were significantly lower in conventionalized (CVz) mice than in germ-free (GF) mice. There was no significant difference in the average number of polyps between GF and gnotobiotic (GB) mice monoassociated with the various strains of intestinal bacteria. However, the incidence of polyposis and the number of polyps per mouse were significantly lower for mice associated with either chloroform-resistant bacteria (CRB) or fusiform bacteria (FB) than for GF mice. This study demonstrated that polyposis was suppressed by FB and CRB in the small intestine of BALB/c mice.

Age-related changes in major lymphocyte subsets in cynomolgus monkeys.

Age-related changes in major lymphocyte subsets were analyzed in 195 cynomolgus monkeys (Macaca fascicularis) aged from one month to 31 years. The percentages of CD20+ B cells in peripheral blood lymphocytes (PBL) decreased with age to five years of age, but after that, no significant change was observed. The percentages of CD16+ NK cells gradually increased during the first five years and reached the peak at from four to ten years of age, whereas the percentages of CD3+ T cells in PBL were relatively constant throughout the life. Among the T cells, the CD4+ CD8- T cells decreased, but CD4- CD8+ T cells increased within the first decade of life. We further analyzed the expressions of CD28 and CD29 molecules on T cells to determine the relation between age-related activation and phenotypic changes. Almost all CD4+ CD8- T cells (> 90%) were CD28+ at all ages analyzed, but a clear age-related decrease in CD28 expression was demonstrated in CD4- CD8+ T cells during the first ten years. In the case of CD29 expression, age-related increases in CD29hi cells were apparent in both CD4+ CD8- and CD4- CD8+ T cells during the first ten years. The percentages of CD29hi cells, however, were higher in CD4- CD8+ T cells than in CD4+ CD8- T cells in all ages analyzed. These results indicated that the age-related changes in percentages of major lymphocyte subsets as well as in phenotypes of T cells might be related to the maturation of the immune system including an increase in memory cells in cynomolgus monkeys.

Histochemical and lectinhistochemical studies on nasal mucosa of pigs with or without respiratory diseases.

Histochemical and lectinhistochemical examinations were carried out on nasal mucosa of pigs with or without respiratory diseases. As the results, both acid and neutral mucins coexisted in nasal mucosa of normal pigs while acid sialomucins were mainly observed in nasal mucosa of pigs infected with Bordetella bronchiseptica and/or Pasteurella multocida. Lectinhistochemistry revealed that the nasal epithelial cells of normal pigs were rich in N-acetylgalactosamine, fucose and N-acetyl-glucosamine residues which showed a tendency to disappear in porcine cytomegalovirus infection and to increase in atrophic rhinitis, respectively.

Age-dependent remodeling of peripheral blood CD4+ CD8+ T lymphocytes in cynomolgus monkeys.

Recently, we have found in adult cynomolgus monkeys that substantial peripheral blood CD4+ CD8+ double-positive (DP) T lymphocytes exhibit a resting memory phenotype and increase in proportion with age. In this study, we investigated whether phenotypic changes occur in the course of the increase in proportion of the DP T cells. The results obtained from 195 clinically healthy monkeys aged from 1 month to 31 years showed that the CD29hi and CD28 subpopulation in the DP T subset increased in proportion with age and that the increase reached a plateau at six years old for the CD29hi subpopulation and at eleven years old for the CD28 one, respectively. The phenotypic alteration preceded the abrupt increase in proportion of the DP T cells and was able to be classified into four phases on the basis of the qualitative and quantitative alteration.

Characterization of an integrase mutant of feline immunodeficiency virus.

The role of the integrase region of feline immunodeficiency virus (FIV) in viral replication was examined using an integrase mutant clone of FIV which carries a frameshift mutation in the region. Upon transfection, although the integrase mutant was able to release virus-like particles into the supernatant from the transfected cells, the virions produced by the mutant contained unprocessed gag precursor protein and undetectable levels of reverse transcriptase activity. Furthermore, the mutant virions were unable to direct the synthesis of viral DNA after infection in target cells. To understand this phenotype of the integrase mutant in more detail, we constructed a gag-pol expression plasmid from an FIV molecular clone and assayed roles of the integrase region on virus particle formation following transfection. When an inframe deletion was introduced into the protease region of the expression plasmid, the mutant was able to efficiently release gag- and gag-pol precursor proteins into the supernatant from the transfected cells. An expression plasmid with mutations in both the protease and integrase regions, however, failed to release the gag-pol precursor protein from the cells. These results suggested an essential role for the integrase region for efficient incorporation of the gag-pol precursor into the virions.

Response of respiratory epithelium of BN and F344 rats to formaldehyde inhalation.

BN rats are well-known for their high capacity for IgE production and hyperresponsiveness to exposure to allergens or other chemicals. We examined the histological changes in the nasal cavity, trachea and lungs of BN and F344 rats after the inhalation of aerosol formaldehyde (HCHO), which exerts direct toxic effects on the respiratory system. The incidence of clinical signs such as sneezing and abnormal respiration in HCHO-treated F344 rats was higher than that in HCHO-treated BN rats. The mean body weight of HCHO-treated F344 rats apparently decreased in comparison with control F344 rats, but that of HCHO-treated BN rats was not significantly different from that of control BN rats. Changes such as squamous metaplasia, stratification, degeneration and desquamation were observed by light microscopy in nasal, tracheal and bronchial mucosa in the lungs of the HCHO-treated F344 rats. In the HCHO-treated BN rats, similar but milder lesions were restricted to the nasal mucosa. Scanning electron microscopic observation supported these light microscopic observations. These results suggest that BN rats have lower sensitivity to HCHO inhalation than F344 rats.

Apoptosis of murine hepatocytes induced by high doses of galactosamine.

Apoptosis induced by high doses of Galactosamine (GalN) was investigated in mice hepatocytes in vivo. In mice intraperitoneally (i.p.) treated with GalN 3 g/kg, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were first observed at 6 hr postadministration (PA). Both acidophilic bodies in hematoxylin and eosin (HE)-stained sections and TUNEL-positive cells were markedly found at 24 hr PA. At 48 hr PA, cellular degeneration and necrosis of hepatocytes were prominently observed, and TUNEL-positive cells were scarcely found. In the mice ip treated with GalN 1.5 g/kg, the lesion was milder than that in those treated with GalN 3 g/kg. Acidophilic bodies and TUNEL-positive cells were scarcely found at 24 hr PA, whereas they were markedly seen at 48 hr PA. In addition, a ladder-like DNA fragmentation pattern by agarose gel electrophoresis was observed most remarkably at 24 hr PA with GalN 3 g/kg and at 48 hr PA with GalN 1.5 g/kg, and less distinctly at 48 hr PA with GalN 3 g/kg. On the other hand, sGOT and sGPT activities increased prominently at 48 hr PA with GalN 3 g/kg. These results suggest that the cell death induced by high dose of GalN may be caused by apoptosis, and subsequently by necrosis in vivo.

Galactosamine-induced apoptosis in the primary mouse hepatocyte cultures.

Galactosamine (GalN)-induced apoptosis was investigated in cultured hepatocytes from mice. The percentage of fragmented DNA measured by the ELISA method increased in a concentration-dependent manner from the very early stage, i.e. 0.5 hrs, after GalN-exposure. In addition, a ladder-like fragmentation pattern by agarose gel electrophoresis appeared first at 3 hr-exposure to 20 mM GalN, at 6 hr-exposure to 10 mM GalN and at 12 hr-exposure to 5 mM GalN, respectively. On the other hand, cytotoxicity indicated by leakage of lactate dehydrogenase from cultured hepatocytes to culture medium was first detected at 24 hrs after GalN-treatment. Morphologically, formation of blebs and apoptotic bodies was observed from 12 hr-exposure to 20 mM GalN and from 24 hr-exposure to 10 mM GalN, respectively. Thus GalN could induce apoptosis in primary hepatocyte cultures from mice.

[The EMG findings of spasmodic torticollis--the character of the EMG findings of neurogenic torticollis].

Spasmodic torticollis is a clinical entity that is hard to treat though various symptomatic therapy have been tried. On the other hand, microvascular decompression operation have been established for cranial nerve vascular compression syndrome such as hemifacial spasm. Case reports on the decompression of the spinal accessory nerve for the treatment of spasmodic torticollis have been published on the basis of the concept of cranial nerve vascular compression syndrome. Thus, spasmodic torticollis related to unilateral accessory nerve has attracted much attention for selecting an optimal treatment, although there have not been any diagnostic criteria with electromyographic study. From the viewpoint of the clinical electrophysiological findings on hemifacial spasm those we have acquired by EMG study, we have examined the EMG findings of various types of spasmodic torticollis and here report the classification of spasmodic torticollis based on the EMG study. Thirty-five patients with spasmodic torticollis were analyzed. The symptoms were classified to the horizontal rotation type, the lateral bending type and the mixed type with the number of each group of 23, 2 and 10, respectively. As we have shown the criteria of the EMG findings on hemifacial spasm, the EMG of the patients with spasmodic torticollis were analyzed on four conditions as follows; (1) distribution of the involved muscles, (2) maximum firing rate of the abnormal spontaneous activity of the sternocleidomastoid muscle, (3) synkinetic discharge between the muscles innervated by unilateral accessory nerve and (4) alteration of the spontaneous muscle discharge by posture change. Abnormal spontaneous muscle discharges were recorded only from the sternocleidomastoid muscle and the trapezius muscle on the same side in twelve patients. Maximum firing rate of spontaneous muscle discharge was higher than that of maximum voluntary contraction in twenty-two patients. Abnormal synkinetic discharge was recorded between the sternocleidomastoid muscle and the trapezius muscle on the same side in twenty-one patients. Spontaneous EMG activities of the muscles innervated by the accessory nerve increased when the patients stood up from the resting supine position in thirty-one patients. Thus, ten patients out of thirty-five subjects had all four conditions mentioned above as typical patients with hemifacial spasm usually had. These ten patients with spasmodic torticollis were thought to have strong similarity to the EMG characteristics of hemifacial spasm that suggested hyperexcitability of unilateral accessory nervous system. This classification with EMG is considered to be useful in diagnosing the spasmodic torticollis related to unilateral accessory nerve and can be applied for selecting an optimal treatment.

Hair follicles of young Wistar strain hairless rats: a histological study.

The histological changes in hair follicles in hairless rats derived from the Wistar strain (hW, hairless Wistar) were examined from birth to maturity and compared with those of age-matched normal Wistar rats. In the 1st hair cycle, the hair follicles of hW rats were shorter and less well developed than those of Wistar rats. In early anagen, eosinophilic bodies were observed in some hair follicles which showed immature histological features. By using Tdt-mediated dUTP nick end labelling (TUNEL) method and electron microscopic examination, these bodies were confirmed to be apoptotic bodies. These follicles seemed to disappear by abnormal regression. In late anagen phase, the follicles in which the apoptosis did not occur showed enlarged hair roots with hypertrophy of the inner root sheath. Subsequently, when the follicles in normal Wistar rats synchronously regressed in the catagen phase, most of the follicles in hW rats similarly entered the catagen phase, but a few follicles did not regress completely and showed aberrant hair root enlargement. Finally, both types of follicle in hW rats formed follicular cysts. These abnormalities in follicle development (abnormal follicular regression and follicular cyst formation) appear to be associated with the hairlessness in this rat strain.