Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Inhibitory Effect of Rotundarpene on Parkinsonian Neurotoxin 1-Methyl-4-Phenylpyridinium-Induced Apoptotic Cell Death

BACKGROUND: The extract and hemiterpene glycosides of Ilex Rotunda Thunb exert antioxidant and anti-inflammatory effects. The effect of rotundarpene on apoptosis in neuronal cells caused by the 1-methyl-4-phenylpyridinium (MPP⁺) has not been reported previously. METHODS: Using differentiated PC12 cells and human neuroblastoma SH-SY5Y cells, we investigated the effect of rotundarpene on MPP⁺-caused apoptosis in relation to the cell death process. RESULTS: MPP⁺-induced cell death was identified using the MTT and neutral red uptake tests. Apoptosis was induced by eliciting decreases in the cytosolic levels of Bid and Bcl-2 proteins, increases in the cytosolic levels of Bax and p53, disruption of the mitochondrial transmembrane potential, and the release of cytochrome c and the activation of caspase-8, -9, and -3 in differentiated PC12 cells and SH-SY5Y cells. Treatment with rotundarpene reduced the MPP⁺-induced changes in the levels of apoptosis-regulated proteins, formation of reactive oxygen species, depletion and oxidation of glutathione, and cell death in both PC12 and SH-SY5Y cells. CONCLUSIONS: Rotundarpene may reduce MPP⁺-induced apoptosis in neuronal cells by suppressing the activation of the mitochondria-mediated pathway and the caspase-8 and Bid pathways. Rotundarpene appears to act by inhibiting the production of reactive oxygen species and by the depletion and oxidation of glutathione.

Antiplasmodial and Cytotoxic Activities of Toad Venoms from Southern Amazon, Brazil

The drug-resistance of malaria parasites is the main problem in the disease control. The huge Brazilian biodiversity promotes the search for new compounds, where the animal kingdom is proving to be a promising source of bioactive compounds. The main objective of this study was to evaluate the antiplasmodial and cytotoxic activity of the compounds obtained from the toad venoms of Brazilian Amazon. Toad venoms were collected from the secretion of Rhinella marina and Rhaebo guttatus in Mato Grosso State, Brazil. The powder was extracted at room temperature, yielding 2 extracts (RG and RM) and a substance ('1') identified as a bufadienolide, named telocinobufagin. Growth inhibition, intraerythrocytic development, and parasite morphology were evaluated in culture by microscopic observations of Giemsa-stained thin blood films. Cytotoxicity was determined against HepG2 and BGM cells by MTT and neutral red assays. The 2 extracts and the pure substance ('1') tested were active against chloroquine-resistant Plasmodium falciparum strain, demonstrating lower IC₅₀ values. In cytotoxic tests, the 2 extracts and substance '1' showed pronounced lethal effects on chloroquine-resistant P. faciparum strain and low cytotoxic effect, highlighting toad parotoid gland secretions as a promising source of novel lead antiplasmodial compounds.

Cytotoxicity assays as tools to assess water quality in the Sinos River basin / Ensaios de citotoxicidade como ferramentas para avaliar a qualidade da água da bacia do Rio do Sinos

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of surface waters and may help to improve the control of water quality. This study compared two methods to prepare culture media for the exposure of Hep-2 cells to water samples collected from the Rolante River, an important affluent of the Sinos River. The toxicity was evaluated using the MTT and neutral red assays. Two methods were used to prepare culture media. In method 1, the sample was diluted at 1:1, 1:10, 1:100, 1:1000, 1:10.000 (v/v, sample/medium) in a standard culture medium; in method 2, water samples were used as the solvent for the culture medium, which was prepared at concentrations of 100, 80, 60, 40 and 20%. Semi-confluent cultures were then exposed to the media test for 24 hours, and cytotoxicity was determined immediately using the MTT and NR assays. Mitochondrial activity (MTT) was significantly lower at all concentrations in both methods, except at 1:1000 in method 1. However, the lysosome viability (NR) results revealed cytotoxicity only in the 1:1 sample of method 1. Both culture preparation methods were efficient and sensitive to the MTT assay, but method 2 seemed to be more adequate for the NR assay. The Rolante River has cytotoxic contaminants to Hep-2 cells, which may be one of the explanations for the poor water quality of the Sinos River basin.

Os ensaios de citotoxicidade utilizando culturas de células constituem uma alternativa para avaliar a toxicidade biológica de águas de superfície e podem auxiliar no controle da qualidade da água. Este estudo comparou dois métodos de preparação dos meios de cultura com amostras de água coletadas no rio Rolante, um importante afluente do Rio dos Sinos, para a exposição de células Hep-2. A toxicidade foi avaliada usando os ensaios do MTT e do vermelho neutro (VN). Dois métodos foram utilizados para preparar os meios de cultura. No método 1, a amostra foi diluída a 1:1, 1:10, 1:100, 1:1000 e 1:10.000 (v/v, amostra/ meio de cultivo) em um meio de cultura padrão; no método 2, as amostras de água foram utilizados como solventes para o meio de cultura, o qual foi preparado em concentração de 100% e nas diluições de 80, 60, 40 e 20%. Culturas semi-confluentes foram então expostas aos meios teste durante 24 horas, e a citotoxicidade foi determinada imediatamente usando os ensaios MTT e VN. A atividade mitocondrial (MTT) foi significativamente menor em todas as concentrações em ambos os métodos, exceto na diluição 1:1000 do método 1. No entanto, os resultados de viabilidade lisossomal (VN) revelaram citotoxicidade apenas no na diluição 1:1 do método 1. Ambos os métodos de preparação do meio cultura foram eficientes e sensíveis para o ensaio do MTT, mas o método 2 foi mais adequado para o ensaio do VN. O rio Rolante possui contaminantes citotóxicos para as células Hep-2, o que pode ser uma das explicações para a baixa qualidade da água da Bacia do Rio dos Sinos.

In vitro cytotoxicity of self-curing acrylic resins of different colors

OBJECTIVE: The aim of this study was to assess the in vitro cytotoxicity of acrylic resins of different colors over time. METHODS: Specimens were divided into 4 groups (n = 6) according to the color of the acrylic resin (Orto Class, Clássico, Campinas, São Paulo, Brazil): Group 1: clear acrylic resin; group 2: pink acrylic resin; group 3: blue acrylic resin and group 4: green acrylic resin. All specimens were fabricated according to the mass manipulation technique and submitted to mechanical polishing protocol. The control was performed with an amalgam specimen (C+), a glass specimen (C-) and cell control (CC). Specimens were immersed in Minimum Eagle's Medium (MEM) and incubated for 24 h at 37o C. The extracts from the experimental material were filtered and mixed with L929 fibroblast. Cytotoxicity was evaluated at 4 different times, 24, 48, 72 and 168 h. After contact, cells were incubated for 24 h and added to 100 µ of 0.01% neutral red dye. The cells were incubated for 3 h for pigment incorporation and fixed. Cells viability was determined by a spectroscopic (BioTek, Winooski, Vermont, USA) with a 492-nm wavelength λ=492 nm). RESULTS: There were no statistical differences between the experimental groups and the CC and C- groups. CONCLUSION: Clear, pink, blue and green self-curing acrylic resins fabricated by means of the mass manipulation technique and mechanically polished are not cytotoxic. Neither the pigment added to the self-curing acrylic resin nor the factor of time influenced the cytotoxicity of the material. .

OBJETIVO: avaliar, in vitro, a citotoxicidade de resinas acrílicas autopolimerizáveis, de diferentes cores, ao longo do tempo. MÉTODOS: os corpos de prova foram divididos em quatro grupos (n = 3), de acordo com a cor da resina acrílica utilizada (Orto Class, Clássico, São Paulo/SP), sendo: grupo 1, acrílica incolor; grupo 2, acrílica rosa; grupo 3, acrílica azul; e, grupo 4, acrílico verde. Todos os corpos de prova foram confeccionados pela técnica de massa e polidos mecanicamente. Um corpo de prova de amálgama, um de vidro e célula constituíram o controle positivo (C+), controle negativo (C-), e controle de célula (CC), respectivamente. Em seguida, esses foram imersos em meio mínimo essencial de Eagle (MEM) por 24h, quando se removeu o sobrenadante e colocou-os em contato com fibroblastos L929. Avaliou-se a citotoxicidade em quatro períodos: 24, 48, 72 e 168h. Após o contato com o meio, as células foram incubadas por 24h e adicionou-se 100µ do corante vermelho neutro a 0,01%. Posteriormente, as células foram incubadas por 3h, para incorporação do corante, e fixadas. A contagem das células viáveis foi realizada em espectrofotômetro (BioTek, Winooski, EUA), com um comprimento de onda de 492nm (λ = 492nm). RESULTADOS: não houve diferença estatística entre os grupos experimentais e os grupos CC e C-. CONCLUSÇÕES: as resinas acrílicas autopolimerizáveis incolor, rosa, azul e verde, manipuladas pela técnica de massa e polidas mecanicamente não são citotóxicas. O corante utilizado em resinas autopolimerizáveis e tempo não influenciam na citotoxocidade do material. .

Preventive Effect of Green Tea Extracts on the Inhibition of Connexin Expression Induced by Carcinogen H2O2 / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Carcinogens result in the impairment of intercellular communication as well as intracellular communication of normal cells. Connexin (Cx) is a main constituent protein of gap junctions that let messengers such as ions communicate between cells. We evaluated the effect of carcinogen H2O2 on the expression of Cxs and gap junction intercellular communication (GJIC) in the human keratinocyte cell line (HaCaT) and analyzed the prevention effect of green tea extracts against H2O2. MATERIALS AND METHOD: We performed neutral red dye uptake tests to determine the optimal concentrations of H2O2, green tea extracts-epicatechin (EC) and epigallocatechin-3-gallate (EGCG) in this study. To analyze the expression change of Cxs, we performed RT-PCR, Western blot, and immunocytochemistry after 24-hour culture of HaCaT cells treated with agents. We also evaluated GJIC quantitatively using the 'scrape loading dye transfer (SLDT)' technique. RESULTS: Cx26, Cx30, Cx31, Cx43, but not Cx29 were expressed in the HaCaT cells. H2O2 (250 uM) down-regulated Cx26 and Cx43 proteins. In HaCaT cells treated with H2O2, EC (175 uM) up-regulated Cx26 and Cx43 proteins, but EGCG (50 uM) up-regulated only Cx43 protein. Immunocytochemistry showed the decreased expression and abnormal location of Cx26 and Cx43 under H2O2, and EC and EGCG (5 uM) inhibited the effect of H2O2, showing similar staining in the control. In SLDT, H2O2 down-regulated GJIC, while EC and EGCG significantly prevented HaCaT cells from the H2O2-induced, down-regulation of GJIC. CONCLUSION: The carcinogen, H2O2, inhibits GJIC in the keratinocyte cell line. Green tea extracts, such as EC and EGCG, prevent GJIC inhibition in the keratinocyte cell line treated with H2O2, suggesting they have a potential anti-cancer properties.

Citotoxidade de três cimentos obturadores do sistema de canais radiculares sobre cultura de células L929 / Citotoxicity of three filling cements of radicular canals system over L929 culture cells

O objetivo desse estudo in vitro foi avaliar a citotoxicidade dos cimentos Intrafill e Pulp Canal Sealer, ambos à base de óxido de zinco eugenol e um outro cimento, o Sealapex, à base de hidróxido de cálcio. Esse experimento foi realizado usando linhagem de células L 929 (de fibroblastos de camundongos). A citotoxicidade foi avaliada in vitro, usando-se o método de difusão em Agar, depois de 48 horas do endurecimento do cimento, o corante utilizado foi o vermelho neutro. Os resultados mostraram diferenças nas médias dos valores da viabilidade celular pelo teste ANOVA. Após o período determinado pela amostra, o Intrafill, e o Sealapex apresentaram alterações morfológicas celulares com maior citotoxicidade quando comparados ao Pulp Canal Sealer.

Comparison of trypsin treatment method and standard laboratory technique for diagnosis of dermatomycosis.

Dermatomycosis is prevalent worldwide. Discrepancy between microscopic examination and culture findings can create problems in the diagnosis of this common infection. In this study, samples from 60 patients were processed after trypsin treatment and examined by neutral red staining to distinguish viable and non-viable fungal elements. The trypsin treatment method was compared with standard laboratory techniques. A higher number of direct-microscopy-positive, culture-negative samples were obtained without trypsin treatment. Trypsin treatment increased the isolation of fungi from clinical samples, and neutral red staining was able to distinguish viable fungal elements.

Microencapsulated Bovine Adrenal Medullary Chromaffin Cells Transplanted into Rat Spinal Cord Alleviated Cold Allodynia / 대한마취과학회지

BACKGROUND: The intrathecal grafting of adrenal chromaffin cells as a potential analgesic source, to delivery analgesic substances such as catecholamines and opioid peptides, is known to be effective at treating acute and chronic pain in several animal pain models. We tested whether the intrathecal implantation of encapsulated bovine chromaffin cells reduces cold allodynia in a rat model of neuropathic pain induced by chronic constriction injury of the sciatic nerve. METHODS: Bovine adrenal medullary chromaffin cells microencapsulated in sodium alginate-poly-l-lysin-alginate (APA) were implanted into the subarachnoid space of rats (n = 10) and foot cold sensitivity was investigated using an acetone test. At the end of the study, histology and capsule catecholamine production were evaluated. RESULTS: A significant reduction in cold allodynia was observed in animals implanted with chromaffin cells. In addition, the suppression of cold allodynia was reversed by naloxone. Abundant clusters of viable chromaffin cells stained with neutral red, were observed in the retrieved implants and after nicotine stimulation, and catecholamine was quantified. An ultrastructural study showed no fibrotic reaction against capsules, or disorganised capsules. CONCLUSIONS: These results suggest that intrathecal encapsulated chromaffin cells act as "mini pumps", which continuously deliver analgesic substances and produce analgesia in this chronic pain model of nerve injury-without immunosuppressant.

Cell-Specific Growth Inhibition of Human Cervical Cancer Cell by Recombinant Adenovirus p53 in vitro and in vivo / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Despite the significance of the p53 adenoviral vector in cancer gene therapy, an advanced strategy for the development of preferential tumor cell-specific delivery and the long-term persistent gene expression control of p53 are required. In this study, the time-course expression patterns of p53 and E6, on cervical cancer cells, were investigated to obtain a molecular level understanding of the cell-dependent tumor growth suppression effects of a recombinant adenovirus expressing p53, both in vitro and in vivo. MATERIALS AND METHODS: The expressions of p53 and E6 in CaSki, SiHa, HeLa, HeLaS3, C33A and HT3 cervical cancer cell lines were examined. After infection with AdCMVp53, the cell growth inhibition was studied via cell count, MTT and Neutral red assays. After transfecting the AdCMVp53 and AdCMVLacZ into the cancer cells-xenografted nude mice, the anti-tumor effects were investigated for one month. RESULTS: The p53 protein levels were more notably expressed in the CaSki and HeLa than in the SiHa and HeLaS3 On day 6, the p53 was only detected in the HeLaS3. In contrast, the p53 expression was highly maintained in the C33A and HT3. The E6 mRNA levels gradually decreased in only the CaSki and HeLa. The growth suppression effects also showed cell-dependent patterns, which were consistent with the reciprocal expression patterns of p53 and E6. After transfection of the AdCMVp53, into the CaSki- and SiHa-xenografted nude mice, the tumor size was remarkably decreased in the SiHa cells as compared to that in the AdCMVLacZ transfected mice, indicating cell-specific growth inhibition patterns. CONCLUSION: The adenovirus-mediated p53 gene transfection was very effective both in vitro and in vivo. Also, the anti-tumor effects were accomplished via the differential role of p53-specific apoptotic cell death, which was dependent on the cervical cancer cell line.

Growth inhibition and induction of apoptosis in cervical cancer cell lines by green tea polyphenols / 대한산부인과학회잡지

OBJECTIVE: The purpose of this article is to estimate the anti-cancer effects of the major components of the green tea (polyphenols, catechin and EGCG) and the mechanism of EGCG on different cervical cancer cell lines. METHODS: Six cervical cancer cell lines (HeLa, HeLaS3, Caski, SiHa, HT3 and C33A) were treated with 20 microgram/ml green tea polyphenols (GTPs), 50 micrometer catechin and various concentrations of (-)-epigallo- catechin-3-gallate (EGCG). The viabilities were determined by trypan blue exclusion assay, neutral red assay and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. DNA fragmentation and nuclear condensation were used to see whether EGCG-induced anti-proliferation effect was due to apoptosis. RESULTS: Both GTPs, catechin and EGCG had growth inhibition effects on cervical cancer cell lines, but EGCG appeared to be the most effective. What's more, the sensitivity of each cell lines to EGCG was different. HT3 cells (HPV negative, mutant type p53) were most sensitive to EGCG (estimate IC50: 10 micrometer). Caski (HPV-16 positive, wild type p53) and HeLaS3 cells (HPV-18 positive, wild type p53) were less sensitive (estimate IC50: 35 and 70 micrometer respectively). EGCG-induced apoptosis can be seen in all the cell lines and it happened as early as 8 hours after EGCG treatment. CONCLUSION: Green tea or EGCG alone will be beneficial to the cervical cancer patients.

Evaluation of Cutaneous Irritancy Using Human Skin Recombinants / 대한피부과학회지

BACKGROUND: There is an increasing need for the development of in vitro models capable of substituting for animals in cutaneous irritancy studies. Until now, various culture models have been developed, including skin organ cultures, conventional and air-exposed cell cultures. The air-exposed culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum corneum layer in these cultures permits the application of potential irritants at the concentrations and formulations which are applied in vivo. Recently, a new human skin recombinant, made of human keratinocytes cultured on de-epidermized dermis with fibroblast-populated collagen matrix, has been developed and appears to represent a better skin equivalent model than previous models. OBJECTIVE: In the present study, monolayer-cultured human keratinocytes and the new human skin recombinants were evaluated for the test models of various skin irritants. METHODS: The extent of skin irritancy induced after application of 3 different irritants (sodium lauryl sulfate, methyl paraben, and polyethylene glycol-400) was evaluated on the basis of (1) MTT assay, (2) neutral red uptake assay, (3) LDH release, and (4) release of IL-1 alpha. In the human skin recombinants, morphological perturbations and changes in the expression of differentiation-specific protein markers (keratin 1, involucrin, filaggrin, and loricrin) were also evaluated. To determine the difference between in vivo and in vitro models for the detection of irritancy, a patch test was performed on 11 normal human volunteers with various concentrations of the different irritants RESULTS: The results of the present study show that irritant cytotoxicity correlates well with irritant concentration in both monolayer-cultured human keratinocytes and the new human skin recombinant. The new human skin recombinant is superior to monolayer culture as an in vitro model for skin irritancy screening in that the concentrations of test irritants are the same as in vivo. With the human skin recombinant, morphological changes were observed according to the irritant concentration. CONCLUSION: The new human skin recombinant can be used as an alternative to animals for skin irritancy screening.

Autoproliferation of Fibroblast by Exposure to Crystalline Silica - Evaluation by H2O2 and PDGF-AA and TGF beta / 대한산업의학회지

OBJECTIVES: The aim of this study is to find out the activity of autoproliferation of ratfibroblast exposed to crystalline silica and the role of mediators secreted from rat fibroblast. METHODS: The effect of alpha-quartz on production of growth factor (platelet-derived growth factor-AA and transforming growth factor beta)from rat fibroblasts were evaluated by ELISA and immunocytochemical analysis. Gene expression of these growth factors in rat fibrobast exposed to crystalline silica was evaluated by RT-PCR. Furthermore, fibroblast proliferation by culture supernatant of rat fibroblast was assayed by the neutral red test. RESULTS: The amounts of H2O2 and growth factors synthesized in rat fibroblasts were significantly increased by the stimulation of crystalline silica(alpha-quartz), which showed the dose-dependent manner to the concentration of alpha-quartz with the maximum response at the dosage of 100 microgram/cm2. The result of RT-PCR demonstrated that alpha-quartz induced gene expression of PDGF-AA and TGFbeta in rat fibroblast. We also found that supernatant of alpha-quartz-cocultured rat fibroblast induced a significant proliferation of fibroblast. CONCLUSION: Crystalline silica directly induce functional change in fibroblast such as increased release of reactive oxygen species and growth factors. The products of these functional change promote fibroblast proliferation via autocrine loop.

Comparison of two colorimetric assays to determine viral infectivity in micro culture virus titration.

Efficacy of two colorimetric assays, viz. MTT (3-4,5-dimethylthiazol-2-(yl-2,5-diphenyl tetrazolium bromide) and neutral red (NR) assays, performed by integrating them to micro culture virus titration (MCVT), was compared with the conventional MCVT method in terms of percentages of infectivity and 50% infectivity end points by employing Polio virus type-3 and Dengue virus type 4 as the candidate viruses. The results suggested that MTT assay has an edge over NR assay as well as conventional MCVT method. For the first time, NR assay has been successfully employed for the determination of virus infectivity titre.

A Study of a Selection of Antidotes for Paraquat induced Skin Damage

BACKGROUND: Paraquat is a widely used herbicide, known to cause lethal toxicity in humans. Most studies about paraquat have concentrated on systemic toxicity, however several cases of paraquat-induced dermatitis have been reported. OBJECTIVE: The purpose of this study was to confirm the cutaneous toxic effect of paraquat and to select potential antidotes in paraquat-induced dermatitis. METHODS: Keratinocyte toxicity due to paraquat and the toxicity reduction capacity of several drugs were investigated in eitro. Topical effects of these drugs on paraquat-induced dermatitis in guinea pig skin was also investigated. RESULTS: Over 50% of keratinocytes failed to survive at a concentration of 2X10-4M paraquat by a neutral red uptake assay. Skin irritation by paraquat was observed at 2% concentration by non-invasive methods as well as a skin biopsy. Dexamethasone, glutathione and tocopherol showed some capacity to reduce paraquat-induced keratinocyte toxicity in vitro. Only dexamethasone, however, showed a reduction of cutaneous blood flow volume and dermal inflammatory cell infiltration in the guinea pig study. CONCLUSION: This result indicates the possible in eitro protective effect of paraquat toxicity in glutathione and tocopherol. Dexamethasone was capable of reducing paraquat-induced cytotoxicity and dermatitis both in vitro and in vivo.

Incidence of Vaculating Toxin Producing Helicobacter pylori from Patients with Gastric Diseases / 대한내과학회지

OBJECTIVES: This study was carried out to survey the prevalence of Helicobacter pylori infection and the incidence of vacuolating toxin producing H. pylori. A further aim of this study was to evaluate the quantitative assay for cell vacuolation on the basis of the rapid uptake of neutral red dye by vaculoes of the cells. METHODS: We studied the gastric biopsy specimens of patients with 154 cases of gastritis, 74 cases of gastric ulcer, and 167 cases of gastric cancer and in 44 cases of healthy persons. One of the biopsy specimen was placed into a CLOtest plate for rapid urease test and the other one of the biopsy spcimen was inoculated on Brain Heart Infusion blood agar for culture. The culture supernatant of isolated H. pylori was serially diluted with BHI broth. After 24 hour incubation of cultured RK-13 cells treated with the culture supernatant of H. pylori, cytoplasmic vacuolation of the cells were observed microscopically. RESULTS: The positivity of urease test and the rate of isolation of H. pylori from urease positive gastric biopsy materials were 34.1% and 93.3% in healthy person, 55.8% and 70.9% in gastritis, 60.8% and 71.1% in gastric ulcer, and 56.3% and 96.8% in gastric cancer. The isolation rate of H. pylori from patients between 20 and 39 years old was 16.8%, for patients between 40 and 59 years old it was 51.9%, and for patients above 60 years old it was 31.2%. The isolation rate of the vacuolating toxin producing H. pylori from gastric biopsy specimens was 66.7% in a healthy person, 76.6% in gastritis, 79.4% in gastric ulcer, and 80% in gastric cancer. CONCLUSIONS: The isolation rate of H. pylori from the patients with gastric diseases is higher than the rate of H. pylori from healthy persons, but the isolation rate of the vacuolating toxin producing H. pylori is not different between the patients with gastric diseases and healthy persons. The titers of vacuolating toxin produced by some H. pylori isolated from the patients with gastric diseases are higher than those from healthy persons.

Necesidad o no del uso de colorantes durante la remoción de la dentina cariada: soporte microbiológico / Caries detector dye, its use during dentinal caries removal: microbiological support

La remoción completa de la dentina cariada es un proceso complejo, por la escasa definición de criterios objetivos para diferenciar la dentina infectada de la afectada. Una de las formas de reducir la subjetividad es la utilización de colorantes, aunque existe controversia respecto a las indicaciones, ventajas y desventajas de su uso. Este estudio comparó la efectividad de la técnica convencional óptica y táctil con la utilización de rojo ácido al 1 por ciento en la remoción de la dentina cariada, y microbiológicamente cuantificó y clasificó los microorganismos de la dentina remanente, en 34 pacientes con caries moderada. Se encontró que el 97 por ciento de las cavidades presentaron tinción de la dentina, siendo más frecuente ésta en la pared pulpar y unión amelodentinaria, pero el colorante no siempre tiñó bacterias, por lo cual llevó a remoción innecesaria de dentina y a varias exposiciones pulpares. El 61.81 por ciento del total de los microorganismos cultivables no se logró indentificar y su papel en el progreso de la caries no se conoce. Los S. mutans y Lactobacillus representaron sólo el 12.79 por ciento del total de los microorganismos. No se encontró correlación entre el frente de decoloración, el sitio de tinción y el frente bacteriano

Application of Neutral Red Staining for the Evaluation ofthe Viability of Dermatophytes / 대한피부과학회지

BACKGROUND: Microsopic examination of potassium hydroxide(KOH) preparation and fungus culture is required for the diagnosis of fungal infection. Sometimes dermatophytes fail to grow on culture medium, although these are observed on microscopic examination of KOH preparation. Recently this discrepancy between microscopic examination and fungus culture can be explained by the hyphothesis that some of the fungal elements are non-viable. OBJECT: This study was made to evaluate the viability of dermatophytes using neutral red(NR) staining for the explaination of discrepancies between microscopic examination and fungus culture. METHODS: After identification of fungus by culture from dermatophytic lesion the hyphae was collected for this study. In order to confirm whether the NR staining is suitable for check the viability of hypae or not, we designed to prepare the preparations of hyphaes by 2 ways. One was killed hyphae by autoclave, the other was kept as viable hyphae. And then we compared the stainability of NR staining and autoradiographic study using (3)H-thymidine. And we compared the results of NR staining and the subsequent culture using the scales which were collected from the lesions 30 dermatophytic patients. RESULTS: The structure inside of cell wall of hyphae stained red color only in case of viable hyphae preparations, but not stained in killed hyphae preparation. Autoradiographic study using (3)H-thymidine confirmed that grain-positive cells(viable cells) were stained with NR, whereas grain negative cells (non viable cells) were not stained. Among the 30 cases with dermatophytosis 27(90.0%) cases showed NR positive and 14(46.6%) cases showed culture-positive. Except the tinea unguium cases which have shown low culture positive rate, 9(75.0%) cases of the 12 NR positive samples were positive on culture. All 14 cases of the culture positive samples were positive on NR staining. And all 3 cases of the NR negative samples were negative on culture. CONCLUSION: NR staining can be a useful method for the evaluation of viability of the fungal elements.

The Effect of Halothane-induced Acidosis on Focal Cerebral Ischemia in Rat

The current study was performed to investigate the influence of acidosis on focal cerebral ischemia in view of morphometric assay and neuropathological examination. The acidosis was induced by increment of halothane concentration and by decreasing respiratory rate. The mean pH were 7.423+/-0.012 in control group and 7.184+/-0.038 in acidosis group. Twenty-four hours after MCA occlusion(MCAO), neutral red staining and perfusion fixation was performed. The ischemic area was measured and morphometric analysis was undertaken. In acidosis group, the infarct area was 25.23+/-4.78% of the total cerebral area;in control group, the infarct area was 27.69+/-4.05%. The histopathological findings were examined under light microscopy, in which the field scanning was carried out from the midline by 0.5mm interval at cortical and basal ganglia levels. These results indicated that although there was no satistically significant difference in infarct area between acidosis and control group, increased acidosis aggravated the extent of histopathologic ischemic neuronal damage.

The Influence of Hyperglycemia on the Extent of Focal Cerebral Ischemia

The present study was undertaken to investigate the influence of hyperglycemia on focal cerebral ischemia in view of morphometric assay and neuropathological examination. Forty Sprague-Dawley rats were divided into two groups of 20 each. Rat MCA occlusion model was used for induction of focal ischemia. Hyperglycemia(20 rats, mean(SEM plasma glucose concentration 378(97.6 mg/dl) was established 30 minutes before MCA occlusion by intraperitoneal injection of 50% dextrose in water;the control group(20 rats, mean(SEM plasma glucose concentration 121(24.9 mg/dl) received normal saline only. Twenty-four hours after MCA occlusion neutral red staining and perfusion fixation was performed and ischemic area were measured using computerized image analysis on cortical surface and coronal cut surface. There was no significant difference on coronal cut surface, but on cortical surface showed increase of non-stained area(infarct core) and decrease of lightly stained area(transitional zone) in hyperglycemic rats(p<0.05) and the sum of two area was not different between two groups. Pathological findings were evaluated under light microscopy, in which the field scanning was carried out from the midline by 0.5 mm interval at cortical and basal ganglia level. There showed no significant difference at basal ganglia level, but at cortical level ischemic transitional zone was decreased in hyperglycemic rats(p<0.05). We conclude that hyperglycemia may worsen the brain from severe, focal ischemic neuronal damage.