Possible mechanism of antifungal phenazine-1-carboxamide from Pseudomonas sp. against dimorphic fungi Benjaminiella poitrasii and human pathogen Candida albicans.

AIM: Investigation of antifungal mechanism of phenazine 1-carboxamide (PC) produced by a Pseudomonas strain MCC2142. METHODS AND RESULTS: An antifungal metabolite produced by a Pseudomonas was purified and identified as PC. Human pathogenic fungi such as Candida albicans, Candida glabrata, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus fumigatus and Aspergillus niger were found to be inhibited by PC (MIC90 32-64 µg ml(-1)). Addition of PC (20 µg ml(-1)) during yeast (Y)-hypha (H) transitions inhibited germ tube formation by >90% and >99% in C. albicans National Collection of Industrial Microorganisms (NCIM) 3471 and nonpathogenic model Benjaminiella poitrasii, respectively. After exposure to PC (20 µg ml(-1)), 75-80% yeast cells of B. poitrasii and C. albicans NCIM 3471 showed rhodamine 123 fluorescence indicating high intracellular reactive oxygen species (ROS) production. ROS further led to hyperpolarization of mitochondrial membrane, subsequently induction of apoptosis as evident by externalization of phosphatidylserine, DNA fragmentation, chromatin condensation and finally death in B. poitrasii. In C. albicans NCIM 3471, PC (20 µg ml(-1)) induced apoptosis. CONCLUSIONS: The antifungal effect of PC in B. poitrasii and C. albicans may be due to ROS-mediated apoptotic death. SIGNIFICANCE AND IMPACT OF THE STUDY: Inhibition of Y-H transition of B. poitrasii and C. albicans by PC indicates that it may prove useful in the control of dimorphic human pathogens.

Synthetic double-stranded RNA oligonucleotides are immunostimulatory for chicken spleen cells.

Toll-like receptors (TLRs) are an integral part of the innate immune system that recognize microbe-derived molecular patterns and initiate innate and adaptive defenses against invading pathogens. TLR3 and TLR7 are involved in sensing virus-associated single-stranded and double-stranded RNA (dsRNA) molecules in cellular endosomes to activate the type I interferon pathway in mammals. Although certain synthetic dsRNA molecules have been identified to show immunostimulation in mammalian cells, very little is known about the ability of these sequences to stimulate avian cells. The current study investigated immunostimulatory properties of four synthetic oligonucleotide sequences using chicken splenocytes. Expression of TLR3 and 7, type I interferons and several other cytokines as well as TLR signaling pathway-related genes at different time points post-stimulation was quantified by real-time PCR. A dose-dependent increase in expression of TLR3 was observed in splenocytes treated with poly-UGUGU (poly-UG) and ß-galactosidase dsRNA molecules. TLR3 and TLR7 gene expression was significantly up-regulated upon stimulation with all four dsRNA molecules. Furthermore, in a time course study, a significant increase was noted in the expression of TLR3, TLR7, interferon (IFN)-α, IFN-ß, interleukin (IL)-1ß, IL-6 as well as 2',5'-OAS in splenocytes treated with poly-UG. In conclusion, the present study demonstrated the immunostimulatory properties of dsRNA oligonucleotides, especially those that contain a poly-UG motif, in chickens.

The invariant NKT cell subset in anti-viral defenses: a dark horse in anti-influenza immunity?

iNKT cells, a small subset of αß TCR+ T cells, are capable of producing large amounts of cytokines upon activation through their TCR. Unlike conventional T cells that express highly diverse TCRs, iNKT cells express a glycolipid-reactive invariant TCR-α chain paired with a limited number of ß chain(s). These cells recognize glycolipid antigens when presented on CD1d molecules found on APC or other cells. Although the immunoregulatory roles of iNKT cells in the context of autoimmune disease are fairly well characterized, several lines of evidence highlight the importance of this cell type in immune responses against microbial insults caused by bacterial, viral, and parasitic pathogens. Recent studies that have investigated the role of iNKT cells in immune responses against influenza virus have suggested an important role for these cells in innate defense mechanisms as well as antibody- and cell-mediated responses. This review highlights the important contributions of iNKT cells to immune responses against viral pathogens with particular emphasis on immunity to influenza infections.

A live oral recombinant Salmonella enterica serovar typhimurium vaccine expressing Clostridium perfringens antigens confers protection against necrotic enteritis in broiler chickens.

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens, and there is currently no effective vaccine for NE. We previously showed that in broiler chickens protection against NE can be achieved through intramuscular immunization with alpha toxin (AT) and hypothetical protein (HP), and we subsequently identified B-cell epitopes in HP. In the present study, we identified B-cell epitopes in AT recognized by chickens immune to NE. The gene fragments encoding immunodominant epitopes of AT as well as those of HP were codon optimized for Salmonella and cloned into pYA3493, and the resultant plasmid constructs were introduced into an attenuated Salmonella enterica serovar Typhimurium chi9352 vaccine vehicle. The expression of these Clostridium perfringens proteins, alpha toxoid (ATd) and truncated HP (HPt), was confirmed by immunoblotting. The protection of broiler chickens against experimentally induced NE was assessed at both the moderate and the severe levels of challenge. Birds immunized orally with Salmonella expressing ATd were significantly protected against moderate NE, and there was a nonsignificant trend for protection against severe challenge, whereas HPt-immunized birds were significantly protected against both severities of challenge. Immunized birds developed serum IgY and mucosal IgA and IgY antibody responses against Clostridium and Salmonella antigens. In conclusion, this study identified, for the first time, the B-cell epitopes in AT from an NE isolate recognized by chickens and showed the partial protective ability of codon-optimized ATd and HPt against NE in broiler chickens when they were delivered orally by using a Salmonella vaccine vehicle.

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations.

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.

Oral immunization of broiler chickens against necrotic enteritis with an attenuated Salmonella vaccine vector expressing Clostridium perfringens antigens.

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens but currently no effective vaccine is available. Our previous study showed that certain C. perfringens secreted proteins when administered intramuscularly protected chickens against experimental infection. In the current study, genes encoding three C. perfringens proteins: fructose-biphosphate-aldolase (FBA), pyruvate:ferredoxin-oxidoreductase (PFOR) and hypothetical protein (HP), were cloned into an avirulent Salmonella enterica sv. typhimurium vaccine vector. Broiler chickens immunized orally with recombinant Salmonella expressing FBA or HP proteins were significantly protected against NE challenge. Immunized birds developed serum and mucosal antibodies to both clostridial and Salmonella antigens. This study showed the oral immunizing ability of two C. perfringens antigens against NE in broiler chickens through an attenuated Salmonella vaccine vector.

Antioxidant and Antiinflammatory Activity of Vitex negundo.

Reactive oxygen species are implicated in various inflammatory disorders. Vitex negundo is mentioned in Ayurveda as useful in treating arthritic disorders. The present work was undertaken to evaluate the antioxidant potential and anti-inflammatory activity of the plant. The total methanol extract of the plant was standardized in terms of total polyphenols. The standardized extract in a dose of 100 mg/kg caused a comparable reduction in edema with that of diclofenac sodium (25 mg/kg) when evaluated for antiinflammatory activity by carrageenan-induced rat paw edema method. The extract also exhibited a strong free radical scavenging activity by 1,1-diphenyl-2-picrylhydrazyl method and caused a significant reduction in the formation of thiobarbituric acid reacting substances when evaluated for its lipid peroxidation inhibitory activity. The results strongly suggest that radical quenching may be one of the mechanisms responsible for its antiinflammatory activity.

Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis.

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens.

Clostridium perfringens antigens recognized by broiler chickens immune to necrotic enteritis.

Little is known about immunity to necrotic enteritis (NE) in chickens. A recent study of broiler chickens showed that protection against NE was associated with infection-immunization with virulent but not with avirulent Clostridium perfringens. In the current study, six secreted antigenic proteins unique to virulent C. perfringens that reacted to serum antibodies from immune birds were identified by mass spectrophotometry; three of these proteins are part of the VirR-VirS regulon.

Live attenuated vaccine-based control of necrotic enteritis of broiler chickens.

A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains. In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize ("infection-immunization") chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.

Ocular manifestations of head injury: a clinical study.

AIM: This prospective study aimed to clinically correlate the various ocular findings with the neurological status in cases of closed head injury. METHODS: A total of 200 consecutive cases of closed head injury admitted to a major teaching hospital underwent a thorough ophthalmic assessment. The Glasgow coma scale (GCS) and the Revised trauma score (RTS) were applied to grade the severity of injury and assess the prognosis. Kendall's tau-b and Fisher's exact test were used in the analysis. RESULTS: The main causes of head injury were road traffic accidents 52.5% followed by assaults in 34%. Ocular involvement was found in 167(83.5%) cases. These included corneal and scleral tears in 2%, subconjunctival haemorrhage or ecchymosis in 46%, orbital fractures 12%, pupillary involvement 6.5%, papilloedema 5.5%, intraocular trauma 5.5%, proptosis 3%, lateral rectus palsy 2%, lacrimal gland prolapse 1%, and optic nerve trauma 0.5%. All 21 patients (10.5%) who died had eye involvement. In all, 150 cases (75%) with a RTS of 12 had a good prognosis. Of these 124 (82.6%) had ocular involvement of no neurological significance. CONCLUSIONS: Although sophisticated imaging techniques are available to localize lesions, early ophthalmic assessment in correlation with the GCS aids in prognosticating outcomes. Pupillary involvement, papilloedema, and ocular motor paresis pointed to a more severe head injury. To our knowledge, this is the only prospective study recording ocular findings in the first few hours and attempting a correlation with the final outcome.

Profile of malignancies at Medical College, Ambajogai--(15 years retrospective study).

Present study comprises of a preliminary analysis of malignant lesions, diagnosed and confirmed by histopathological examination, carried out in Department of Pathology, S. R. T. R. Medical College, Ambajogai, from August 1976 to July 1991. 30,168 specimens were received for histopathological examination out of which 2,663 cases were diagnosed as malignant lesions (8.82%). 163 cases of leukemias were diagnosed by peripheral smear and bone marrow examination. Our of 2826 patients with malignant lesions 1,566 (55.41%) were males and 1,260 (44.59%) were females with M:F ratio as 1.24:1. Analysis of all malignant lesions showed more predilection towards genital organs (26.22%), gastrointestinal tract (13.72%) and breast (10.47%). Penis (11.36%), gastrointestinal tract (16.73%), oral cavity and tongue with pharynx (20.81%) and skin (11.11%) were leading sites in males where as cervix (30.63%) breast (22.46%), gastrointestinal tract (10.00%), skin (6.82%) and lymphoma, leukaemias (7.30%) were common sites for malignancies in females. The higher incidence of genital and skin tumours is discussed in the paper. Available data is compared with data from Tata Memorial Cancer Hospital Bombay and Cancer Hospital at Barshi in Solapur District of Maharashtra.

Position of the axillary nerve in the deltoid muscle.

The relationship of the anterior branch of the axillary nerve to the deltoid muscle was studied on both sides in 66 adult cadavers. The nerve runs a course on the deep surface of the deltoid muscle about 2.2 cm above the midpoint on the vertical plane of the muscle.

The scalene tubercle.

One hundred and thirty right and 114 left first ribs from a random collection have been studied for the characters of the scalene tubercle and the related structures. The tubercle is not always present but the muscular impression for the insertion of the scalenus anterior muscle on the superior surface of the first rib is almost constant and it generally extends upto the medial third of this surface. The arterial groove, generally shallow, lies behind the tubercle or the muscular impression and generally extends only upto the medial 2/3 of the superior surface. Likewise, the venous groove, mostly shallow, lies in front of the tubercle or the muscular impression and extends, generally, only upto the medial 2/3 of the surface. Normally, there is no separate groove for the lower trunk of the brachial plexus.

Treatment of osteoarthritis with a herbomineral formulation: a double-blind, placebo-controlled, cross-over study.

The clinical efficacy of a herbomineral formulation containing roots of Withania somnifera, the stem of Boswellia serrata, rhizomes of Curcuma longa and a zinc complex (Articulin-F), was evaluated in a randomized, double-blind, placebo controlled, cross-over study in patients with osteoarthritis. After a one-month single blind run-in period, 42 patients with osteoarthritis were randomly allocated to receive either a drug treatment or a matching placebo for a period of three months. After a 15-day wash-out period the patients were transferred to the other treatment for a further period of three months. Clinical efficacy was evaluated every fortnight on the basis of severity of pain, morning stiffness, Ritchie articular index, joint score, disability score and grip strength. Other parameters like erythrocyte sedimentation rate and radiological examination were carried out on a monthly basis. Treatment with the herbomineral formulation produced a significant drop in severity of pain (P less than 0.001) and disability score (P less than 0.05). Radiological assessment, however, did not show any significant changes in both the groups. Side effects observed with this formulation did not necessitate withdrawal of treatment.