Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

Histopathology of a wavy medaka.

Wavy medakas are medakas that exhibit spinal curvature characterized by dorsoventrally curved vertebrae. We found a spontaneous wavy medaka in our experimental stock and subjected it to a histopathological examination. Macroscopically, the wavy medaka's spine formed an M shape, and its vertebrae displayed a dorsoventral curvature that started at the third vertebral bone. Microscopically, the vertebral cavities were filled with fibrous tissue, which was similar to that seen in the central parts of the intervertebral discs of a normal medaka. The vertebral joints were composed of vacuolated notochord cells without intervertebral disc formation. These changes were also observed in the caudal region, which exhibited less curvature. In the normal medaka, the intervertebral discs form via the regression of the notochord that plays a key role in the development of vertebrae and disc formation. We concluded that notochordal subinvolution had induced intervertebral disc dysplasia, leading to lordokyphosis, in the wavy medaka.

Histomorphological comparison of rat placentas by different timing of chlorpromazine-administration.

The effects of chlorpromazine-treatment timing on the development of the placenta in pregnant rats were examined. Chlorpromazine was administered intraperitoneally at 100mg/kg on gestation day (GD) 11 (GD11-treated group), GD 13 (GD13-treated group) or GD 15 (GD15-treated group) into pregnant rats. All treated dams exhibited decreased body weight, prone position, hypothermia, loss or decrease of locomotor activity, etc. The fetal mortality rates were increased up to 42.9% in the GD11- and GD13-treated groups and up to 16.7% in the GD15-treated group. The embryo/fetal weight was on a declining trend from GD 16 onward, and the intrauterine growth retardation (IUGR) rates on GD 21 were increased in all treated groups. The placental weight showed a declining trend from GD 15 onward in all treated groups. Histopathologically, apoptosis was detected 1 or 2 days after treatment, and led to hypoplasia in the labyrinth zone and metrial gland, and cystic degeneration in the basal zone on GD 21 in all treated groups. There was no difference in the histopathological lesions on GD 21 among the treated groups. Thus, it is considered that chlorpromazine-induced placental toxicity is characterized in that there is no obvious specific sensitive period from GD 11 to GD 15. Chlorpromazine induced a non-specific transient development retardation of the placenta by apoptosis independently of the cell proliferation period in each part/zone.

Evaluation of repeated dose micronucleus assays of the liver using N-nitrosopyrrolidine: a report of the collaborative study by CSGMT/JEMS.MMS.

The repeated dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens, and can be integrated into a general toxicological study. To assess the performance of the assay, N-nitrosopyrrolidine (NPYR), a genotoxic hepatocarcinogen, was tested in 14- or 28-day RDLMN assays. NPYR was orally administered to rats at a daily dose of 25, 50 or 100 mg/kg. One day after the last administration, a portion of the liver was removed and hepatocyte micronucleus (MN) specimens were prepared by the new method recently established by Narumi et al. In addition, a bone marrow MN assay and a histopathological examination of the liver were conducted. The detection of Phospho-Histone H3 was performed by immunohistochemistry to evaluate the proliferation rate of hepatocytes. The results showed significant increase in the number of micronucleated hepatocytes and Phospho-Histone H3-positive cells from the lowest dose in both 14- and 28-day RDLMN assays. On the other hand, the bone marrow MN assay yielded a negative result, which was in accordance with the existing report of the bone marrow MN assay using mice. Upon histopathological examination, inflammatory lesions and hypertrophy were noted, which may explain the increase in the hepatocyte proliferation and the enhancement of MN induction by NPYR. Our findings indicate that the RDLMN assay could be a useful tool for comprehensive risk assessment of carcinogenicity by providing information on both genotoxicity and histopathology when integrated into a general repeat dosing toxicity assay.

Pancreatic Ductal Adenocarcinoma in a Wistar Hannover GALAS Rat.

There are no reported spontaneous cases of pancreatic ductal adenocarcinoma (PDAC), and there are few reports about chemically-induced PDAC in rats. We encountered a PDAC in a Wistar Hannover GALAS rat that had been subjected to a medium-term multiorgan carcinogenicity bioassay. This article describes the histological and histochemical findings of the tumor. The tumor was located in the pancreatic tissue and had not invaded the liver parenchyma or the mucosal layer of the alimentary tract. The tumor cells were atypical and were mainly arranged in small tubules. In addition, abundant stroma and mucus production were observed in the tumor. In an immunohistochemical examination, the tumor cells were positive for cytokeratin, Sox9 and pancreas duodenum homeobox 1 and negative for amylase 2A and insulin. Therefore, the tumor was diagnosed as a PDAC based on its histological and histochemical findings. We considered that the tumor was caused by the carcinogens administered during the abovementioned bioassay.

Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test.

Recently, the liver micronucleus (MN) assay using young adult rats with repeated administrations has been investigated by employing a new method without partial hepatectomy or in situcollagenase perfusion as the repeated dose liver MN (RDLMN) assay by Narumi et al. (2012). In our study, in order to investigate the possibility of the RDLMN assay using young adult mice instead of rats and the feasibility of employing some genotoxicity assays along with the RDLMN assay as a combination test, two genotoxic carcinogens (N,N-diethylnitrosoamine (DEN) and cisplatin (CIS)) and a nongenotoxic carcinogen (phenobarbital sodium (PHE)) were administered to mice for 15 or 29 days. Then, the liver MN assay, peripheral blood (PB) MN assay and comet assay using the liver and kidney were concurrently performed as a combination test. DEN showed positive responses to all endpoints except MN induction in PB after 15 days of repeat administration. A cross-linking agent, CIS, showed MN induction in liver after 29 days of repeat administration, and in PB after 15 and 29 days of repeat administration, although the comet assay yielded negative responses for both organs at both sampling times. PHE yielded negative responses for all endpoints. In conclusion, it is suggested that the RDLMN assay using mice is a feasible method to be integrated into the general repeated toxicity test along with the combination assays, i.e., comet assay or PB MN assay, which would help in risk assessment for carcinogenicity by comparing the results of combination assays with each other.

Effect of chlorpromazine on rat placenta development.

We examined the sequential histopathological changes in the placentas from rats exposed to chlorpromazine. Chlorpromazine was intraperitoneally administered on GD 14 at 50 and 100 mg/kg and the placentas were sampled on GDs 14.5, 15, 17 and 21. The incidence of dams with complete fetal resorption was increased from GD 17 up to 20% at 50 mg/kg and 44.4% at 100 mg/kg. The embryo/fetal weights reduced on GDs 15 and 17 at 50 mg/kg and during GDs 15-21 at 100 mg/kg. The placental weights reduced on GD 17 at 50 mg/kg and during GDs 14.5-21 at 100 mg/kg. Histopathologically, in the labyrinth zone, apoptotic cells were scattered in the trophoblastic septa without inhibition of cell proliferation on GDs 14.5 and 15 at 50 and 100 mg/kg in a dose-dependent manner. A decrease in trophoblasts led to labyrinth zone hypoplasia. In the basal zone, apoptotic cells were scattered on GDs 14.5 and 15 at 100 mg/kg, and most of them appeared to be glycogen cells. A decrease in glycogen cells induced the delayed development of glycogen cell islands and the subsequent remaining glycogen cell islands, and led to the cystic degeneration of glycogen cells. In addition, failure of development of the glycogen cell islands led to the impaired interstitial invasion of the glycogen cells, and then metrial gland hypoplasia occurred.

Effect of cisplatin on rat placenta development.

We examined the sequential histopathological changes in the placenta from rats exposed to cisplatin. Cisplatin was intraperitoneally administered at 2 mg/kg/day during GDs 11-12 (GD11,12-treated group), or GDs 13-14 (GD13,14-treated group), and the placentas were sampled on GDs 13, 15, 17 and 21. Fetal mortality rates were increased up to approximately 65% from GD 17 onward, and fetal weights were decreased on GD 21 in the GD11,12-treated group. A reduction in placental weights was detected from GD 15 onward, and the placentas on GD 21 were macroscopically small and thin in both treated groups. Histopathologically, in the GD13,14-treated group, an increase in apoptotic cells was detected on GDs 15 and 17 in the labyrinth zone, and on GD 21 in the basal zone, resulting in labyrinth zone hypoplasia. By contrast, in the GD11,12-treated group, an increase in apoptotic cells was detected on GDs 13, 15 and 17 in the labyrinth zone, and during the experimental period in the basal zone. A decrease in Phospho-Histone H3 positive cells was detected on GD 13 in the labyrinth zone and basal zone, resulting in hypoplasia of the labyrinth zone and basal zone. In addition, a marked decrease in glycogen cell-islands in the basal zone was also detected on GDs 15 and 17. There was a reduction in interstitial invasion of glycogen cell-like trophoblasts into the metrial gland on GD 15, resulting in metrial gland hypoplasia. Therefore, we consider that cisplatin administration in pregnant rats induces growth arrest of the labyrinth zone and basal zone, leading to small placenta. It is assumed that metrial gland hypoplasia is secondarily induced by the failure of glycogen cell island development associated with basal zone hypoplasia.

Effect of estrogen on rat placental development depending on gestation stage.

We examined the sequential histopathological changes in the placenta from rats exposed to estrogen. 17 ß-estrogiol-3-benzoate was intraperitoneally administered at 100 µg/animal/day during GD 6 to GD 8 (GD6-8 treated group), GD 9 to GD 11 (GD9-11 treated group) and GD 12 to GD 14 (GD12-14 treated group), and the placentas were sampled on GDs 11, 13, 15, 17, and 21. Fetal mortality rates were increased up to approximately 50% in the GD6-8 and 9-11 treated groups, but there was no change of fetal weight on GD 21. An increase in placental weight and a reduction in fetal/placental weight ratio were detected during GD 17 to GD 21 in the GD6-8 treated group. Histopathologically, hypoplasia of metrial gland was detected with defective development of spiral arteries in the GD6-8 and GD9-11 treated groups. A decrease in the thickness of metrial gland was observed from GD 11 onwards in the GD6-8 treated group and from GD 13 onwards in the GD9-11 treated group. The endovascular trophoblasts invaded into the spiral arteries in the deep part of metrial gland in these treated groups. The number of phospho-histone H3 positive cells was decreased on GD 11 or GD 13 in these groups. In the decidua basalis, transitory necrosis was observed with hemorrhage on GD 13 in the GD6-8 and GD9-11 treated groups. In the labyrinth zone, cystic dilatation of the sinusoid was observed with congestion in the GD6-8 treated group, resulting in an increased placental weight. Therefore, we consider that estrogen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland, and leads to metrial gland hypoplasia with less development of the spiral arteries. The reduced utero-placental blood flow is supposed to be one of the important factors for poor reproductive performance.

Carcinogen-induced Thyroid Proliferative Lesions in Wistar Hannover GALAS Rats with Thyroid Dysplasia.

Incidences and morphological features of thyroid proliferative lesions induced by carcinogens in Wistar Hannover GALAS rats (GALAS rats) showing normal growth with or without thyroid dysplasia were examined. All thyroid tissue samples were obtained from our recently conducted study using male GALAS rats treated with 5 carcinogens according to the medium-term multiorgan carcinogenicity bioassay protocol (called DMBDD treatment). In the DMBDD-treated rats, thyroid dysplasia was found in 9 out of 114 rats. Follicular cell adenomas were found in 5 out of 9 rats with thyroid dysplasia and in 7 out of 105 rats without thyroid dysplasia. The incidence of adenoma was significantly increased in rats with thyroid dysplasia (55.6%) compared with that in rats without thyroid dysplasia (6.7%). Adenomas in rats with thyroid dysplasia were observed as single or multiple nodules, well demarcated and composed of variously sized vacuolated cells or unvacuolated cells. These histopathological features and staining profiles of luminal colloid for PAS and thyroglobulin, together with PCNA-positive cells, were fundamentally similar to those of rats without thyroid dysplasia. On the other hand, the luminal colloid in adenomas of rats with thyroid dysplasia had a tendency to be poorly stained for T(4) compared with that of rats without thyroid dysplasia. From these findings, it appears that dysplastic thyroids of rats showing normal growth are more sensitive to carcinogens than normal thyroids. In addition, the morphological features of carcinogen-induced thyroid proliferative lesions in GALAS rats with thyroid dysplasia were fundamentally similar to those of rats without thyroid dysplasia, except for the vacuoles and T(4) staining profile.

The relationship between fetal growth restriction and small placenta in 6-mercaptopurine exposed rat.

In order to investigate the effect of placental size on fetal intrauterine growth retardation (IURG), we examined the morphology and alterations in the expression of glucose transporter in the placentas of rats exposed to 6-mercaptopurine (6-MP). 6-MP was administered orally at 0 and 60 mg/kg/day on gestation day (GD) 9, 11, 13 or 15, and the placentas were sampled on GDs 17 and 21. The main findings in the treated groups were small placenta caused by mitotic inhibition and apoptosis, fetal resorption and IUGR with or without some malformations. The most sensitive period to 6-MP-induced fetal mortality was found to be in the GD9-treated group, and the small placenta and fetal abnormalities in the GD11-treated group, respectively. However, the litters in a quarter of the dams with the treatment on GD 11 had no fetotoxicity despite 25% decline in the placental weight. Histopathologically, the expression of glucose transporter GLUT3 was increased in the trophoblastic septa in all treated groups, particularly remarkable with proliferation of trophoblasts in the above litters, where the fetal-placental weight ratio was increased. Thus, we consider that the normal fetal growth and development can be maintained caused by adaptive change, even if the placental weight decreased by approximately 25% in 6-MP exposed rats.

Multi-endpoint genotoxic assay using L5178Y (Tk(+/-) -3.7.2c) cells.

When the mouse lymphoma Tk assay (MLA) provides a positive result, its cause can be roughly estimated by examining colony sizes. An increase in the number of large colonies means that the compound tested has point mutational potential, while an increase in small colonies indicates the potential for chromosome aberration. However, it was found to be difficult to clearly judge this in the case of caffeine known as a clastogen lacking the potential of point mutation. In our study, caffeine significantly increased the thymidine kinase (Tk) mutation frequencies derived from large colonies as well as those from small colonies in the standard protocol, although the frequencies derived from a small colony were higher than those from large colonies at higher doses. Therefore, we prolonged the expression period from 2 days, a standard period, to 6 days after treatment and then examined the Tk and Hprt mutations simultaneously. The result showed that caffeine gave a completely negative result on a mutation test for both Tk and Hprt. On the other hand, ethyl methanesulfonate (EMS), a genotoxic carcinogen, showed a positive result for both. Moreover, caffeine and EMS significantly increased the frequencies of micronucleated cells. In conclusion, when MLA gives a positive result and the cause is ambiguous, in order to identify the exact cause of the positive response, it is helpful to perform a confirmatory test investigating the potential of Tk and Hprt gene mutation simultaneously after 6-day expression and to perform an in vitro micronucleus assay during the expression period.

Enhancement of lung carcinogenesis initiated with 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone by Ogg1 gene deficiency in female, but not male, mice.

The present study was conducted to assess involvement of oxidative stress in lung adeno-carcinogenesis, using mice deficient in the 8-hydroxyguanine DNA glycosylase 1 (Ogg1) gene encoding an enzyme that repairs an oxidative DNA injury 8-oxoguanine (8-oxoG). Furthermore, for comparison with the human case, mutations of mouse epidermal growth factor receptor (Egfr) and K-ras genes were examined. The homo- and heterozygously Ogg1 gene-deficient and wild-type mice (C57BL6/J origin), 6 weeks old, were administered 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by continuous subcutaneous infusion using an osmotic pump at a total dose of 6 mg/mouse for 1 week, then treated with one of 4 antioxidants (phenyl N-tert-butyl nitrone 0.13% in drinking water, resveratrol 20 ppm in diet, lactoferrin 2% in diet and bilberry powder 2% in diet) or no supplement for 33 weeks. Development of lung adenomas and preneoplastic atypical hypreplasias was significantly enhanced by the homo- and heterozygous Ogg1 gene deficiency only in female mice with intralesion accumulation of 8-oxoG. All antioxidants tended to inhibit enhanced adeno-carcinogenesis. The Egfr and K-ras gene mutations were detected at sites also found in human lung cancers with low incidences, while the Egfr gene mutation was detected for the first time in chemical lung carcinogenesis of animals. It is indicated that the Ogg1 gene deficiency enhances lung adeno-carcinogenesis in mice by virtue of accelerated oxidative stress. The presently utilized Ogg1 gene-deficient mice model may be useful to draw mechanism-based strategies to control human lung adenocarcinomas, especially in women.

Histopathological effect of ketoconazole on rat placenta.

In order to investigate the morphological effects of ketoconazole on hypertrophied placentas, we examined the sequential histopathological changes in the placenta from rats exposed to ketoconazole. Ketoconazole was administered orally at 0 and 25 mg/kg/day during gestation days (GDs) 12 to 14, and the placentas were sampled on GDs 15, 17 and 21. All dams showed neither effect on body weight nor any abnormal clinical signs during the experimental period. In the treated group, the placentas appeared more hypertrophic with increases in the weight, diameter and thickness on GD 21. Histopathologically, increased thickness was noted in the labyrinth zone and basal zone on GDs 17 and 21, while on GD 15 the change had been already evident in the former zone. In the labyrinth zone, the mitotic figures of the trophoblasts were significantly elevated on GD 15. A multiple cystic dilatation of maternal sinusoids was observed in some placentas on GDs 15, 17 and 21. In the basal zone, an increase in spongiotrophoblasts and clusters of glycogen cells were detected on GDs 17 and 21. In the decidua basalis, there were no significant changes in either histology or thickness between the control and treated group during GDs 15 to 21. In conclusion, ketoconazole increased the population of composed cells in the labyrinth and basal zone, leading to placental hypertrophy in pregnant rats.

Carcinogenic risk of copper gluconate evaluated by a rat medium-term liver carcinogenicity bioassay protocol.

Carcinogenic risk and molecular mechanisms underlying the liver tumor-promoting activity of copper gluconate, an additive of functional foods, were investigated using a rat medium-term liver carcinogenicity bioassay protocol (Ito test) and a 2-week short-term administration experiment. In the medium-term liver bioassay, Fischer 344 male rats were given a single i.p. injection of N-nitrosodiethylamine at a dose of 200 mg/kg b.w. as a carcinogenic initiator. Starting 2 weeks thereafter, rats received 0, 10, 300 or 6,000 ppm of copper gluconate in diet for 6 weeks. All rats underwent 2/3 partial hepatectomy at the end of week 3, and all surviving rats were killed at the end of week 8. In the short-term experiment, rats were given 0, 10, 300 or 6,000 ppm of copper gluconate for 2 weeks. Numbers of glutathione S-transferase placental form (GST-P) positive lesions, single GST-P-positive hepatocytes and 8-oxoguanine-positive hepatocytes, and levels of cell proliferation and apoptosis in the liver were significantly increased by 6,000 ppm of copper gluconate in the medium-term liver bioassay. Furthermore, hepatic mRNA expression of genes relating to the metal metabolism, inflammation and apoptosis were elevated by 6,000 ppm of copper gluconate both in the medium-term liver bioassay and the short-term experiments. These results indicate that copper gluconate possesses carcinogenic risk toward the liver at the high dose level, and that oxidative stress and inflammatory and pro-apoptotic signaling statuses may participate in its underlying mechanisms.

Possible carcinogenic risks of copper gluconate and their prevention by co-administered green tea catechins evaluated by a rat medium-term multi-organ carcinogenicity bioassay protocol.

Carcinogenic risks of copper gluconate, green tea catechins and their combined exposure were evaluated using a rat medium-term multi-organ carcinogenicity bioassay protocol. Male BrlHan:WIST@Jcl (GALAS) rats were given N-nitrosodiethylamine (DEN), N-methylnitrosourea (MNU), 1,2-dimethylhydrazine (DMH), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) for a total multiple initiation period of 4 weeks (DMBDD treatment). Rats were then given a diet containing copper gluconate at a concentration of 0, 10, 300, 3000 or 6000 ppm with or without a co-administration of catechins starting 1 week later by admixing in the drinking water at a concentration of 5000 ppm. All survivors were sacrificed at the end of week 29. Number of putatively preneoplastic, glutathione S-transferase placental form-positive, liver lesions significantly increased by copper gluconate of 300 ppm or greater. In addition, both incidence and grade of hyperplasia in the forestomach significantly increased by copper gluconate of 6000 ppm. Catechins, exerting no effects by themselves, inhibited these effects of copper gluconate. The present results indicate that copper gluconate may possess carcinogenic risks for the liver and forestomach at its high dose level, and that co-administered green tea catechins may exert preventive effects.

Comparative study to elucidate the mechanism underlying the difference in airway hyperresponsiveness between two mouse strains.

The mechanism underlying airway hyperresponsiveness (AHR), a characteristic feature of asthma, remains obscure. We attempted to elucidate the mechanism responsible for the different degrees of AHR in two mouse strains, BALB/c and C57BL/6, following exposure to an anaphylactic trigger. When ovalbumin (OVA)-sensitized mice were challenged daily with OVA for up to three consecutive days, the BALB/c mice showed a higher degree of airway responsiveness to methacholine than did C57BL/6. Following the OVA challenge, eosinophils and macrophages in bronchoalveolar lavage fluid (BALF) from BALB/c increased significantly in number compared to those from C57BL/6. BALB/c mice also exhibited a higher serum IgE level than that of C57BL/6 after OVA challenge. The enhanced AHR and eosinophilic infiltration in BALF were significantly reduced by pretreatment with a selective cysteinyl-leukotriene type 1 receptor (cysLT(1)R) antagonist, montelukast. In the in vitro study, cysLT production was significantly lower in the dissected lung tissue from BALB/c than in tissue from C57BL/6 when both groups were stimulated with saline. The lungs from BALB/c generated significantly larger amounts of cysLTs on incubation with OVA rather than with saline, while the lungs from C57BL/6 did not show any significant increase in cysLTs with antigen stimulation. Significant upregulation of cysLT(1)R and cysLT(2)R mRNA expression was induced by OVA challenge in the lungs of BALB/c, but not in those of C57BL/6. It is suggested that, after an anaphylactic reaction, the degree of AHR is dependent on the genetic background and that cysLTs play an important role in the mechanism involved.

[Study for an allergic inflammation model using human lungs and its pharmacological application].

The complement system, which plays an important role in inmate immunity, is considered to be important in the pathophysiology of allergic asthma. A patient with allergic asthma shows the reversible characteristic system of bronchoconstriction, increased mucus secretion, and complicated airway inflammation. Various cytokines secreted from Th2 cells contribute to the system. Cysteinyl-leukotrienes (CysLTs) are also considered to be one of the important mediators involved in asthmatic pathophysiology. However, the effects of a drug on humans may not be the same as those on animals due to species differences in complement-related molecules. In this series of experiments, we tried to establish a model in which the effects of a drug on the production of CysLTs from human lung preparations were evaluated following an anaphylactic reaction. CysLT production increased when the passively sensitized lung tissues were stimulated with anti-IgE antibody. The coaddition of anaphylatoxin, C5a, with the anti-IgE antibody potentiated CysLT production. The response to C3a was weaker when compared with that to C5a. In addition, increased production of CysLTs by adding serum at a specific ratio was dose dependently inhibited by nonpeptide C5a receptor antagonist, W-54011, or a novel complementary peptide inhibitor of C5a, acetyl peptide A. From these results, it is suggested that C5a potentiates cysLT production from human lung tissues and contributes to allergic inflammation like asthma, and thus acetylated peptide A and W-54011 are useful for suppressing allergic inflammation in the lungs.

[Complement activation and inflammation].

The complement system not only plays an important role in the defense system, but also contributes to the amplification of inflammation if activated in excess or inappropriately controlled. Complement activation through one of three pathways is tightly controlled by various regulators of complement activation (RCA) which are constitutively expressed on various cells in order to restrict excessive activation. Complement activation may generate polypeptides, so-called anaphylatoxins, and membrane attack complex (MAC) with large molecular mass. Anaphylatoxins (C3a, C4a, and C5a) produced by activation of the complement is considered to bridge innate and acquired immunity. Considering that C5a is more potent than C3a, but the serum concentration of C3 is 10 times higher than that of C5, the overall effects of C3a may be comparative with those of C5a. Since both anaphylatoxins are considered to exert their actions through rhodopsin-typed receptors, their receptor antagonists are targets for the discovery of anti-inflammatory and immune-modulating drugs. Complement activation may be related to the pathophysiology of various refractory disorders including ARDS, asthma, septic syndrome, SLE, rheumatoid arthritis, ischemia-reperfusion injury, and psoriasis etc. Pharmacological manipulation of the complement system may consist of various strategies including (1) inhibitors of complement activation at various levels, (2) receptor antagonists of anaphylatoxins, C3a and C5a, and (3) inhibitors of C5a including monoclonal antibody. Candidate agents concerning the above-mentioned manipulations have being produced and some are now in progress toward clinical trials in patients with certain diseases.

A novel method for preserving human lungs using a super-cooling system.

PURPOSE: To ensure the suitable preservation of isolated lungs, a super-cooling system was used to cool water to temperatures as low as -5 degrees C without freezing. DESCRIPTION: After lung tissues were obtained from patients with lung cancer, they were kept at -5 degrees C or 4 degrees C for as many as 5 days, and then they were histologically and biochemically examined. To evaluate biochemical stability, tissues after storage were passively sensitized with immunoglobulin E and then incubated with anti-immunoglobulin-E antibody. EVALUATION: Although tissues preserved at -5 degrees C for 5 days had an almost normal appearance with intact cilia on bronchial epithelium and normal endothelium, tissues stored at 4 degrees C showed degradation of these structures. Single-stranded DNA, a sign of DNA cleavage, was frequently noted in tissues stored at 4 degrees C, but only rarely observed at -5 degrees C. A significant amount of cysteinyl-leukotrienes was generated from tissues stored at -5 degrees C for 3 days, but there was no response to antibody stimulation from tissues stored at 4 degrees C. CONCLUSIONS: Super-cooling systems may provide useful applications as a novel preserving method.