P-coumaric acid ameliorates fipronil induced liver injury in mice through attenuation of structural changes, oxidative stress and inflammation.

Fipronil is a broad-spectrum phenylpyrazole insecticide and has been used effectively in the agriculture. Due to its widespread use and bioaccumulation in the environment, it possesses significant threat to human and animals. P-coumaric acid is a natural dietary polyphenolic compound that has anti-oxidant and anti-inflammatory property. The present study was aim to investigate the ameliorative effect of p-coumaric acid on fipronil induced liver injury. The mice were divided into five groups (SHAM, FPN, FPN/PCA/50, FPN/PCA/100 and PCA/100) and challenged with fipronil @ 25 mg/kg bw (half of LD50). Haematological, liver function biomarkers (ALT, AST, ALP, GGT), biochemical parameters (MPO, oxidative, nitrosative stress and anti-oxidant enzyme activity), levels of serum and liver inflammatory cytokines (TNF-α, IL-1ß and IL-10), histopathology were monitored. Fipronil administration caused a significant increase in liver enzymes with concomitant significant increase in inflammatory cytokines (TNF-α, IL-1ß, IL-10) and myeloperoxidase activity. A significant increase in oxidative stress (lipid peroxidation, nitric oxide) as well as down regulation of anti-oxidant enzymes like superoxide dismutase (SOD) and catalase (CAT) along with histopathological changes such as microsteatosis, hypertrophy of the hepatocytes and necrosis were observed on fipronil administration. Administration of p-coumaric acid against fipronil caused decreased serum liver enzymes, inflammatory cytokines, myeloperoxidase activity and oxidative stress along with improvement in anti-oxidant enzyme levels and structural changes induced by fipronil. Thus p-coumaric acid ameliorates the FPN induced liver injury in mice through attenuation of structural changes, oxidative stress, and inflammation.

Effect of feeding graded doses of citrinin on apoptosis and oxidative stress in male Wistar rats through the F1 generation.

The objective of the present study was to study the effect of graded doses of citrinin (CIT) on apoptosis and oxidative stress in male Wistar rats till F1 generation. The animals were divided into four groups comprising 25 males and 25 females each, that is, group I: 1 ppm CIT; group II: 3 ppm CIT; group III: 5 ppm CIT; and group IV was kept as a control. The male and female animals of all the groups were kept separately and were fed basal rations containing the above-mentioned concentrations of CIT for 10 weeks. After 10 weeks, male and female animals of respective groups were kept for mating (one male/two females). After getting 10 pregnant females, the males were killed. These 10 pregnant females were allowed to give birth to young ones (F1 generation) naturally which were fed CIT in the above-mentioned doses till the age of 6 weeks and then were killed. Apoptosis was analysed in kidneys, liver and testes by DNA ladder pattern, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labelling assay and Bcl-2/Bax ratio. Besides, tissue oxidative stress was also analysed. It was concluded in the present study that CIT induces its toxic effects till F1 generation, and apoptosis and oxidative stress both play a very important role in toxicity. The effect of CIT was observed in a dose-dependent manner. However, in kidneys, both the mechanisms (apoptosis and oxidative stress) play their role in inflicting renal damage, while in liver only reactive oxygen species play a major role. Finally, the CIT toxicity did not lead to apoptosis and oxidative stress in male gonads till F1 generation.

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 µg/mL and 6.5 µg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 µg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials.

Amelioration of bleomycin induced pulmonary fibrosis by administration of Salvianolic acid B in mice.

Pulmonary fibrosis is the end­stage manifestation of wide range of respiratory diseases and during pulmonary fibrosis, pulmonary inflammation and epithelial­mesenchymal transition (EMT) play important roles. Salvianolic acid B (SAB) from the herb Salviae miltiorrhiza has been reported to possess an excellent anti­inflammatory, antifibrotic and antioxidant activity. The present study aimed to investigate the ameliorative effect of SAB on bleomycin induced pulmonary fibrosis in mice. Adult albino mice were divided as SHAM/control group (saline alone), BLM group (bleomycin @ 1mg/kg intratracheally once) and SAB groups (BLM challenged once and SAB administration in three dosages @ 5, 10 and 15 mg/kg intraperitoneally daily for 30 days). Lungs wet/dry ratio and protein concentration in bronchoalveolar lavage fluid, MPO activity, oxidative stress markers, hydroxyproline assay, levels of inflammatory cytokines (TNF­α, IL­6 and TGF­ß1), NF­κB activity, histopathology, immunostaining (E­cadherin, vimentin and alpha ­smooth muscle actin) and ultrastructural changes were studied. SAB showed anti­inflammatory and anti­fibrotic effects through inhibition of inflammatory cell infiltration, alveolar structure disruption, and collagen deposition and the expression of several fibrogenic cytokines. SAB also up­regulate E­cadherin and down­regulated vimentin and alpha­smooth muscle actin expression. In conclusion, Salvianolic acid B is effective in alleviating the BLM induced lung fibrosis through suppression of oxidative stress, inflammation, histological, ultrastructural changes and EMT.

Beneficial effects of high dose of L-arginine on airway hyperresponsiveness and airway inflammation in a murine model of asthma.

BACKGROUND: Disturbance in the delicate balance between L-arginine-metabolizing enzymes such as nitric oxide synthase (NOS) and arginase may lead to decreased L-arginine availability to constitutive forms of NOS (endothelial NOS), thereby increasing the nitro-oxidative stress and airway hyperresponsiveness (AHR). OBJECTIVE: In this study, we investigated the effects of high doses of L-arginine on L-arginine-metabolizing enzymes and subsequent biological effects such as cyclic guanosine monophosphate production, lipid peroxidation, peroxynitrite, AHR, and airway inflammation in a murine model of asthma. METHODS: Different doses of L-arginine were administered to ovalbumin-sensitized and challenged mice. Exhaled nitric oxide, AHR, airway inflammation, T(H)2 cytokines, goblet cell metaplasia, nitro-oxidative stress, and expressions of arginase 1, endothelial NOS, and inducible NOS in lung were determined. RESULTS: L-arginine significantly reduced AHR and airway inflammation including bronchoalveolar lavage fluid eosinophilia, T(H)2 cytokines, TGF-beta1, goblet cell metaplasia, and subepithelial fibrosis. Further, L-arginine increased ENO levels and cyclic guanosine monophosphate in lung and reduced the markers of nitro-oxidative stress such as nitrotyrosine, 8-isoprostane, and 8-hydroxy-2'-deoxyguanosine. This was associated with reduced activity and expression of arginase 1, increased expression of endothelial NOS, and reduction of inducible NOS in bronchial epithelia. CONCLUSION: We conclude that L-arginine administration may improve disordered nitric oxide metabolism associated with allergic airway inflammation, and alleviates some features of asthma.

A case of an incidental Strongyloides stercoralis infection in the intestine of an Indian monitor lizard (Varanus bengalensis).

The present study reports a case of an incidental Strongyloides stercoralis infection in the intestine of an Indian monitor lizard (Varanus bengalensis). Nematode larvae were embedded in the mucosa of the small intestine. The nematodes were small, whitish in colour and measures (2.16 ± 69.85) mm length and (206.75 ± 38.85) µm width. Histopathologically, the intestine showed tufting of degenerated surface epithelium. There was presence of sections of the nematodes within the mucosa of the intestinal epithelium.

Cell free mitochondrial DNA in serum and milk associated with bovine mastitis: a pilot study.

Mastitis is inflammation of mammary gland affecting all the species of domestic animals. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that bovine mastitis would be associated with increased cell free mitochondrial DNA (mtDNA) content in serum and milk. Milk and serum samples were collected from sub-clinical mastitic and normal animals. Mastitis was confirmed by California mastitis test and bacterial isolation. Oxidative stress, nitric oxide and inflammatory cytokines were also estimated. Real time polymerase chain reaction was conducted in serum and milk from sub-clinical mastitic animals and compared with healthy animals targeting the mtDNA genes cytochrome b. Mastitis animals showed higher oxidative stress markers and nitric oxide along with higher level of inflammatory cytokines. Cell free mtDNA was significantly higher in serum and milk of mastitic animals comparing to that of healthy control. The higher cell free relative mtDNA content in mastitis animals indicates injury to the mammary epithelial cells and thereby releasing the mtDNA in the milk and blood. This mtDNA may be a bio-marker of oxidative stress and tissue injury in bovine mastitis.

Hepatoprotective Effect of Auricularia delicata (Agaricomycetes) from India in Rats: Biochemical and Histopathological Studies and Antimicrobial Activity.

Auricularia delicata, an edible mushroom, has been used as a traditional medicine in Manipur, India, for various gastrointestinal and liver ailments. This study evaluates the antioxidant, antimicrobial, and hepatoprotective potential of A. delicata. A. delicata fruiting bodies were extracted with hexane, chloroform, ethyl acetate, and methanol. All these extracts were examined for in vitro antioxidant activity. To study antimicrobial activity, minimum inhibitory concentrations (milligrams per milliliter) were determined through the use of the broth dilution method. In vivo hepatoprotective activity against acetaminophen-induced hepatic injury in rats was investigated by evaluating serum biochemistry, antioxidant enzymes, and histopathology. With regard to antioxidant activity, 21 and 48 µg/mL were the lowest half-maximal effective concentrations, obtained for the methanol and ethyl acetate fractions, respectively. In the antimicrobial study, the ethyl acetate fraction showed the lowest inhibition of Bacillus subtilis, Enterococcus feacium, Streptococcus aureus, B. cereus, and Escherichia coli, with minimum inhibitory concentrations of 0.03, 0.015, 0.03, 0.11, and 0.5 mg/mL, respectively. Further, in in vivo studies, elevated levels of biochemical markers were significantly returned to near normal values; this was supported by histopathological changes. Thus A. delicata showed antimicrobial, antioxidant, and protective roles in induced hepatic injury. Phytochemical analysis using high-performance liquid chromatography revealed the presence of chlorogenic acid in the extracts. Its protective property might be due to the presence of a mitochondrial-targeted antioxidant effect of chlorogenic acid. The antimicrobial activity accounts for its use against diarrhea. Hence, A. delicata could be one of the best sources for natural gastrointestinal and hepatoprotective medicines in the future.

Molecular and pathological studies on natural cases of bovine theileriosis.

The present communication is a part of study conducted on 32 cases of bovine lymphadenopathies. Out of which, six cases of bovine theileriosis were diagnosed on the basis of peripheral blood smear examination and gross lesions in lymph nodes and visceral organs. Nested PCR using two primer sets N516/N517 and 14136/294 was conducted on samples collected from affected lymph nodes confirms Theileria annulata infection in five out of six cases. Sequencing analysis of amplified product showed 92, 94 and 93 % homology of isolate TH1_Bovine, TH2_Bovine and THEN_Bovine for T. annulata with T. annulata Tamilnadu and Pant Nagar. An upregulation of Th2 cytokines in the cases of theileriosis was observed as the level of TNF-α in individual animals varies from higher value (1028 pg/100 µg protein) to as low as 500 pg/100 µg protein. An increase in level of Th2 cytokines IL-4 and IL-10 was also observed. The present study concluded that pathological studies and cytokine analysis of lymph nodes are of paramount importance in disease diagnosis and associated immune response of the animal with lymphadenopathies.

Detection of Mycobacterium tuberculosis and Mycobacterium bovis in Sahiwal cattle from an organized farm using ante-mortem techniques.

AIM: The aim of this study was to investigate the prevalence of bovine tuberculosis (TB) and detection of Mycobacterium bovis in cattle from an organized dairy farm. MATERIALS AND METHODS: A total of 121 animals (93 females and 28 males) of 1 year and above were studied for the prevalence of bovine TB using single intradermal comparative cervical tuberculin (SICCT) test, bovine gamma-interferon (γ-IFN) enzyme immunoassay, and polymerase chain reactions (PCRs). RESULTS: Out of total 121 animals, 17 (14.04%) animals were positive reactors to SICCT test while only one (0.82%) animal for γ-IFN assay. By PCR, Mycobacterium TB complex was detected in 19 (15.70%) animals out of which 4 (3.30%) animal were also positive for M. bovis. CONCLUSIONS: Diagnosis of bovine TB can be done in early stage in live animals with multiple approaches like skin test followed by a molecular technique like PCR which showed promising results.

Subcutaneous Dirofilaria repens infestation in non-descript canines.

Dirofilaria repens is a filarial nematode which cause subcutaneous dirofilariosis. Dogs, foxes and cats are the definitive hosts and principal reservoirs of the parasite. We report cases of D. repens infestation in non-descript canines from Goa, India. The nematodes were enclosed within fibrous capsule or freely present in the tunica vaginalis of the testes, in sub-cutaneous tissue of foreleg and body cavity. The parasite showed well-developed thick multilayered cuticular ridges in the outermost layer, followed by transverse smooth muscles striations.

Ascaridia galli induced ulcerative proventriculitis in a poultry bird.

Various possible causes of proventriculitis include virus, bacteria, fungus, protozoans, nematodes, biogenic amines and excessive copper sulphate. In the present case, parasites were found in the lumen of the proventriculus, gizzard and duodenum of a poultry bird. Characteristic features of the parasite were studied and confirmed as Ascaridia galli. An ulcerative proventriculitis evident as denuded superficial epithelium, sub-epithelial hemorrhages, infiltration of the inflammatory cells and fibrosis were seen at histopathology. Proventriculitis caused by A. galli has not been reported till date. Here, we report a case of ulcerative proventriculitis in a poultry bird caused by nematode, A. galli.

Histopathological and immunohistochemical approaches for the diagnosis of Pasteurellosis in swine population of Punjab.

AIM: Infectious porcine bronchopneumonia, caused by Pasteurella multocida, is a widespread disease of major economic significance. Thus, the aim of the present study was to diagnose swine Pasteurellosis using gross, histopathological, and immunopathological approaches in the swine population of Punjab and to compare the efficacy of immunohistochemical (IHC) techniques with conventional diagnostic techniques. MATERIALS AND METHODS: A total of 71 adult swine lung samples showing gross pneumonic changes were collected along with the associated lymph nodes to carry out the study. The collected samples were then processed for histopathological and IHC studies. RESULTS: Out of the total 71 lung samples, 26 samples were found to be suspected for Pasteurellosis as per the microscopic changes observed, and out of these 26 samples, 16 cases were confirmed to be positive for Pasteurellosis by IHC. Varied macroscopic changes noted in lungs were pneumonic patches with consolidation of many lobes, congestion, and focal hemorrhages. Main lesions associated with lymph nodes were its enlargement and hemorrhages. Histologically, the lung showed fibrinous and suppurative bronchopneumonia, multifocal suppuration, thickening of septa with fibrin combined with cellular infiltration and edema. The higher IHC expression of P. multocida was seen in the bronchial epithelium besides in alveolar and bronchial exudate. Moreover, on comparing the histopathological and IHC scores which were calculated on the basis of characteristic microscopic lesions and number of antigen positive cells, respectively, a significant positive correlation (r=0.4234) was found. CONCLUSION: It was concluded that swine population of Punjab is having P. multocida infection. The gross and histopathological lesions can be helpful in the preliminary diagnosis of Pasteurellosis but needs to be supplemented by other immunodiagnostic tests. Moreover, IHC technique proved to be a specific, reliable, precise, and rapid technique to supplement these conventional methods of diagnosis for Pasteurellosis.

Ultrastructural changes of airway in murine models of allergy and diet-induced metabolic syndrome.

Studying ultrastructural changes could reveal novel pathophysiology of obese-asthmatic condition as existing concepts in asthma pathogenesis are based on the histological changes of the diseased airway. While asthma is defined in functional terms, the potential of electron microscopy (EM) in providing cellular and subcellular detail is underutilized. With this view, we have performed transmission EM in the lungs from allergic mice that show key features of asthma and high-fat- or high-fructose-fed mice that mimicked metabolic syndrome to illustrate the ultrastructural changes. The primary focus was epithelial injury and metaplasia, which are cardinal features of asthma and initiate airway remodeling. EM findings of the allergically inflamed mouse lungs correlate with known features of human asthma such as increased mitochondria in airway smooth muscle, platelet activation and subepithelial myofibroblasts. Interestingly, we found a clear and unambiguous evidence to suggest that ciliated cells can become goblet cells using immunoelectron microscopy. Additionally, we show for the first time the stressed mitochondria in the bronchial epithelia of high-fat- or high-fructose-fed mice even without allergen exposure. These results may stimulate interest in using EM in understanding novel pathological mechanisms for different subtypes of asthma including obese asthma.

12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

The mechanisms underlying asthmatic airway epithelial injury are not clear. 12/15-lipoxygenase (an ortholog of human 15-LOX-1), which is induced by IL-13, is associated with mitochondrial degradation in reticulocytes at physiological conditions. In this study, we showed that 12/15-LOX expressed in nonepithelial cells caused epithelial injury in asthma pathogenesis. While 12/15-LOX overexpression or IL-13 administration to naïve mice showed airway epithelial injury, 12/15-LOX knockout/knockdown in allergic mice reduced airway epithelial injury. The constitutive expression of 15-LOX-1 in bronchial epithelia of normal human lungs further indicated that epithelial 15-LOX-1 may not cause epithelial injury. 12/15-LOX expression is increased in various inflammatory cells in allergic mice. Though non-epithelial cells such as macrophages or fibroblasts released 12/15-LOX metabolites upon IL-13 induction, bronchial epithelia didn't release. Further 12-S-HETE, arachidonic acid metabolite of 12/15-LOX leads to epithelial injury. These findings suggested 12/15-LOX expressed in non-epithelial cells such as macrophages and fibroblasts leads to bronchial epithelial injury.

Baicalein reduces airway injury in allergen and IL-13 induced airway inflammation.

BACKGROUND: Baicalein, a bioflavone present in the dry roots of Scutellaria baicalensis Georgi, is known to reduce eotaxin production in human fibroblasts. However, there are no reports of its anti-asthma activity or its effect on airway injury. METHODOLOGY/PRINCIPAL FINDINGS: In a standard experimental asthma model, male Balb/c mice that were sensitized with ovalbumin (OVA), treated with baicalein (10 mg/kg, ip) or a vehicle control, either during (preventive use) or after OVA challenge (therapeutic use). In an alternate model, baicalein was administered to male Balb/c mice which were given either IL-4 or IL-13 intranasally. Features of asthma were determined by estimating airway hyperresponsiveness (AHR), histopathological changes and biochemical assays of key inflammatory molecules. Airway injury was determined with apoptotic assays, transmission electron microscopy and assessing key mitochondrial functions. Baicalein treatment reduced AHR and inflammation in both experimental models. TGF-ß1, sub-epithelial fibrosis and goblet cell metaplasia, were also reduced. Furthermore, baicalein treatment significantly reduced 12/15-LOX activity, features of mitochondrial dysfunctions, and apoptosis of bronchial epithelia. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that baicalein can attenuate important features of asthma, possibly through the reduction of airway injury and restoration of mitochondrial function.

L-arginine reduces mitochondrial dysfunction and airway injury in murine allergic airway inflammation.

Bronchial epithelial injury is the hall mark of asthma which is a chronic airway inflammatory disease. We have shown the mitochondrial ultrastructural changes and dysfunction in bronchial epithelia of OVA induced mice. Reduced L-arginine bioavailability in asthma leads to increased formation of peroxynitrite which could induce mitochondrial dysfunction. We have also shown that L-arginine administration attenuates experimental asthma and reduces peroxynitrite. In this study, we wanted to determine the effect of L-arginine on mitochondrial dysfunction and airway injury in allergic airway inflammation. To determine this, L-arginine was administered to ovalbumin sensitized and challenged mice during allergen challenges. Mitochondrial and cytosolic fractions were purified from the lung to determine key mitochondrial functions, and mitochondrial ultrastructural changes in bronchial epithelia of first generation bronchi were determined. It was found that L-arginine administration increased mitochondrial cytochrome c oxidase activity, reduced cytosolic cytochrome c, increased lung ATP levels, reduced DNA fragmentation in bronchial epithelia and restored the ultrastructural changes of mitochondria of bronchial epithelia. In addition, L-arginine administration reduced the widening of intercellular spaces between adjacent bronchial epithelia. These findings indicated that L-arginine administration reduced airway injury and restored mitochondrial dysfunction in murine allergic airway inflammation.

Effects of vitamin E on mitochondrial dysfunction and asthma features in an experimental allergic murine model.

We showed recently that IL-4 causes mitochondrial dysfunction in allergic asthma. IL-4 is also known to induce 12/15-lipoxygenase (12/15-LOX), a potent candidate molecule in asthma. Because vitamin E (Vit-E) reduces IL-4 and inhibits 12/15-LOX in vitro, here we tested the hypothesis that Vit-E may be effective in restoring key mitochondrial dysfunctions, thus alleviating asthma features in an experimental allergic murine model. Ovalbumin (OVA)-sensitized and challenged male BALB/c mice showed the characteristic features of asthma such as airway hyperresponsiveness (AHR), airway inflammation, and airway remodeling. In addition, these mice showed increase in the expression and metabolites of 12/15-LOX, reduction in the activity and expression of the third subunit of mitochondrial cytochrome-c oxidase, and increased cytochrome c in lung cytosol, which indicate that OVA sensitization and challenge causes mitochondrial dysfunction. Vit-E was administered orally to these mice, and 12/15-LOX expression, key mitochondrial functions, ultrastructural changes of mitochondria in bronchial epithelia, and asthmatic parameters were determined. Vit-E treatment reduced AHR, Th2 response including IL-4, IL-5, IL-13, and OVA-specific IgE, eotaxin, transforming growth factor-beta1, airway inflammation, expression and metabolites of 12/15-LOX in lung cytosol, lipid peroxidation, and nitric oxide metabolites in the lung, restored the activity and expression of the third subunit of cytochrome-c oxidase in lung mitochondria and bronchial epithelia, respectively, reduced the appearance of cytochrome c in lung cytosol, and also restored mitochondrial ultrastructural changes of bronchial epithelia. In summary, these findings show that Vit-E reduces key mitochondrial dysfunctions and alleviates asthmatic features.