Development and validation of analytical method by HPLC-DAD for determination of vasodilator active in pharmaceutical ophthalmic forms

The substance 4-Aminobenzamidine dihydrochloride (4-AD) is one of the degradation products of diminazene aceturate and has demonstrated antiglaucomatous potential. Glaucoma is the second leading cause of blindness worldwide; thus, new therapeutic alternatives must be studied, for example, the molecule 4-AD vehiculated into polymeric inserts for prolonged release. The present work aims to develop and validate an analytical method to quantify 4-AD in pharmaceutical ophthalmic forms. A HPLC was used with UV-Vis detector, at 290 ƞm and ACE® C18 column (125 × 4.6 mm, 5 µm), in which the mobile phase consists of phosphate buffer (pH 7.4) and triethylamine (30 mmol/L), under an isocratic flow of 1.0 mL/min. The retention time of 3.2 minutes was observed. The method was developed and validated in accordance with ANVISA recommendations and ICH guides. The linearity range was established between the concentrations 5 and 25 µg/mL (correlation coefficient r = 0.993). The accuracy, repeatability, and intermediate precision tests obtained a relative standard deviation less than or equal to 5%. In addition, the method was considered selective, exact. and robust, with pH being its critical factor. Therefore, the HPLC analysis method is robust and can be used to quantify 4-AD in pharmaceutical forms for ocular application.(AU)

Clinical and therapeutic aspects of an outbreak of canine trypanosomiasis / Aspectos clínicos e terapêuticos de um surto de tripanossomíase canina

Abstract Trypanosomiasis caused by Trypanosoma evansi can seriously affect both domestic and wild animals. This article reports on an outbreak of canine trypanosomiasis on a farm in the Pantanal region of Brazil. The farm had 38 dogs, 20 of which died before receiving veterinary care. The remaining 18 dogs were underwent anamnesisn, clinical examination, hematological and biochemical evaluations. Blood smears and PCR analysis were performed for the diagnosis. The treatment protocols used according to the clinical recovery or parasitological cure of the dogs, using diminazene diaceturate, isometamidium chloride or quinapyramine sulfate. Post-treatment parasitological evaluation was performed by the microhematocrit technique. 7/18 dogs were PCR positive for T. evansi (confirmed by sequencing). There was clinical findings, which were consistent with both the acute and chronic stages of the disease in dogs. The infected dogs all exhibited at least one clinical sign of the disease. The hematological findings were compatible with trypanosomiasis, highlighting the hypochromic microcytic anemia as the main outcome. No treatment protocol was fully effective and the prolonged use of diminazene diaceturate caused the death of an animal. The trypanosomiasis can cause high rates of morbidity and mortality in dogs and difficulty in establishment an effective and safe therapeutic protocol.

Resumo A tripanossomíase causada por Trypanosoma evansi pode acometer gravemente os animais domésticos e selvagens. Este artigo relata um surto de tripanossomíase canina em uma fazenda na região do Pantanal, Brasil. Na fazenda havia 38 cães, 20 dos quais morreram antes de receber cuidados veterinários. Os 18 cães restantes foram submetidos a anamnese, exame clínico, avaliação hematológica e bioquímica. Esfregaços de sangue e análise da PCR foram realizados para o diagnóstico. Os protocolos de tratamento foram utilizados de acordo com a recuperação clínica ou cura parasitológica dos cães, utilizando diaceturato de diminazeno, cloreto de isometamídio ou sulfato de quinapiramina. A avaliação parasitológica pós-tratamento foi realizada pela técnica de microhematócrito. 7/18 cães foram PCR positivos para T. evansi (confirmado por sequenciamento). Os achados clínicos encontrados, foram consistentes com os estágios agudo e crônico da doença em cães. Todos os cães infectados exibiram pelo menos um sinal clínico da doença. Os achados hematológicos foram compatíveis com a tripanossomíase, destacando a anemia microcítica hipocrômica como principal consequência. Nenhum protocolo de tratamento foi totalmente eficaz e o uso prolongado de diaceturato de diminazeno causou a morte de um animal. A tripanossomíase pode causar altas taxas de morbidade e mortalidade em cães e dificultar o estabelecimento de um protocolo terapêutico eficaz e seguro.

Aspectos patológicos da intoxicação por aceturato de diminazeno em camelídeos sul-americanos / Pathological aspects of diminazene aceturate toxicosis in american camelids

Aceturato de diminazeno é um fármaco quimioterápico sintético comumente usado na medicina veterinária para o tratamento de doenças causadas por parasitos hematozoários. Entretanto, seu uso pode levar a efeitos colaterais, como alterações neurológicas graves e morte. A criação de camelídeos é uma atividade recente no Brasil, fazendo-se necessário conhecer mais sobre as doenças que acometem essas espécies. De dez camelídeos (seis lhamas e quatro alpacas) da propriedade, seis tiveram sinais clínicos e, destes, apenas uma lhama com manifestações leves recuperou-se. Os sinais clínicos incluíam apatia, andar cambaleante, fraqueza, sialorreia, cabeça baixa e pendida lateralmente, dificuldade em levantar e dispneia, observados a partir de 18 horas após o uso do medicamento. À necropsia e ao exame histopatológico foram observadas alterações de encefalopatia hemorrágica bilateral e simétrica, mais graves em tronco encefálico e tálamo. Este trabalho descreve as principais lesões observadas em um surto de intoxicação por diminazeno em alpacas (Lama pacos) e lhamas (Lama glama) e alerta criadores e veterinários sobre o risco de intoxicação por aceturato de diminazeno em camelídeos sul americanos.(AU)

Diminazene aceturate is a synthetic chemotherapeutic drug commonly used in veterinary medicine for the treatment of diseases caused by hematozoan parasites. However, side effects as severe neurological disorders and death can occur. The raising of american camelids is a recent activity in Brazil, requiring knowledge about diseases that affect these species, in order to avoid misguided conducts. In a herd of ten camelids (six llamas and four alpacas) six showed clinical signs and five died; only a llama with mild signs recovered. The clinical signs included apathy, difficulty to stand up, staggering gait, weakness, down head and drooping the head laterally, dyspnea and drooling of saliva, observed from 18 hours after use of the drug. At necropsy and histopathological examination was found bilateral and symmetrical hemorrhagic encephalopathy, more severe in brainstem and thalamus. This paper describes the main lesions observed in an outbreak of diminazene aceturate poisoning in alpacas (Lama pacos) and llamas (Lama glama) and alert breeders and veterinarians about the risk of poisoning by this drug in american camelids.(AU)

Association of exercise training and angiotensin-converting enzyme 2 activator improves baroreflex sensitivity of spontaneously hypertensive rats

The present study sought to determine cardiovascular effects of aerobic training associated with diminazene aceturate (DIZE), an activator of the angiotensin converting enzyme 2, in spontaneously hypertensive rats (SHRs). Male SHRs (280–350 g) were either subjected to exercise training or not (sedentary group). The trained group was subjected to 8 weeks of aerobic training on a treadmill (five times a week, lasting 60 min at an intensity of 50–60% of maximum aerobic speed). In the last 15 days of the experimental protocol, these groups were redistributed into four groups: i) sedentary SHRs with daily treatment of 1 mg/kg DIZE (S+D1); ii) trained SHRs with daily treatment of 1 mg/kg DIZE (T+D1); iii) sedentary SHRs with daily treatment of vehicle (S+V); and iv) trained SHRs with daily treatment of vehicle (T+V). After treatment, SHRs were anesthetized and subjected to artery and femoral vein cannulation prior to the implantation of ECG electrode. After 24 h, mean arterial pressure (MAP) and heart rate (HR) were recorded; the baroreflex sensitivity and the effect of double autonomic blockade (DAB) were evaluated in non-anesthetized SHRs. DIZE treatment improved baroreflex sensitivity in the T+D1 group as compared with the T+V and S+D1 groups. The intrinsic heart rate (IHR) and MAP were reduced in T+D1 group as compared with T+V and S+D1 groups. Hence, we conclude that the association of exercise training with DIZE treatment improved baroreflex function and cardiovascular regulation.

Aspectos epidemiológicos, clínicos e anatomopatológicos da intoxicação por aceturato de diminazeno em cães / Epidemiological, clinical, and pathological aspects of diminazene aceturate toxicosis in dogs

Os aspectos epidemiológicos, clínicos e anatomopatológicos da intoxicação espontânea por aceturato de diminazeno foram estudados em 10 cães. Em todos os casos, os cães afetados demonstraram sinais de síndrome tálamo-cortical, principalmente alteração do nível de consciência, tetraparesia, rigidez extensora e crise convulsiva. Em alguns casos, os cães acometidos apresentaram sinais de síndrome cerebelar, como tremores musculares generalizados de alta frequência e baixa amplitude, e/ou de síndrome vestibular, como ataxia, inclinação de cabeça e quedas. Esses sinais ocorreram entre 24 e 48 horas após o uso do fármaco injetável por via intramuscular e se mantiveram até a morte ou eutanásia dos cães (entre 1 e 7 dias). Tais sinais clínicos refletiam encefalomalacia hemorrágica focal simétrica, que afetava a medula oblonga, a ponte, a medular do cerebelo, o tálamo, o mesencéfalo, os pedúnculos cerebelares e os núcleos da base. Esse artigo: 1) descreve e discute essa forma de intoxicação medicamentosa tão pouco citada na literatura internacional e desconhecida da maior parte dos clínicos e patologistas veterinários brasileiros, 2) estabelece critérios clínicos e anatomopatológicos para o seu diagnóstico e, principalmente, 3) atenta para os riscos da utilização desse princípio ativo na terapêutica canina.

The epidemiological, clinical, and pathological aspects of diminazene aceturate (DA) spontaneous toxicosis were evaluated in 10 dogs. All affected dogs developed signs of thalamic-cortical syndrome, characterized mainly by neurological changes in the conscience levels, tetraparesis, extensor stiffness, and seizures. In some cases there was also evidence of cerebellar syndrome, characterized by generalized muscle tremors (high-frequency and low-amplitude) and/or vestibular syndrome, characterized by or ataxia, head tilt, and falling. These clinical signs occurred between 24 and 48 hours following intramuscular administration of DA and persisted until spontaneous death or euthanasia occurred between 1 and 7 days after the onset of clinical signs. The mentioned clinical signs reflected lesions that consisted of focal symmetrical hemorrhagic encephalomalacia affecting medulla oblongata, pons, cerebellar medulla, thalamus, midbrain, cerebellar peduncles, and basal nuclei. This article (1) describes and discusses DA toxicosis in dogs, a poorly-described clinical entity that is unknown by most clinicians and pathologists in Brazil; (2) establishes the clinical and pathological criteria for the diagnosis of DA toxicosis in dogs; and (3) calls up the attention for the risks of using DA in dogs in clinical settings.

Effect of diminazene aceturate, levamisole and vitamin C combination therapy in rats experimentally infected with Trypanosoma brucei brucei

OBJECTIVE@#To investigate the effect of diminazene aceturate (DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei.@*METHODS@#Thirty adult male albino rats, randomly assigned into 6 groups (A-F) of 5 rats each were used. They were either infected with 1×10(6) trypanosomes intraperitoneally (groups A-E) or uninfected (group F). The different groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B-3.5 mg/kg DA and 7.5 mg/kg levamisole; group C-3.5 mg/kg DA and 100 mg/kg vitamin C; and group D-3.5 mg/kg DA and 7.5 mg/kg levamisole and 100 mg/kg vitamin C. Group E was left untreated. Parameters assessed include: rectal temperature, body weight changes, packed cell volume (PCV), Haemoglobin concentration (Hb), total leucocyte count (TLC) differential leucocyte count (DLC), parasitaemia, clinical signs and survivability.@*RESULTS@#Average pre-patent period of 5 days was recorded. Parasites in the blood were cleared in all treated groups (A-D) within 48 hours post treatment (PT). Untreated rats in group E died between 25 and 32 days post infection (PI). Relapse was not recorded in all the treated groups (A-D). The initial reduction in PCV, Hb, TLC and increases in rectal temperature following infection were reversed by the treatments. The rats that received drug combinations (groups B, C and D) showed faster and higher recovery rates than the uninfected control and group A.@*CONCLUSIONS@#Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei.

Cerebellar encephalopathy from diminazene aceturate (beneril) toxicity in a dog / 대한수의학회지

A 2-year old castrated male Alaskan malamute was referred with primary complaints of marked anemia, hemeglobinuria and depression. Laboratory tests revealed canine babesiois with severe anemia. The dog was treated by blood transfusion and beneril (diminazene aceturate, 3.5 mg/kg IM). Two days after Beneril injection, the dog suddenly showed ataxia progressing to paresis. MRI revealed irregularly diffused lesions in the cerebellum. The case was tentatively diagnosed as cerebellar encephalopathy caused by diminazene toxicity. The dog successfully recovered following steroid therapy.

Cerebellar encephalopathy from diminazene aceturate (beneril) toxicity in a dog / 대한수의학회지

A 2-year old castrated male Alaskan malamute was referred with primary complaints of marked anemia, hemeglobinuria and depression. Laboratory tests revealed canine babesiois with severe anemia. The dog was treated by blood transfusion and beneril (diminazene aceturate, 3.5 mg/kg IM). Two days after Beneril injection, the dog suddenly showed ataxia progressing to paresis. MRI revealed irregularly diffused lesions in the cerebellum. The case was tentatively diagnosed as cerebellar encephalopathy caused by diminazene toxicity. The dog successfully recovered following steroid therapy.

Immunostimulatory Effects of beta-glucan Purified from Paenibacillus polymyxa JB115 on Mouse Splenocytes

We investigated the effects of beta-glucan purified from Paenibacillus polymyxa JB115 on the viability and proliferation of splenocytes. Splenocytes play a critical role in host immunity. MTT assays and trypan blue exclusion tests revealed that beta-glucan significantly promoted the viability and proliferation of splenocytes over a range of concentrations. However, there was no specific subset change. beta-glucan protected splenocytes from cytokine withdrawal-induced spontaneous cell death. For further mechanistic studies, ELISA assay revealed that beta-glucan enhanced the expression of anti-apoptotic molecules and interleukin 7 (IL-7), a cytokine critical for lymphocyte survival. We also investigated the IL-2 dependency of beta-glucan-treated splenocytes to determine if treated cells could still undergo clonal expansion. In flow cytometric analysis, beta-glucan induced increased levels of the activation marker CD25 on the surface of splenocytes and beta-glucan-treated splenocytes showed higher proliferation rates in response to IL-2 treatment. This study demonstrates that beta-glucan can enhance the survival of splenocytes and provides valuable information to broaden the use of beta-glucan in research fields.

Immunostimulatory effects of BCG-CWS on the proliferation and viability of mouse spleen cells / 대한수의학회지

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been tried to use for immunotherapy. The CWS of Mycobacterium bovis bacillus Calmette-Guerin (BCG-CWS) was studied as an universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccine. Although a variety of biological activities of BCG-CWS have been studied, the effects of BCG-CWS on spleen cells are not fully elucidated. Using MTT assay and trypan blue exclusion test, we found that BCG-CWS significantly enhanced the viability and proliferation of cells. Multiple clusters, indicating proliferation, were observed in BCG-CWS-treated spleen cells and surface marker staining assay revealed that BCG-CWS promoted the proliferation of CD19+ B lymphocyte rather than CD4+ or CD8+ T lymphocyte. In addition, BCG-CWS up-regulated the expression of anti-apoptotic molecules such as bcl-2, bcl-xL. BCG-CWS increased the surface expression of CD25 and CD69 as well as IL-2 production of spleen cells, suggesting increased activation. Furthermore, BCG-CWS enhanced the antigen-specific cell proliferation and interferon-gamma production of spleen cells. Taken together, these results demonstrate the immunostimulatory effects of BCG-CWS on spleen cells via multiple mechanisms, providing valuable information to broaden the use of BCG-CWS in clinical and research settings.

Trypan Blue-Assisted Measurements of Anatomical Positions of the Superior Rectus Muscle and Superior Oblique Muscle in Enucleated Eyes

PURPOSE: To recognize the anatomical positions of the superior oblique muscle in enucleated eyes using trypan blue. METHODS: Twenty-two surgically-enucleated eyes of 11 bodies were studied. The shortest distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon, the distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion, and the greatest width of superior oblique tendon insertion were measured by caliper 3 consecutive times. The average values in each of the above 3 points were calculated, and values prior to and after trypan blue staining were compared. RESULTS: Prior to staining with trypan blue, the average distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon was 4.97 mm and the average distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion was 7.57 mm; after staining with trypan blue, the average values were 5.09 mm and 7.65 mm, respectively. There was no statistically meaningful difference in values prior to and after staining (p > 0.05). Prior to staining, the average value of the greatest width of the superior oblique tendon was 10.32 mm, and after staining with trypan blue, the average value increased to 10.76 mm. There was a statistically meaningful difference between the values (p = 0.02). CONCLUSIONS: Trypan blue staining helped to recognize the location and the width of the superior oblique tendon more precisely.

In vitro and in vivo antileishmanial efficacy of a combination therapy of diminazene and artesunate against Leishmania donovani in BALB/c mice / Estudo in vitro e in vivo da eficácia anti leishmaniótica de terapêutica combinada de Diminazene e Artesunate contra Leishmania donovani em camundongos Balb/c

The in vitro and in vivo activity of diminazene (Dim), artesunate (Art) and combination of Dim and Art (Dim-Art) against Leishmania donovani was compared to reference drug; amphotericin B. IC50 of Dim-Art was found to be 2.28 ± 0.24 µg/mL while those of Dim and Art were 9.16 ± 0.3 µg/mL and 4.64 ± 0.48 µg/mL respectively. The IC50 for Amphot B was 0.16 ± 0.32 µg/mL against stationary-phase promastigotes. In vivo evaluation in the L. donovani BALB/c mice model indicated that treatments with the combined drug therapy at doses of 12.5 mg/kg for 28 consecutive days significantly (p < 0.001) reduced parasite burden in the spleen as compared to the single drug treatments given at the same dosages. Although parasite burden was slightly lower (p < 0.05) in the Amphot B group than in the Dim-Art treatment group, the present study demonstrates the positive advantage and the potential use of the combined therapy of Dim-Art over the constituent drugs, Dim or Art when used alone. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.

A atividade in vitro e in vivo de Diminazene (Dim), Artezunate (Art) e a combinação Dim e Art (Dim-Art) contra Leishmania donovani foi comparada com a droga de referência Anfotericina B. IC50 da Dim-Art foi 2,28 ± 0,24 µg/mL enquanto aquelas de Dim e Art foram 9,16 ± 0,3 µg/mL e 4,64 ± 0,48 µg/mL respectivamente. O IC50 da Anfotericina B foi 0,16 ± 0,32 µg/mL contra a fase estacionária de promastigotas. A avaliação in vivo do modelo de L. donovani em camundongos Balb/c indicou que os tratamentos com a terapêutica de drogas combinadas em doses de 12,5 mg/kg por 28 dias consecutivos significantemente (p < 0,001) reduziu a carga parasitária no baço quando comparada a tratamentos com uma única droga dada nas mesmas dosagens. Embora a carga parasitária tenha sido levemente mais baixa (p < 0.05) no grupo Anfotericina B quando comparada com o grupo tratado Dim-Art, o estudo presente demonstra a vantagem positiva do uso potencial da terapêutica combinada Dim-Art sobre drogas como Dim ou Art quando usadas isoladamente. Posterior avaliação é recomendada para determinar a média de combinação mais eficaz dos dois compostos.

Optimal Media Conditions for the Detection of Extracellular Cellulase Activity in Ganoderma neo-japonicum

To determine the optimal media conditions for the detection of the extracellular cellulase activity in Ganoderma neo-japonicum, we varied three media conditions: dye reagent, pH, and temperature. We evaluated the use of four dyes, Congo red, phenol red, remazol brilliant blue, and trypan blue. To observe the effect of pH on the chromogenic reaction, we tested media ranging from 4.5 to 8.0. To research the effect of temperature on the clear zone and the fungus growing zone, we tested temperatures ranging from 15 to 35degrees C. On the whole, the best protocol called for Ganoderma neo-japonicum transfer onto media containing Congo red with a pH of 7.0, followed by incubation at 25degrees C for 5 days. Our results will be useful to researchers who study extracellular enzyme activity in Ganoderma neo-japonicum.

Role of Clusterin and Tumor Necrosis Factor Receptors on the Apoptosis of Prostate Cancer Cells / 대한남성과학회지

PURPOSE: In prostate cancer, the anti-apoptotic mechanism of sulfated glycoprotein-2 (clusterin) against tumor necrosis factor-alpha (TNF-alpha) receptors and the action of type 2 TNF-alpha receptor (TNFR2) were investigated. MATERIALS AND METHODS: TNF-alpha, agonistic-TNF type 1 receptor (TNFR1) antibody, agonistic-TNF-R2 antibody and their combination were treated in PC3 cell line with or without anti-clusterin. Cytotoxicity was assessed by trypan blue dye exclusion assay. By using flowcytometric analysis, the exact amount of apoptosis and their changes were assessed. RESULTS: Apoptosis was significantly increased in both agonistic-TNFR1 antibody and TNF-alpha treated cases after blocking the activity of clusterin. The more the anti-clusterin antibody added, the more the apoptosis occurred. The increase of total apoptosis was greater in TNF-alpha treated cells than in agonistic-TNFR1 antibody treated ones. However, there was no increase of apoptosis in agonistic-TNFR2 antibody and TNF-alpha with agonistic-TNFR2 antibody treated cases, respectively. CONCLUSIONS: Clusterin prevents TNF-alpha induced apoptosis by affecting TNFR1. The difference in degree of apoptosis between agonistic-TNFR1 antibody treated cells and TNF-alpha treated ones suggests the possibility of the action of TNFR2. It may be associated with affinity of TNF-alpha to the tumor cell surface.

Cryopreservation of Collected Peripheral Blood Hematopoietic Stem Cell Product with 5% DMSO by Adding Nontoxic Natural Cryoprotectants / 대한수혈학회지

BACKGROUND: Cryopreservation of hematopoietic stem cells has become an important process due to the therapeutic protocol, which includes stem cell transplantation after chemotherapy, for many hematological malignancies. The conventional medium contains 10% dimethyl sulfoxide (DMSO) as a cryoprotectant, but this has been reported to be related with many complications. We analyzed the usefulness of trehalose, catalase and zVAD-fmk for cryopreservation along with using a reduced concentration of DMSO to 5%. METHODS: Peripheral blood stem cells were frozen in 10% DMSO as a control and also in 5% DMSO with trehalose and catalase. After 3 weeks of storage in a liquid nitrogen tank, the viability of the thawed hematopoietic stem cells was measured using Trypan blue staining and 7-AAD analysis via conducting flow cytometry. The colony forming potential was assessed using methylcellulose culture. We measured the viability of cells in 5% DMSO medium with or without addition of 30 uM zVAD-fmk right after thawing, and we also did this 6 and 24 hours after incubation. RESULTS: Cryopreserved cells in 5% DMSO with trehalose and catalase showed similar survival (50.42%) compared with the control (49.78%). The viability of cells that were also treated with added zVAD-fmk showed a better result (13.12%) than without it (5.5%) after 24 hours of incubation. Colony forming assay showed similar colony formation in 5% DMSO with the natural cryoprotectants. CONCLUSION: According to the results, lowering the DMSO concentration to 5% is significant and we can expect better cell viability and prevent many side effects of high dose DMSO when adding natural cryprotectants in the cryopreservation medium or by adding caspase-inhibitor right after thawing.

Biodynamic parameters of micellar diminazene in sheep erythrocytes and blood plasma

In this work, we used a preparation of diminazene, which belongs to the group of aromatic diamidines. This compound acts on the causative agents of blood protozoan diseases produced by both flagellated protozoa (Trypanosoma) and members of the class Piroplasmida (Babesia, Theileria, and Cytauxzoon) in various domestic and wild animals, and it is widely used in veterinary medicine. We examined the behavior of water-disperse diminazene (immobilized in Tween 80 micelles) at the cellular and organismal levels. We assessed the interaction of an aqueous and a water-disperse preparation with cells of the reticuloendothelial system. We compared the kinetic parameters of aqueous and water-disperse diminazene in sheep erythrocytes and plasma. The therapeutic properties of these two preparations were also compared. We found that the surface-active substances improved intracellular penetration of the active substance through interaction with the cell membrane. In sheep blood erythrocytes, micellar diminazene accumulated more than its aqueous analog. This form was also more effective therapeutically than the aqueous analog. Our findings demonstrate that use of micellar diminazene allows the injection dose to be reduced by 30%.

Ejaculate characteristics of rabbits infected with Trypanosoma congolense and changes caused after treatment with Diminazene aceturate (Diminaveto®) / Características eyaculatorias de conejos infectados con Trypanosoma congolense y cambios causados después del tratamiento con Diminazeno aceturato (Diminaveto®)

The effects of experimental Trypanosoma congolense infection on the ejaculate of rabbits and changes caused after treatment with Diminaveto® were investigated using 24 New Zealand White rabbits (bucks). The bucks were housed singly in standard rabbit cages and fed on specialized ration containing 10 percent Protein supplement, grains, legume, salt and fresh water ad libitum during the study. Data on ejaculate characteristics were collected from all the bucks in the first phase (i.e. before infection) and in the second phase (i.e. during infection, with 4.8x10(5) Trypanosoma congolense, intraperitoneally). Similar data were collected from 12 randomly selected bucks treated with 7.0mg/kg Diminaveto® following reconstitution during the third phase. Data collected were analysed using the Paired T- Test and Analysis of Variance. The infection led to significant (P< 0.05) reduction in spermatozoa motility, concentration and mass activity, with a significant (P< 0.05) increase in percentage of sperm cells with morphological abnormalities. Treatment with Diminaveto® led to improvement in all ejaculate parameters investigated. However, it was observed that the ejaculate did not attain the "before-infection" status following treatment with Diminaveto®. The study showed that infection with Trypanosoma congolense in rabbits caused significant reduction in ejaculate characteristics. Treatment with Diminaveto® however led to improvement in the ejaculate though at a rate slower than that at which the infection caused the reduction.

Fue estudiado en 24 conejos Nuevo zelandeses blancos, machos, los efectos de la infección experimental de Trypanosoma congolense sobre la eyaculación y los cambios producidos después del tratamiento con Diminaveto® . Los machos fueron colocados individualmente en jaulas de conejos estándar. Durante el estudio fueron alimentados con ración especializada, con 10 por ciento de suplemento de proteínas, granos, legumbres, sal y agua fresca ad libitum. Los datos sobre las características de la eyaculación se obtuvieron de todos los machos en la primera fase (es decir, antes de la infección) y en la segunda fase (es decir, durante la infección, con 4,8x10(5) Trypanosoma congolense, por vía intraperitoneal). Datos similares se obtuvieron de 12 machos al seleccionados al azar, tratados con Diminaveto® 7,0mg/kg después de la reconstitución durante la tercera fase. Los datos fueron analizados mediante t de student y análisis de varianza. La infección fue significativa (P <0,05) habiendo reducción de la motilidad de los espermatozoides, la concentración y actividad de masas, con un efecto significativo (P <0,05) aumento en el porcentaje de espermatozoides con anomalías morfológicas. El tratamiento con Diminaveto® condujo a una mejoría en todos los parámetros investigados del eyaculado. Sin embargo, se observó que el eyaculado no alcanzó el "antes de la infección" tras el tratamiento con Diminaveto®. Además, el estudio mostró que la infección con Trypanosoma congolense en conejos causó una reducción significativa en las características del eyaculado. Sin embargo, el tratamiento con Diminaveto ® condujo a una mejoría en la eyaculación aunque a un ritmo más lento que en la infección causada por la reducción.

Development of Detection Methods for Cellulolytic Activity of Auricularia auricula-judae

To obtain basic information on the detection of cellulolytic activity in Auricularia auricula-judae, the influences of dye reagent, pH, and temperature were assessed. Chromogenic dye (congo red, phenol red, remazol brilliant blue, and trypan blue) was individually incorporated into a medium containing either carboxymethyl-cellulose, Avicel, or D-cellobiose as a polysaccharide carbon substrate. The other assessments utilized pHs ranging from 4.5 to 8.0 and temperatures from 15~35degrees C. Overall, when A. auricula-judae species were transferred onto media contained Congo red and adjusted pH 7.0 and then incubated at 25degrees C for 5 days, the clear zone indicative of cellulolytic activity was more pronounced.

Effects of Knockout Serum Replacement in the Culture Medium on the Proliferation of Porcine Fetal Fibroblasts In Vitro

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells (1.09 x 10(5) cells/ml). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR: 64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% (3.0 x 10(4) cells/ml) or 20% KSR (4.8 x 10(4) cells/ml) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.

Stimulatory Effects of Ginsan on the Proliferation and Viability of Mouse Spleen Cells

Ginsan is an acidic polysaccharide purified from Panax ginseng, a famous oriental herb. Although a variety of biological activities of ginsan have been studied, the effects of ginsan on spleen cells are not fully elucidated. We investigated the effect of ginsan on the viability and proliferation of spleen cells. Using Cell Counting Kit-8(R) solution and trypan blue solution, we found that ginsan significantly enhanced viability and proliferation. Multiple clusters, indicating proliferation, were observed in ginsan-treated spleen cells and, carboxyfluorescein succinimidyl ester and surface marker staining assay revealed that ginsan promoted proliferation from CD19+ B cells rather than CD4+ or CD8+ T cells. In addition, ginsan decreased the percentage of late apoptotic cells. Ginsan increased the surface expression of CD25 and CD69 as well as production of interleukin-2 from spleen cells, suggesting increased activation. Taken together, these results demonstrate that ginsan increases the viability and proliferation of spleen cells via multiple mechanisms, valuable information for broadening the use of ginsan in clinical and research settings.