Stem cell intervention ameliorates epigallocatechin-3-gallate/lipopolysaccharide-induced hepatotoxicity in mice.

Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor ß1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.

Oxidized low-density lipoprotein and tissue factor are involved in monocrotaline/lipopolysaccharide-induced hepatotoxicity.

These studies were aimed at characterizing an animal model of inflammation-induced hepatotoxicity that would mimic features of idiosyncratic liver toxicity observed in humans. An attempt was made to identify oxidative damage and the involvement of coagulation system in liver after monocrotaline (MCT) administration under the modest inflammatory condition induced by lipopolysaccharide (LPS) exposure. Mice were given MCT (200 mg/kg) or an equivalent volume of sterile saline (Veh.) po followed 4 h later by ip injection of LPS (6 mg/kg) or vehicle. Mice co-treated with MCT and LPS showed increased plasma alanine aminotransferase (ALT), decrease in platelet number, and a reduction in hematocrit. Accumulation of oxidized low-density lipoprotein (ox-LDL) was remarkably higher in the liver sections of mice co-treated with MCT and LPS compared to those given MCT or LPS alone. A similar trend was observed in the expression of CXCL16 receptor in the same liver sections. Elevated expression of tissue factor (TF) and fibrinogen was also observed in the liver sections of MCT/LPS co-treated mice. The in vitro results showed that incubation of HepG2 cells with CXCL16 antibody strongly diminished uptake of ox-LDL. Expression of ox-LDL, CXCL16, and TF represents an early event in the onset of hepatotoxicity induced by MCT/LPS; thus, it may contribute to our understanding of idiosyncratic liver injury and points to potential targets for protection or intervention.

Benzothieno[3,2-b]quinolinium and 3-(phenylthio)quinolinium compounds: Synthesis and evaluation against opportunistic fungal pathogens.

Substitution around 5-methyl benzothieno[3,2-b]quinolinium (2) ring system was explored in order to identify positions of substitution that could improve its antifungal profile. The 3-methoxy (10b) was active against C. albicans, C. neoformans, and A. fumigatus and the 4-chloro (10f) analog showed moderate increases in anti-cryptococcal and anti-aspergillus activities. The effectiveness of 10b and 10f were validated in murine models of candidiasis and cryptococcosis, respectively. The efficacy of 10f in reducing brain cryptococcal infection and its observation in the brain of mice injected with this quaternary compound confirm the capacity of these compounds to cross the blood-brain barrier of mice. Overall, several of the chloro and methoxy substituted compounds showed significant improvements in activity against A. fumigatus, the fungal pathogen prevalent in patients receiving organ transplant. Opening the benzothiophene ring of 2 to form 1-(5-cyclohexylpentyl)-3-(phenylthio)quinolinium compound (3) resulted in the identification of several novel compounds with over 50-fold increases in potency (cf. 2) while retaining low cytotoxicities. Thus, compound 3 constitutes a new scaffold for development of drugs against opportunistic infections.

The in vitro effect of aqueous extract of Nigella sativa seeds on nitric oxide production.

The in vitro effect of aqueous extract of Nigella sativa seeds on nitric oxide (NO) production by murine macrophages was studied. Murine peritoneal macrophages were pre-incubated with the extract and then activated with Escherichia coli lipopolysaccharride. NO production was measured after 24 hours by spectrophotometry. The plant extract caused a dose-dependent decrease in NO production. Dialyzed preparation of the extract did not affect NO production. However, the boiled fraction of the extract resulted in a dose-dependent inhibition of NO apparently comparable to that of the whole extract. These results indicate that the aqueous extract of N. sativa seeds exhibits an inhibitory effect on nitric oxide production by murine macrophages and the active component(s) is/are non-protein in nature. In view of the fact that nitric oxide is a pro-inflammatory mediator, this study validates the traditional use of the Nigella sativa seeds for the treatment of rheumatism.

The in vivo antifungal activity of the aqueous extract from Nigella sativa seeds.

The effect of an aqueous extract of Nigella sativa seeds was studied on candidiasis in mice. An intravenous inoculum of Candida albicans produced colonies of the organism in the liver, spleen and kidneys. Treatment of mice with the plant extract (6.6 mL/kg equivalent to 5 mg of estimated protein, once daily for 3 days) 24 h after the inoculation caused a considerable inhibitory effect on the growth of the organism in all organs studied. A 5-fold decrease in Candida in kidneys, 8-fold in liver and 11-fold in spleen was observed in the groups of animals post-treated with the plant extract. Histopathological examination of the respective organs confirmed these findings. These results indicate that the aqueous extract of Nigella sativa seeds exhibits inhibitory effect against candidiasis and this study validates the traditional use of the plant in fungal infections.

Nitric oxide mediated effect of cyclo-oxygenase inhibitors.

OBJECTIVES: Non-Steroidal anti-inflammatory drugs (NSAIDS) have long been used as anti-inflammatory agents, yet their mode of action is not entirely clear. The inhibitory effects of NSAIDS on prostaglandin production can only partly explain their anti-inflammatory actions. This study was aimed at defining the role of cycl-oxygenase (COX) inhibitors on nitric oxide (NO) production in murine macrophages in vitro. METHODS: Murine macrophages were obtained from the peritoneum and after exposure, in vitro to lipopolysaccharide (LPS) produced nitrite, measured after 24 hours by Griess reaction. The macrophages were pre-incubated with aspirin or indomethacin before activation with LPS. RESULTS: Treatment with aspirin resulted in an increase in nitric oxide production. A similar response was obtained with indomethacin treatment. CONCLUSION: This study shows that COX inhibitors significantly increase NO production in murine macrophages in vitro and this may be one of the mechanisms by which they exert their anti-inflammatory effects.

Changing pattern of antimicrobial susceptibility of organisms causing community acquired urinary tract infections.

OBJECTIVE: To assess common organisms causing Urinary Treat Infection (UTI) in this community and to see antimicrobial susceptibility pattern of these isolates. DESIGN: Prospective study on urine samples. SETTING: Tertiary care hospital in Karachi. METHODS: Over a period of 8 years (1990-97) 9,892 urine samples grew significant bacteriuria for various organisms. All Gram negative rods and entercocci was identified by using API 20E and API 32 strips respectively. Staphylococci were identified by catalase, coagulase and D'Nase tests. Antimicrobial sensitivity testing of all isolates was performed on Diagnostic Sensitivity Test plates by Kerby Bauer method. The discs used were ampicillin, trimethoprim-sulfamethoxazole, cefotaxime, ceftriaxone, aztreonam, ofloxacin, carbenicillin, amikacin, gentamicin, penicillin, clindamycin, methicillin, vancomycin, ceftazidime, cefuroxime, Nalidixic acid, pipemedic acid and Nitrofurantoin. RESULTS: Our results indicate that E. coli and Klebsiella aerogenes are the most common organisms causing UTI in this community. Other organisms involved are Pseudomonas aeroginosa, Enterobacter species, Enterococcus, Proteus mirabillus, Staphylococcus aureus and Staphylococcus saprophyticus. Organisms resistant to various antimicrobial agents such as gentamicin, Amikacin, Ofloxacin, Cefotaxime and Ceftazidime are increasing. CONCLUSION: In conclusion, E. coli and Klebsiella aerogenes are the most common organisms causing UTI in this community. Pattern of antibiotic susceptibility to first line antibiotics is changing. Antimicrobial susceptibility testing of all isolates is crucial for the treatment of UTI.

Vitamin E and beta-carotene affect natural killer cell function.

Vitamin E supplementation has been shown to contribute in immunoregulation, antibody production, and resistance to implanted tumors. Similarly beta-carotene has been shown to down-regulate growth factors which contribute towards proliferation of pre-malignant cells. We embarked upon a study to evaluate the effect of vitamin E and beta-carotene on natural killer (NK) cells, which perform tumor surveillance role in the mammalian body. Mouse splenocytes or human peripheral blood lymphocytes were used as NK cells with murine YAC-1 lymphoma or human K-562 lymphoma cells, respectively, as target cells. The NK cells were treated with vitamin E or beta-carotene while target cells were labeled with sodium 51chromate. Both cell types were then reacted for 4 hours. The NK cell tumorolytic activity was measured by the chromium release assay. Oral administration of alpha-tocopherol at a dose of 100 mg/d in mice showed a significant increase in NK cell activity. Similarly, treatment of NK cells with alpha-tocopherol in vitro at doses 0.5 mg/ml, 1-0 mg/ml, and 2.0 mg/ml increased the tumorolytic activity of NK cells. Tocotrienol showed a similar response at ten times lower dose. When NK cells were treated with varying concentrations of palm vitee (mixture of alpha-tocopherol and tocotrienol), maximum effect was observed at the dose mixture of 12 micrograms and 24 micrograms alpha-tocopherol and tocotrienol, respectively. When murine NK cells were treated in vitro with beta-carotene at doses ranging from 2 ng/mg to 200 ng/ml, a decrease in tumorolytic effect was observed. However, human NK cells after treatment with beta-carotene at doses ranging from 0.1 microgram/ml to 10 micrograms/ml showed a significant increase in tumorolytic function. NK cells were also obtained from mice that had been parenterally administered beta-carotene and alpha-tocopherol. These experiments showed no significant increase in the NK cell function.

Prevalence of chlamydial infection in females attending antenatal and family planning clinics in Karachi Pakistan.

A prospective study was undertaken to investigate the relative prevalence of Chlamydia trachomatis in asymptomatic pregnant women of 2 socioeconomic groups and those attending the family planning clinics. Group 1 consisted of women attending the antenatal clinics of the Aga Khan University Hospital which caters to the affluent strata of our society (n = 100). Group 2 comprised women attending the antenatal clinics of Lady Dufferin Hospital which provides free obstetric care to women belonging to the lower socioeconomic groups of Karachi (n = 100). Group 3 consisted of sexually active women attending the family planning clinics of Lady Dufferin Hospital (n = 100). Endocervical swabs were taken from women assigned to each group. Chlamydiazyme, an enzyme linked immunoassay, was used to detect chlamydia antigen. The positive samples were retested by using the direct fluorescent monoclonal antibody technique. Chlamydia positive patients and their sexual partners were treated with Erythromycin stearate 500 mg 8-hourly for 7 days. These patients were retested after antibiotic therapy to assess the efficacy of the therapy. In groups 1 and 2, 2% and in Group 3, 12% of the females tested positive. Selective screening of sexually active women for chlamydial infection is advocated as a cost-effective public health measure.

Pharmacokinetics of aditoprim and trimethoprim in buffalo calves.

Pharmacokinetic parameters of two antifolates, trimethoprim and aditoprim, were studied in buffalo calves. The elimination half-life of aditoprim (6.14 h) was nearly twice as long as that of trimethoprim (3.08 h) and compares well with values observed in heifers. This longer half-life of aditoprim is a result of its much larger distribution volume (four to five times larger) because the clearance of aditoprim was about twice as high as that of trimethoprim. The longer half-life of aditoprim is expected to give a longer duration of in vivo bacteriostatic activity than that of trimethoprim.

Comparative yield of Salmonella typhi from blood and bone marrow cultures in patients with fever of unknown origin.

Over three years, a comparative study on 100 selected patients with fever of unknown origin was undertaken to determine the yield of Salmonella typhi from their blood and bone marrow cultures. The results indicate that in patients who had an infection with S typhi the organism was isolated from the bone marrow in all of them and from the blood in only 66%. This suggests that bone marrow cultures may be attempted when blood cultures are negative for bacterial growth after three to four days of incubation.

Therapeutic implications of ofloxacin in the treatment of typhoid fever caused by multiply resistant Salmonella typhi.

The antibiotic of choice for the treatment of typhoid fever in most parts of the world is still chloramphenicol. Ampicillin and cotrimoxazole have been used in recent years. Selection of antimicrobials for therapy has been complicated by the emergence of Salmonella typhi strains resistant to the above mentioned antibiotics. Blood and/or bone marrow cultures of 30 adult patients grew S. typhi that was resistant to chloramphenicol, ampicillin and cotrimoxazole. However, these strains were sensitive to cefotaxime, ceftrioxone, aztreonam and ofloxacin. Ofloxacin 400 mg twice a day was given orally to these patients for 14 days. All patients recovered with no untoward side effect. We concluded that ofloxacin can be used as a drug of choice for typhoid fever, in those adult patients who are infected with S. typhi resistant to chloramphenicol, ampicillin and cotrimoxazole.

In vivo efficacy of antifungal oxoaporphine alkaloids in experimental disseminated candidiasis.

The efficacy of three antifungal oxoaporphine alkaloids, liriodenine, liriodenine methiodide, and oxoglaucine methiodide, was determined in a mouse model of disseminated candidiasis. Mice infected with a lethal dose of Candida albicans NIH B311 were administered varying doses of each drug intraperitoneally or intravenously 7 hr postinfection. Reductions in the number of colony-forming units (CFU) recovered per milligram of kidney tissue were observed in drug-treated animals compared to vehicle-treated control mice. Significance was determined by the Wilcoxon nonparametric rank sum test. Intravenous administration of both liriodenine and liriodenine methiodide resulted in a significant reduction in the number of recovered CFU, while there was no significant response to treatment with oxoglaucine methiodide.

Efficacy of a streptomycin-dependent, live Pasteurella haemolytica vaccine against challenge exposure to Pasteurella haemolytica in cattle.

A streptomycin-dependent, live Pasteurella haemolytica vaccine was given in 1 or 2 doses to 2 groups of weaned calves; 2 other groups of calves were not vaccinated. All calves in the vaccinated groups and calves in 1 of the nonvaccinated groups were stressed by transport, intratracheally inoculated with bovine herpesvirus type-1 (Cooper strain), and then intratracheally inoculated with P haemolytica type A1. The 4th group of calves (nonvaccinated controls) was not stressed and were not intratracheally inoculated with virus or bacteria. Mean daily weight gains, total clinical sign scores, lung lesion scores, plasma fibrinogen concentrations, and antibody titers against P haemolytica were determined at various intervals. Calves that had been vaccinated twice had greater mean daily weight gains and lower total clinical sign scores and lung lesion scores than did nonvaccinated, challenge-exposed calves, but the difference was not significant (P greater than 0.05). Calves vaccinated once had the greatest mean daily weight gains, the lowest total clinical sign scores, and the lowest lung lesion scores when compared with the other 2 challenge-exposed groups of calves. Mean daily weight gains and total clinical sign scores of calves vaccinated once were significantly different (P less than 0.05) than those of calves vaccinated twice. Nonvaccinated, nonchallenge-exposed control calves did not develop clinical signs of disease, did not develop lung lesions, and had consistently positive daily weight gains, and had scores in these areas that were significantly different (P less than 0.05) from those of all challenge-exposed groups of calves. Increases in plasma fibrinogen concentrations corresponded to infection with P haemolytica.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of subacute marijuana smoke inhalation on experimentally induced dermonecrosis by S. aureus infection.

Dermonecrosis was induced in ICR mice by subcutaneous implantation of Staphylococcus aureus absorbed onto sterile cotton pellets. This model was used to assess the effects of marijuana smoke, marijuana placebo smoke and delta 9-tetrahydrocannabinol (delta 9-THC) on the local immune response to bacterial infection. Mice were exposed to 40 or 80 "puffs" of marijuana smoke, marijuana placebo smoke or air daily for 4 consecutive days. The estimated dose of delta 9THC per day generated from 40 or 80 puffs of marijuana smoke was 3.2 and 6.4 mg/kg, respectively. A group of sentinel (Shelf) control mice were included in each experiment. The necrotic index (NI) of mice exposed to 40 or 80 puffs of marijuana smoke were 67% and 44% of control, respectively. Air exposed mice showed a necrotic index comparable to the shelf control group. In chronically (60 days) exposed mice (80 puffs per day) the necrotic index was about 12% of control, while air-exposed mice were about 40% of control. Placebo marijuana smoke exposed mice had a NI comparable to that of marijuana smoke exposed mice which suggested that the reduction in NI was unrelated to the pychomimetic component delta 9THC. To further explore which of the constituents of marijuana were responsible for the decreased NI, the ethanol extract from marijuana leaves was partioned between water (cannabinoid free) and chloroform (cannabinoid rich). Injection of the cannabinoid free fraction produced comparable decrease in the NI as observed with whole marijuana smoke, while the cannabinoid rich fraction produced no effect. delta 9THC at a dose of 10 mg/kg per day did not alter the NI.

Spectrophotometric evaluation of carboxyhemoglobin in blood of mice after exposure to marijuana or tobacco smoke in a modified Walton horizontal smoke exposure machine.

Carboxyhemoglobin (COHb) values were determined in mice exposed to varying amounts of marijuana and tobacco cigarette smoke utilizing a spectrophotometric technique. Mice were exposed to smoke inhalation in a modified Walton horizontal smoke exposure machine, whereby rodents can be exposed to multiples of 1-min smoke exposure cycles. Smoke exposure was intermittent; during the first 30 sec of each 1-min cycle, the subjects were exposed to smoke diluted either 1:10 or 1:5 with air. During the second half of the cycle the animals were given fresh air. There was a positive linear relationship between COHb values obtained and the number of puffs of marijuana smoke administered via either 2, 4, 6, or 8 "puffs" of marijuana smoke. COHb levels in plasma did not increase in animals given multiple 8-puff episodes of smoke daily as long as a 60-min period was interposed between smoking episodes. COHb values in mice exposed to tobacco smoke were significantly higher than those in mice receiving equal numbers of exposures to marijuana smoke. Mean COHb values of mice receiving 8 consecutive puffs of marijuana smoke were 18.6 and 22.0% saturation, but CO was rapidly cleared from the blood. This rapid clearance suggests that the binding affinity of CO for mouse hemoglobin may be be weaker than that of human hemoglobin. Mice similarly exposed to 6 or 8 puffs of tobacco smoke had mean COHb values of 24.6 and 28.5% saturation, respectively. No acute lethal effects were observed in mice receiving multiple daily episodes of 8 puffs per episode of marijuana smoke, whereas mice exposed to a single 8-puff episode of tobacco smoke suffered about 50% acute lethal effects.

The influence of opsonins on the bactericidal effect of bovine alveolar macrophages against Pasteurella multocida.

Monolayers of bovine alveolar macrophages were infected with smooth and rough strains of Pasteurella multocida to determine their bactericidal function. Heat inactivated normal bovine serum, fresh bovine serum, specific immune serum and a combination of specific immune serum and fresh bovine serum were used as opsonins. A substantial difference in the bactericidal effect of macrophages against the logarithmic and stationary phase p. multocida was observed. Opsonization of the rough strain of P. multocida with the combination of specific immune and fresh serum increased their association with alveolar macrophages compared to those opsonized with either normal serum, fresh serum or specific immune serum. Opsonization of the smooth strain of P. multocida with normal bovine serum resulted in substantially fewer bacteria associated with macrophages at time zero, compared with other opsonins. Regardless of the kind of opsonins used in these experiments, the number of the rough strain of P. multocida killed by macrophages was always higher than those of the smooth strain.

Experimentally induced caseous lymphadenitis in goats.

Caseous lymphadenitis was experimentally induced in goats by inoculating 1 x 10(6) Corynebacterium pseudotuberculosis by 3 routes: subcutaneous, intradermal, and submucosal. The incubation period for the development of abscesses in the present study ranged from 41 to 147 days, the average being 95.2 days. The shedding period for C pseudotuberculosis from opened abscesses ranged from 9 to 37 days, with an average of 20.3 days. The infection spread to mediastinal lymph nodes in 1 goat and to lumbar lymph nodes in 5 goats. None of the goats had abscesses in the mesenteric lymph nodes. Corynebacterium pseudotuberculosis was recovered from all the abscessed lymph nodes but not from the feces or nasal secretions.