A Review of the Cytokine IL-17 in Ocular Surface and Corneal Disease.

Aim: To investigate the role of interleukin-17 in ocular surface and corneal disease. Ocular surface and corneal disease is a leading cause of blindness and is an ongoing challenge for the public health sector to implement effective therapies. The majority of cells in corneal lesions are derived primarily from neutrophils that induce inflammatory events that lead to tissue damage. One of the key pro-inflammatory cytokines is IL-17, and it has been investigated in order to facilitate the understanding of the pathogenesis of ocular surface lesion development. Method: A review of the literature was performed through a systematic approach. Results: IL-17 has been shown to exacerbate dry eye disease, viral and bacterial keratitis lesion severity, although it was found to be protective for Acanthamoeba. Antibodies developed to neutralize IL-17 have shown some promise in reducing the severity of some diseases. Conclusion: IL-17 plays a role in the pathogenesis of ocular surface and corneal disease and targeting this cytokine may provide a useful treatment option in the future.

Functional block of IL-17 cytokine promotes bone healing by augmenting FOXO1 and ATF4 activity in cortical bone defect model.

We determine the effect of interleukin (IL)-17 neutralizing antibody on new bone regeneration. Anti-IL-17 antibody promoted new bone regeneration in cortical bone defect model by augmenting FOXO1 and ATF4 activity thereby decreasing oxidative stress. Our study demonstrates the bone healing and regeneration potential of neutralizing IL-17antibody in osteoporotic fractures. INTRODUCTION: The immune system plays important role in the fracture healing process. However, fracture healing is prolonged in disorders associated with systemic inflammation. Fracture healing is decelerated in osteoporosis, condition linked with systemic inflammation. Bone regeneration therapies like recombinant human BMP2 are associated with serious side effects. Studies have been carried out where agents like denosumab and infliximab enhance bone regeneration in osteoporotic conditions. Our previous studies show the osteoprotective and immunoprotective effects of neutralizing IL-17 antibody. Here, we determine the effect of IL-17 neutralizing antibody on new bone regeneration and compare its efficacy with known osteoporotic therapies. METHODS: For the study, female BALB/c mice were ovariectomized or sham operated and left for a month followed by a 0.6-mm drill-hole injury in femur mid-diaphysis. The treatment was commenced next day onwards with anti-IL-17, anti-RANKL (Receptor activator of nuclear factor kappa-B ligand), parathyroid hormone (PTH), or alendronate for a period of 3, 10, or 21 days. Animals were then autopsied, and femur bones were dissected out for micro-CT scanning, confocal microscopy, and gene and protein expression studies. RESULTS: Micro-CT analysis showed that anti-IL-17 antibody promoted bone healing at days 10 and 21, and the healing effect observed was significantly better than Ovx, anti-RANKL antibody, and ALN, and equal to PTH. Anti-IL-17 also enhanced new bone regeneration as assessed by calcein-labeling studies. Additionally, anti-IL-17 therapy enhanced expression of osteogenic markers and decreased oxidative stress at the injury site. CONCLUSION: Overall, our study demonstrates bone healing and regeneration potential of neutralizing IL-17 antibody in osteoporotic fractures.

In vitro rapid clearance of infectious bursal disease virus in peripheral blood mononuclear cells of chicken lines divergent for antibody response might be related to the enhanced expression of proinflammatory cytokines.

Infectious bursal disease (IBD) is an acute and highly contagious viral disease of young chickens caused by infectious bursal disease virus (IBDV). An effective way to control IBDV would be to breed chickens with a reduced susceptibility to IBDV infection. In the present work, we used chickens selected for high and low specific responses to sheep red blood cells (SRBC) (H and L, respectively) to assess the susceptibility of differential immune competent animals to IBDV infection. The peripheral blood mononuclear cells (PBMCs) of high SRBC line (HL) and low SRBC line (LL) were infected with IBDV and viral RNA loads were determined at different time post-IBDV infection. Chicken orthologues of the T helper 1 (Th1) cytokines, interferon-γ (IFN-γ) and interleukin-2 (IL-2); a Th2 cytokine, IL-10; a pro inflammatory cytokine, IL-6; the CCL chemokines, chCCLi2, chCCLi4 and chCCLi7; colony stimulating factor, GM-CSF; and a anti-inflammatory cytokine, transforming growth factor ß-2 (TGFß-2) were quantified. The expression of chCCLi2, chCCLi4 and chCCLi7 was significantly higher in L line as compared to H line. However, in H line the viral RNA loads were significantly lower than in L line. Therefore, the upregulated chemokines might be associated with the susceptibility to IBDV. The expression of IFN-γ, IL-2 and IL-6 was significantly higher in H line as compared to L line. We assume that the higher proinflammatory cytokines expression in H line might be related to the rapid clearance of virus from PBMCs. Significantly higher levels of IL-10 and TGFß-2 mRNAs in L line might be related to the pathogenesis of IBDV. In conclusion, selection for antibody responses appears to influence the expression profiles of chemokines and cytokines against IBDV. Further, the selection for high SRBC response might improve the immuno-competence of chickens against IBDV.

The role of the male cloacal gland in reproductive success in Japanese quail (Coturnix japonica).

The adult male Japanese quail has a well developed cloacal gland that produces meringue-like white foam. The physiological significance of the cloacal gland and its foam is still unclear. Therefore, we conducted two experiments to establish the functional role of the cloacal gland and its foam in natural mating and oviducal sperm transport. In the first experiment, artificial insemination of equal numbers of spermatozoa diluted in foam extract and normal saline once in a week were used to determine the role of foam in sperm transport in the female oviduct. After artificial insemination, eggs were collected to measure fertility, the duration of the fertile period, sperm holes and attached spermatozoa in the perivitelline membrane. Higher (P<0.05) fertility and greater duration of the fertile period were observed when semen was inseminated along with foam extract compared with normal saline. Further, the sperm holes and trapped spermatozoa in the perivitelline membrane were also higher (P<0.05) in the presence of foam extract. In the second experiment, two males with bigger and smaller cloacal gland areas were allowed to mate with a female. The mating attempts of males with larger cloacal gland were more successful (P<0.05) than males with smaller cloacal glands. Our results indicated that cloacal foam improves sperm transport in the female oviduct and that males with larger cloacal gland areas are preferred during mating.

Cytokines Expression and Nitric Oxide Production under Induced Infection to Salmonella Typhimurium in Chicken Lines Divergently Selected for Cutaneous Hypersensitivity.

In the present study, the impact of Salmonella Typhimurium on cell-mediated immunity (CMI) was investigated in 5 week-old immuno divergent broiler lines selected for the high and low response to phytohemagglutinin-P. The immune response was assessed in peripheral-blood mononuclear cells (PBMCs) induced with Salmonella Typhimurium at different time intervals (0 h, 0.5 h, 2 h, 4 h, 6 h, 12 h and 24 h). The differential mRNA expression patterns of IFN-γ, IL-2 and iNOS were evaluated by quantitative real time PCR. In-vitro production of nitric oxide (NO) was also estimated in the culture supernatant and correlated with iNOS mRNA expression. Present study showed higher production of NO in the high cell-mediated line (HCMI) as compared to the low cell-mediated line (LCMI) upon stimulation with Salmonella Typhimurium. Correspondingly, higher mRNA expression of iNOS and IFN-γ were observed in high response birds (HCMI); but IL-2 was down regulated in this line compared to the low response birds (LCMI). Significantly (p<0.05) higher expression of iNOS, IFN-γ and higher production of NO in high line indicated that the selection for PHA-P response might be employed for increasing the immune competence against Salmonella Typhimurium in chicken flocks.

Cloacal gland foam enhances motility and disaggregation of spermatozoa in Japanese quail (Coturnix japonica).

The adult male Japanese quail produces white foam from the cloacal gland, which is transferred to the female proctodeum during natural mating. The physiological role of foam on quail spermatozoa is still unclear. Therefore, attempts have been made to understand the effect of cloacal foam on motility and metabolism of quail spermatozoa. The profile of various biochemical constitutes in the foam extract was investigated. The addition of foam extract to neat semen completely disaggregated the clumps of spermatozoa leading to vigorous motility. The metabolic rate (MBRT) of the spermatozoa was significantly increased with the addition of foam extract. The foam extract was sub fractionated into seven different fractions by using the molecular cut off devices. Among all the seven sub-fractions from the foam extract, the addition of < 1 KDa sub-fraction contained lactate and has enhanced sperm motility and metabolism. Another fraction (3-10 KDa) has non-protein and non-heparin components which completely disaggregated the clumped quail spermatozoa. However, the remaining fractions did not show any effect on quail spermatozoa. It can be concluded from the present investigation that the lactate present in foam might be a fuel for sperm metabolism and motility. Furthermore, low molecular weight (3-10 KDa) components in the foam may responsible for sperm disaggregation.

Characterization of lactate dehydrogenase enzyme in seminal plasma of Japanese quail (Coturnix coturnix japonica).

Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential.

Impact of rainfall on residual subsidence in old coal mine workings.

Subsidence over old coal mine workings can not be avoided if the underground workings are not fully filled. Existence of fire, illegal mining operation and seasonal impact (rainfall) aggravate proneness of subsidence over old workings. This paper deals with the causative factors of subsidence over old workings and its relation with rainfall with reference to Jharia and Raniganj Coalfields, India during the year 2007. The impact of subsidence has also been dealt in this paper.

Appraisal of carbon monoxide emission at surface due to long standing underground fires in Jharia coalfield, India.

Several locations of Jharia coalfield are affected by fire, and carbon monoxide (CO) is a product of active fires in coal mines. The paper deals with the investigation on emission of CO concentration at surface due to long standing fire at Lodna and Kusunda areas of Jharia coalfield. CO level was high at the point of emission from the surface (> 1000 ppm). CO concentration was confined within the smoke at the areas where only smoke was existing. The lateral spreading of the CO concentration was high all around the outcrop of fire.

Immune response to Newcastle disease virus in chicken lines divergently selected for cutaneous hypersensitivity.

This paper describes for the first time the differential immune response to virulent Newcastle disease virus (NDV) in birds differing in cell-mediated immunity, as measured by response to phytohaemagglutinin-P. To explore potential host-pathogen interactions, peripheral blood mononuclear cells (PBMC) were collected from 40 extreme responder birds (20 birds each from high and low cell-mediated immunity lines). PBMC cultures were stimulated by virulent NDV and temporal expression profiles of interferon-gamma (IFN-gamma), and inducible nitric oxide synthase (iNOS) mRNA was evaluated by semiquantitative reverse transcription polymerase chain reaction (PCR). To further explore the correlation of iNOS mRNA expression and nitric oxide (NO) production, we assayed the culture supernatants for NO. NO production, as well as iNOS and IFN-gamma mRNA expression, was significantly (P < 0.05) higher in the line with higher cell-mediated immunity. In our study, a significant (P < 0.05) difference was observed between the lines for IFN-gamma promoter polymorphism for the TspEI site. The high cell-mediated immunity line mostly revealed the genotype (GG) with a 168-bp fragment. On the other hand, this genotype was not predominant in the low cell-mediated immunity line. Later, quantitative real-time PCR demonstrated higher (P < 0.01) IFN-gamma mRNA transcription in the genotype GG in response to NDV. This difference in promoter region may be responsible for differential IFN-gamma mRNA transcription in chicken lines. Furthermore, birds of high cell-mediated immunity line showed better adaptive immunity to booster NDV vaccination as revealed by an enhanced antibody titre. Thus, this study provides baseline data on the effect of phytohaemagglutinin-P response-based selection on immune responses to virulent NDV and the data could be of immense importance to poultry geneticist and immunologist attempting to breed poultry for disease resistance.

Characterization of resistance to multiple aphid species (Hemiptera: Aphididae) in Medicago truncatula.

Aphids are phloem-feeding insects that damage many important crops throughout the world yet, compared to plant-pathogen interactions, little is known about the mechanisms by which plants become resistant to aphids. Medicago truncatula (barrel medic) is widely considered as the pre-eminent model legume for genetic and biological research and in Australia is an important pasture species. Six cultivars of M. truncatula with varying levels of resistance to two pests of pasture and forage legumes, the bluegreen aphid Acyrthosiphon kondoi Shinji and the spotted alfalfa aphid Therioaphis trifolii f. maculata. (Buckton) are investigated. Two resistance phenotypes against T. trifolii f. maculata are described, one of which is particularly effective, killing most aphids within 24 h of infestation. Each resistance phenotype provided a similar but somewhat less effective degree of resistance to the closely-related spotted clover aphid Therioaphis trifolii (Monell). In the case of A. kondoi only one resistance phenotype was observed, which did not vary among different genetic backgrounds. None of the observed resistance against A. kondoi or T. trifolii f. maculata significantly affected the performance of green peach aphid Myzus persicae (Sulzer) or cowpea aphid Aphis craccivora Koch. The existence of multiple aphid resistance mechanisms in similar genetic backgrounds of this model plant provides a unique opportunity to characterize the fundamental basis of plant defence to these serious agricultural pests.

Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response.

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1beta in monocyte culture, whereas T(H)1 cytokines (IL-2 and IFN-gamma) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-gamma and the chemokine MIP-1beta, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T(H)1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.

Estimation of mortality and morbidity due to strokes in India.

In order to control the stroke problem, its magnitude should be assessed. India is ranked among the countries where the information on stroke is minimal. We decided to review the information available in order to estimate the mortality and morbidity due to stroke in India. Information was collected through electronic search, hand search and contact with experts. Each article was reviewed for relevance and epidemiological rigor. The demographic data were as derived from published government figures. The prevalence from individual studies was pooled and weighted based on sample size. Analysis was done separately for males and females at 10-year intervals (20 years onwards). A total of 7 studies was located, but 2 were discarded. All were done in rural areas except 2 which also included urban areas. The prevalence was estimated as 203 per 100,000 population above 20 years amounting to a total of about 1 million cases. The male to female ratio was 1.7. Around 12% of all strokes occurred in population below 40 years. The estimation of stroke mortality was seriously limited by the method of classification of cause of death in the country. The best estimate derived was 102,000 deaths; which represented 1.2% of total deaths in the country. There is need to initiate steps to collect data on morbidity and mortality due to stroke in the country as a first step towards control measures.

Characterization of salicylic acid-responsive, arabidopsis Dof domain proteins: overexpression of OBP3 leads to growth defects.

Dof proteins are unique to plants and contain a single zinc finger DNA-binding domain called the Dof domain. OBP1, an Arabidopsis Dof protein, was previously isolated through an interaction with OBF4, an ocs element-binding protein. Two additional Dof proteins, called OBP2 and OBP3, were isolated through homologous screening. All three OBP proteins contain transcriptional activation domains in their C-terminal region. While no significant differences were found between the OBP proteins in terms of their DNA and protein-binding properties, tissue-specific RNA expression patterns were found. The RNA expression levels of all three OBP proteins increased following treatment with auxin, salicylic acid (SA) or cycloheximide, although the level of induction varied among the different proteins and in the tissues tested. These results suggest that even though they have similar DNA binding and protein-protein interaction properties, the different OBP proteins are likely to have distinct functions in specific parts of the plant. There is a good correlation between the expression of the OBP proteins and the ocs element, a stress-response element which is also induced by auxin, SA and cycloheximide. To begin to analyze the function of the OBP proteins, transgenic lines overexpressing OBP3 were generated. These plants showed a severe growth defect with altered root development and yellowish leaves. The severity of the growth defects correlated with OBP3 expression levels and in some cases led to death, suggesting that some Dof proteins play important roles in plant growth and development.

The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element.

Glutathione S-transferases (GSTs) are multi-functional enzymes that play critical roles in a number of plant stress responses. Ocs elements are enhancer sequences found in some pathogen and GST promoters. We previously isolated an Arabidopsis GST gene, called GST6, which contained an ocs-like element. Here we tested the role this putative ocs element plays in GST6 expression in response to auxin, salicylic acid (SA) and hydrogen peroxide (H2O2) using a transient assay and transgenic Arabidopsis. The requirement for the ocs element for basal and/or induced expression varied between the transient and transgenic studies, and in different plant tissues. The GST6 promoter showed the largest induction in roots following treatment with SA (84-fold) or H2O2 (122-fold), and the effect of mutating the ocs element was most pronounced in root tissue. However, in no case did mutation of the ocs element abolish GST6 expression, suggesting that other promoter element(s) are also important. Using the transient assay, other promoter element(s) critical for GST6 expression were identified that may also help mediate the activity of the ocs element. The finding that the ocs element plays a role in the transcriptional response of a plant GST gene to SA and H2O2 has potential significance for the plant defense response, where SA and H2O2 play critical roles.

A glucocorticoid-inducible transcription system causes severe growth defects in Arabidopsis and induces defense-related genes.

A glucocorticoid-inducible transcription system was employed to control the expression of AtEBP, an Arabidopsis transcription factor. A number of the transgenic AtEBP lines had developmental and growth defects when grown on dexamethasone (DEX), a strong synthetic glucocorticoid. However, these growth defects were not confined to the AtEBP lines but were observed with other transgenic lines that were generated using the same system, including empty vector lines. In about 25% of the AtEBP or empty vector transgenic lines, these growth defects were severe and in some cases led to death. As AtEBP has been linked to the plant defense response, the expression of specific defense-related genes, including a number of pathogenesis-related (PR) genes was also examined. PDF1.2, a plant defensin gene, was strongly induced in all transgenic lines examined following treatment with DEX, including empty vector lines that did not show any observable DEX-induced growth defect. PR-5 was induced to a lesser extent in all the lines, while the expression of PR-1, PR-2 and phenylalanine ammonia-lyase 3 (PAL3) did not change significantly. While the induction of the AtEBP transgene and PDF1.2 had similar DEX concentration requirements, the kinetics of induction differed significantly, with the AtEBP transgene being induced within 1 h and PDF1.2 only being induced between 24 and 48 h. Although the molecular mechanisms underlying the growth defects and changes in gene expression remain to be determined, these changes appear to result from the glucocorticoid-inducible system itself, and may therefore limit the usefulness of this system for controlling gene expression in Arabidopsis.