Recombinant Human erythropoietin reduces viability of MCF-7 breast cancer cells from 3D culture without caspase activation.

Recombinant human erythropoietin (rHuEPO) is the erythropoiesis-stimulating hormone that is being used concurrently with chemotherapeutic drugs in the treatment of anemia of cancer. The effect of rHuEPO on cancer cells in 3-dimensional (3D) cultures is not known. The objective of the study was to determine the effect of rHuEPO on the viability of MCF-7 breast cancer cells from 2-dimensional (2D) and 3D cell cultures. The monolayer MCF-7 cells from 2D culture and MCF-7 cell from 3D culture generated by ultra-low adhesive microplate technique, were treated with 0, 0.1, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The effects of rHuEPO on MCF-7 cell viability and proliferation were determined using the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), neutral red retention time (NRRT), trypan blue exclusion assay (TBE), DNA fragmentation, acridine orange/propidium iodide staining (AO/PI) assays. The MCF-7 cells for 3D culture were also subjected to caspase assays and cell cycle analysis using flow cytometry. rHuEPO appeared to have greater effect at lowering the viability of MCF-7 cells from 3D than 2D cultures. rHuEPO significantly (p < 0.05) decreased viability and down-regulated the caspase activities of 3D MCF-7 cells in dose- and time-dependent manner. The cell cycle analysis showed that rHuEPO caused MCF-7 cells to enter the subG0/G1 phase. Thus, the study suggests that rHuEPO has a cytostatic effect on the MCF-7 breast cancer cells from 3D culture.

Infectious bronchitis associated with Escherichia coli infection in commercial broiler chickens: a case report.

OBJECTIVE: In this case report, we have investigated the infectious bronchitis (IB) virus (IBV) outbreak with the co-infection of Escherichia coli in 28-33-day-old broiler chickens in Malaysia. MATERIALS AND METHODS: A farmer complained that Cobb 500 chickens, raised in the open house, were having bloody diarrhea, open mouth breathing, non-uniform growth, and ruffled feathers. The mortality was about 100 birds (from about 7000 birds) per day. The sick birds were isolated and subjected to physical examination, postmortem, and histopathological analyses. Gross lesions were observed and recorded. The lung samples have proceeded with histopathological evaluations. The lungs, kidneys, trachea, air sac, and heart samples were collected to isolate bacteria and fungi through a series of conventional cultural methods, followed by molecular confirmation of the IBV. RESULTS: Postmortem examination revealed air sacculitis, hemorrhagic tracheitis, pulmonary congestion, fibrin deposition in the liver and air sac, hemorrhagic enteritis, and renomegaly. The bacterial culture and biochemical tests revealed E. coli in the lungs, trachea, liver, intestine, and kidney samples. However, no fungus could be isolated from those samples. Histological evaluation of lung samples demonstrated infiltration of inflammatory cells in the pulmonary tissues. Apart from this, reverse transcription-polymerase chain reaction confirmed the presence of avian coronavirus responsible for infectious bronchitis (IB). CONCLUSION: The chickens were diagnosed with IB concurrent with E.coli. The chickens exhibited typical nephropathogenic strain of IBV infection, causing high mortality.

Comparative Efficacy of Selected Phytobiotics with Halquinol and Tetracycline on Gut Morphology, Ileal Digestibility, Cecal Microbiota Composition and Growth Performance in Broiler Chickens.

The current experiment was designed to estimate the comparative efficacy of selected phytobiotics Persicaria odorata leaf meal (POLM) and Piper betle leaf meal (PBLM) with halquinol, and tetracycline in broiler chickens. The 150-day-old broiler chickens were randomly assigned to five dietary groups. The dietary supplementation groups were the basal diet (BD), which served as the negative control (NC), and BD + 0.2 g/kg tetracycline, which served as the positive control (PC); BD + 0.03 g/kg halquinol (HAL), BD + 8 g/kg POLM (Po8), and BD + 4 g/kg PBLM (Pb4) were the treatment groups. Growth performance, gut morphology, ileal digestibility, and cecal microbiota composition were measured. On day 21, the body weight gain (BWG) was enhanced (p < 0.05) in the broiler chickens fed on phytobiotics (Po8 and Pb4) relative to the NC group, however, on day 42 and in terms of overall growth performance, BWG was enhanced (p < 0.05 in diets (Po8, Pb4, HAL and PC) in comparison with the NC group. Conversely, feed conversion ratio (FCR) was recorded reduced (p < 0.05) in Pb4, Po8, HAL, and PC group in comparison with the NC group. Supplementation of phytobiotics (Po8 and Pb4), HAL and PC, positively improved the gut morphology compared to the NC group. Furthermore, the maximum (p < 0.05) villus height (VH) in duodenum and jejunum was observed in broilers fed on diet Pb4. Supplementation of phytobiotics, HAL and PC, improved (p < 0.05) the digestibility of dry matter (DM) (except for HAL), organic matter (OM), crude protein (CP), ether extract (EE), and ash compared to the NC group. Dietary supplementation of phytobiotics (Po8 and Pb4), HAL and PC, significantly reduced the E. coli, Salmonella, and Staphylococcus aureus (except for HAL) counts compared to the NC group. However, supplementation of Pb4 resulted in significantly decreased total anaerobic bacteria and Clostridium spp. counts compared to the NC group. In addition, supplementation of phytobiotics significantly increased the Lactobacillus count compared to HAL, PC, and NC groups. In conclusion, dietary supplementation of phytobiotics improved the gut morphology, positively modulated and maintained the dynamics of cecal microbiota with enhanced nutrient digestibility, thus, increased the growth performance. Based on current results, phytobiotics could be used as an alternative to AGPs for sustainable broiler chicken production.

Modelling the immunopathophysiology of Brucella melitensis and its lipopolysaccharide in mice infected via oral route of exposure.

Brucella melitensis is one of the leading zoonotic pathogens with significant economic implications in animal industry worldwide. Lipopolysaccharide, however, remains by far the major virulence with substantial role in diseases pathogenesis. Nonetheless, the effect of B. melitensis and its lipopolysaccharide on immunopathophysiological aspects largely remains an enigma. This study examines the effect of B.melitensis and its lipopolysaccharide on immunopathophysiological parameters following experimental infection using mouse model. Eighty four (n = 84) mice, BALB/c, both sexes with equal gender distribution and 6-8 weeks-old were randomly assigned into three groups. Group 1-2 (n = 72) were orally inoculated with 0.4 mL containing 109 CFU/mL of B. melitensis and its LPS, respectively. Group 3 (n = 12) was challenged orally with phosphate buffered saline and served as a control group. Animals were observed for clinical signs, haematological and histopathological analysis for a period of 24 days post-infection. We hereby report that B.melitensis infected group demonstrated significant clinical signs and histopathological changes than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1ß and IL-6) and antibody levels (IgM and IgG) with varying degrees of predominance in LPS infected group than B. melitensis infected group. For hormone analysis, low levels of progesterone, estradiol and testosterone were observed in both B. melitensis and LPS groups throughout the study period. Moreover, in B. melitensis infected group, the organism was re-isolated from the organs and tissues of gastrointestinal, respiratory and reproductive systems thereby confirming the infection and transmission dynamics. This report is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in a mouse model after oral inoculation with B. melitensis and its lipopolysaccharide.

Antibacterial and cytotoxic activities of the Syzygium polyanthum leaf extract from Malaysia.

BACKGROUND AND AIM: The increasing prevalence of drug resistance eventually leads scientist to discover new drugs that could solve the problem. Since ancient immemorial times, medicinal plants generally known as herbs were widely used in every culture throughout the world. In fact, currently up to 70,000 plant species have been screened for biological activities and about 70% ends up for commercialization. Therefore, this study was aimed to evaluate the potential cytotoxic and antibacterial effect of Syzygium polyanthum leaves which are local Malaysia plants, against 4T1 and MCF-7 mammary carcinoma cells, respectively, and also against bacteria causing mastitis in cows. MATERIALS AND METHODS: The cytotoxic effect of hydromethanolic extract of S. polyanthum against 4T1 and MCF-7 mammary carcinoma cells was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cells were treated with the concentration of extracts ranging from 15.63 µg/mL to 1000 µg/ml for 72 h, and the percentage of cell survivability was determined based on minimum concentration that was able to allow at least 50% growth of cancer cells (IC50) after 72 h. The antibacterial activity was tested against common bacteria causing mastitis in cow. The bacteria were isolated from milk samples. The antibacterial activity of the extract was determined by disk diffusion method and susceptibility test based on minimum inhibitory concentration (MIC). RESULTS: Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius were isolated from the milk samples that positive for mastitis. The MIC values range from 7.12 mm to 13.5 mm. The extract exhibits the widest zone of inhibition (13.5±0.20 mm) at 1000 mg/ml of concentrations. The extract relatively has low cytotoxicity effect against 4T1 and MCF-7 cells with IC50 values ranging from 672.57±59.42 and 126.05±50.89 µg/ml, respectively. CONCLUSION: S. polyanthum exerts weak antibacterial activity and cytotoxic effect to mammary carcinoma cells. The extract does not toxic to cells. However, further study is recommended, especially, this plant should be tested for in vivo.

Comparative experimental study of Brucella melitensis and its lipopolysaccharide in mouse model infected via subcutaneous route of exposure.

Brucella melitensis is a major zoonotic pathogen in which lipopolysaccharide (LPS) is believed to play a major role in the diseases pathogenesis. To study the immunopathophysiological aspects, we established a mouse model experimentally infected with whole cell of B. melitensis and its lipopolysaccharide via subcutaneous route of exposure. Eighty four mice, BALB/c, both sexes with equal gender distribution and 6-8 weeks-old were randomly assigned into 3 groups. Group 1 (n = 36) were subcutaneoulsy inoculated with 0.4 mL 109 of B. melitensis while group 2 (n = 36) were subcutaneously challenged with 0.4 mL 109 of LPS. Group 3 (n = 12) was challenged subcuatneously with phosphate buffered saline and served as a control group. Animals were observed for clinical signs, haematological and histopathological analysis for a period of 24 days post-inoculation. Our results revealed that B. melitensis infected group demonstrated significant clinical signs and histopathological evidence than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1ß & IL6), antibody levels (IgM & IgG) as early as 3 days post-infection with predominance in LPS infected group. For hormone analysis, low levels of progesterone, estradiol and testosterone were observed in both B. melitensis and LPS challenged groups throughout the study period. Moreover, in B. melitensis infected groups, the organism was re-isolated from the organs and tissues of gastrointestinal, respiratory and reproductive systems; thereby confirming the possible transmission of the disease dynamics. Moreover, LPS stimulated significantly the innate and acquired immune system without significant systemic dysfunction suggesting the potentiality of the protective properties of this component as an alternative vaccine for brucellosis infection. This report is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in mouse model after subcutaneous inoculation with B. melitensis and its lipopolysaccharide.

Comparative study of immunopathophysiological responses induced by B. melitensis and its lipopolysaccharide in mouse model infected via intranasal route of exposure.

In this study, we developed a mouse model and characterized the effects of intranasal inoculation of virulent Brucella melitensis strain 16M and its lipopolysaccharide (LPS). The effects of the exposure were compared with respective control groups. Both Brucella melitensis-infected and LPS-infected groups showed no significant clinical presentation with minor relevance in the mortality associated with the infection. In Brucella melitensis-infected group, significant histopathological changes in comparison to the LPS infected group with increase bacterial burden in the lungs, reproductive and reticuloendothelial organs were observed. However, both infected groups showed elevated levels of pro-inflammatory cytokine expression (IL-1ß and IL6) and antibody production (IgM an IgG) as early as 3 days post-infection with predominance in LPS infected group. In contrast, low levels of sex related hormonal changes was recorded in both infected groups throughout the experimental period. This is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in mouse model after intranasal inoculation with B. melitensis and its lipopolysaccharide. The study revealed a significant difference between infected and control groups with overlap in clinical, pathological, and immunological responses as well as sex related hormonal changes resulting from the infections.

Possible mechanisms of antinociception of methanol extract of Melastoma malabathricum leaves

ABSTRACT Melastoma malabathricum L., Melastomaceae, has been traditionally used to relieve diverse pain-related ailments. The objectives of the present study were to determine the antinociceptive activity of methanol extract of M. malabathricum leaves and to elucidate the possible mechanisms of antinociception involved using various rats' models. The extract (100, 250, and 500 mg/kg) was administered orally 60 min prior to subjection to the respective test. The in vivo acetic acid-induced abdominal constriction, formalin-induced paw licking, and hot plate tests were used as the models of nociception to evaluate the extract antinociceptive activity. Further studies were carried out to determine the role of opioid and vanilloid receptors, glutamate system and nitric oxide/cyclic guanosine phosphate (NO/cGMP) pathway in modulating the extract antinociceptive activity. From the results obtained, M. malabathricum exhibited significant (p < 0.05) antinociceptive activity in all the chemical- and thermal-induced nociception models. Naloxone (5 mg/kg), a non-selective opioid antagonist, failed to significantly affect the antinociceptive activity of MEMM when assessed using the abdominal constriction-, hot plate- and formalin-induced paw licking-test. M. malabathricum also significantly (p < 0.05) reversed the nociceptive response in capsaicin- and glutamate-induced paw licking test. Furthermore, only L-arginine (a nitric oxide precursor) alone, but not, NG-nitro-L-arginine methyl esters (L-NAME; an inhibitor of NO synthase), methylene blue (MB; an inhibitor of cGMP), or their combination thereof, significantly (p < 0.05) block the antinociceptive activity of M. malabathricum. In conclusion, M. malabathricum exerted a non-opioid antinociceptive activity at the central and peripheral levels partly via the inhibition of vanilloid receptors and glutamatergic system, and activation of the NO-mediated/cGMP-independent pathway.

Immuno-pathophysiological responses of mouse model to experimental infection with Brucella melitensis and its lipopolysaccharides via intraperitoneal route.

Brucella melitensis is one of the major zoonotic pathogens with significant economic implications worldwide. The pathogenicity is complex and not always well understood. Lipopolysaccharide (LPS) remains the major virulent factor of B. melitensis and responsible for the mechanism by which the pathogen causes its deleterious effects. In this study, 84 mice of 6-8 weeks old of both sexes were divided equally into 3 groups; namely Brucella melitensis infected group, lipopolysaccharide (LPS) infected group and control group. The former two groups contained 36 mice each with equal gender distribution. The control group consisted of 12 mice only. Animals in B. melitensis infected group, a single inoculum of 0.4 ml containing 109 of B. melitensis were intraperitoneally challenged while animals in LPS group, a single dose of 0.4 ml containing LPS extracted from the B. melitensis were intraperitoneally inoculated. Animals in control group received intraperitoneally, a single dose of 0.4 ml phosphate buffered saline (PBS) of pH7. Animals that were infected intraperitoneally with B. melitensis demonstrated significant clinical presentation; gross and histo-pathological evidence than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1ß and IL6), antibody levels (IgM an IgG) as early as 3 days post-infection with predominance in LPS infected group. In contrast, low levels of sex related hormonal changes in which LPS infected group showed the least concentration were also detected throughout the experimental period. In conclusion, B. melitensis can be transmitted via gastrointestinal, respiratory and reproductive tract. Moreover, LPS stimulated significantly the innate and acquired immune system without significant systemic dysfunction, suggesting potentiality of the protective properties of this component as alternative vaccine for brucellosis infection.

Anti-inflammatory and anti-pyretic properties of Spirulina platensis and Spirulina lonar: a comparative study.

Spirulina spp. is a blue-green algae belongs to the family of Oscillatoriaceae, which having diverse biological activity. The aim of this current study was to evaluate and compare the anti-pyretic and anti-inflammatory activity of Spirulina platensis/SP and Spirulina lonar/SL extracts. In the anti-pyretic study, the ability to reduce the rectal temperature of rats induced pyrexia with 2g/kg Brewer's Yeast (BY) was performed. Rats were dosed either 2 or 4 mg/kg SP or SL. Rectal temperature was taken every hour for 8 hours. Results shown that there were significant dose-dependent (p<0.05) reduction of both treatments. However, SP treatment revealed faster reduction in rectal temperature. For anti-inflammatory activity, the reduction in the volume of paw edema induced by Prostaglandin E2 (100 IU/rat intraplantar) was measured. Rats were dosed orally with 2 or 4 mg/kg SP or SL. The paw edema was measured every 30 minutes for 4 hours using plethysmometer. Results had shown a significant dose dependent reduction in diameter of paw edema (p<0.05). The finding suggests that SP and SL extracts have anti-pyretic and anti-inflammatory properties. However, SP was found to be more effective than SL as anti-pyretic and anti-inflammatory agent.

Two new chemical constituents from the stem bark of Garcinia mangostana.

A detailed chemical study on the ethyl acetate and methanol extracts of the stem bark of Garcinia mangostana resulted in the successful isolation of one new prenylated xanthone, mangaxanthone B (1), one new benzophenone, mangaphenone (2), and two known xanthones, mangostanin (3) and mangostenol (4). The structures of these compounds were elucidated through analysis of their spectroscopic data obtained using 1D and 2D NMR and MS techniques.

Chemical constituents and antihistamine activity of Bixa orellana leaf extract.

BACKGROUND: Bixa orellana L. has been traditionally used in Central and South America to treat a number of ailments, including internal inflammation, and in other tropical countries like Malaysia as treatment for gastric ulcers and stomach discomfort. The current study aimed to determine the major chemical constituents of the aqueous extract of B. orellana (AEBO) and to evaluate the antihistamine activity of AEBO during acute inflammation induced in rats. METHODS: Acute inflammation was produced by subplantar injection of 0.1 mL of 0.1% histamine into the right hind paw of each rat in the control and treatment groups. The degree of edema was measured before injection and at the time points of 30, 60, 120, 180, 240 and 300 min after injection. Changes of peritoneal vascular permeability were studied using Evans blue dye as a detector. Vascular permeability was evaluated by the amount of dye leakage into the peritoneal cavity in rats. To evaluate the inhibitory effect of AEBO on biochemical mediators of vascular permeability, the levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) were determined in histamine-treated paw tissues. The major constituents of AEBO were determined by gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: AEBO produced a significant inhibition of histamine-induced paw edema starting at 60 min time point, with maximal percentage of inhibition (60.25%) achieved with a dose of 150 mg/kg of AEBO at 60 min time point. Up to 99% of increased peritoneal vascular permeability produced by histamine was successfully suppressed by AEBO. The expression of biochemical mediators of vascular permeability, NO and VEGF, was also found to be downregulated in the AEBO treated group. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the major constituent in AEBO was acetic acid. CONCLUSIONS: The experimental findings demonstrated that the anti-inflammatory activity of AEBO was due to its inhibitory effect on vascular permeability, which was suppressed as a result of the reduced expression of biochemical mediators (NO and VEGF) in tissues. Our results contribute towards the validation of the traditional use of Bixa orellana in the treatment of inflammatory disorders.

Free radical scavenging activity of conjugated linoleic acid as single or mixed isomers.

CONTEXT: Conjugated linoleic acids (CLAs) are a mixture of positional and geometric isomers of linoleic acid (LA) and believed to have many positive biological activities. OBJECTIVE: The present study was undertaken to assess the antioxidant activity of cis-9, trans-11 and trans-10, cis-12 as single or mixed CLA isomers at two ratios, 1:6 and 1:13 (trans-10, cis-12/cis-9, trans-11). MATERIALS AND METHODS: A microplate reader was used to determine the free radical scavenging properties of CLAs against DPPH radical in ethanol. RESULTS: The kinetic reactions of CLA-DPPH(•) showed that all tested CLAs have exerted radical scavenging activities in a dose-dependent manner and observed to immediately react and quench DPPH radicals at all tested levels and no lag phase was noticed in CLA-DPPH(•) reactions. The median inhibitory concentration (IC50) value for cis-9, trans-11 CLA was observed to be more effective than other tested CLA. Total antioxidant capacity (TAC) of all tested CLAs were less effective radical scavengers as compared to vitamin E and butylated hydroxytoluene, although all tested CLAs were quenched a high amount (P < 0.05) of DPPH free radicals. DISCUSSION AND CONCLUSION: All tested CLAs have the ability to directly react and quench DPPH free radicals in ethanol. Furthermore, trans-10, cis-12 CLA has greater maximal efficacy than other tested CLAs as free radical scavenger, while cis-9, trans-11 CLA is the most potent isomer to directly react and quench free radicals at low concentrations in the system, suggesting that the free radical scavenging activity of CLA isomers may contribute to their diverse biological activities.