Club cell protein 16 as a biomarker for early detection of silicosis.

Background & objectives: Clinically silicosis is diagnosed by chest X-ray showing specific opacities along with history of silica dust exposure. Diagnosis is invariably made at an advanced or end stage when it is irreversible. Moreover, silicosis patients are susceptible to develop tuberculosis. Therefore, a suitable biomarker for early detection of silicosis is needed. This study evaluated the suitability of club cell protein (CC16) as a biomarker for early detection of silicosis. Methods: This pilot study included 121 individuals from X-ray-confirmed/advanced silicosis, moderate silica dust-exposed workers and healthy controls from western India. CC16 levels were quantified in serum samples through ELISA. Sensitivity and specificity of CC16 values at different cut-off points were calculated in both non-smokers and smokers. Results: Serum CC16 level was significantly (P <0.01) decreased in X-ray confirmed advanced silicosis patients (4.7±3.07 ng/ml) followed by moderately exposed workers (10.2±1.77 ng/ml) as compared to healthy non-exposed individuals (16.7±3.81 ng/ml). Tobacco smoking also caused a significant decrease of serum CC16 concentration in both healthy (10.2±1.12 ng/ml) and advanced silicosis workers (2.6±2.28 ng/ml) compared to non-smokers. Sensitivity and specificity of CC16 values were also found to be ≥83 per cent for screening all categories of individuals. Interpretation & conclusions: Because of high sensitivity and specificity, serum CC16 could be used as predictive biomarker for suspicion and early detection of silicosis, which would help in reducing/delaying premature deaths caused by silicosis. It would also control silicotuberculosis additionally.

Poisoning Cases Reported to Poison Information Centre, Ahmedabad, India: A Three Year Observational Study.

Introduction: Morbidity and mortality associated with pesticide poisoning is a major public health issue, especially in lower and middle income countries, including India. Timely understanding of poisoning trends is required for improved prevention. The objective of the present study was to analyze the trend of poisoning cases in Ahmedabad, India in the period of 2015-2017. Methods: Detailed history, including demographic data, risk factors, poisoning history, agents involved, and occupational influence were collected for poisoning cases reported to the Poison Information Centre in Ahmedabad. Cholinesterase activity and HPTLC method for detection of sanguinarine in urine were used to investigate the agents of poisoning. Non-parametric tests, such as Chi-square test and Mann-Whitney U Test were applied to test statistical significance between the groups. All statistical analysis was carried out using IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp. Results: A total 1373 poisoning cases were investigated. The incidence and fatality rate was found to be higher in males compared to females (M/F ratio 1.89:1). About 91.62% of the poisoning were through the oral route. Erythrocyte cholinesterase activity assay results indicated that 41.29% of the cases were due to organophosphorus/carbamate poisoning. Insecticides were found to be the agent of poisoning in 26.29% cases, and 11.07% of all the cases were agricultural workers. Poisoning with medications, household pesticides and chemicals were also reported. Few cases of food poisoning with sanguinarine were detected. Conclusions: The data presented here suggest that pesticides used for agriculture are the major source of poisonings. Implementation of usage guidelines, educating farmers and vulnerable population, and finding novel alternatives for highly toxic chemicals may be helpful in decreasing the number of poisoning cases.

Hepatoprotective effect of Lobelia alsinoides Lam. in Wistar rats.

BACKGROUND: Traditional healing practitioners of South India use fine paste (an Ayurvedic dosage form known as 'kalka') of Lobelia alsinoides Lam., an ethno medicinal plant for curing hepatic diseases. OBJECTIVE: To evaluate in-vivo hepatoprotective effect of a candidate formulation viz. kalka containing whole plant (L. alsinoides Lam.) in rat model of Carbon-tetrachloride (CCl4) induced hepatotoxicity. MATERIALS & METHODS: Hepatotoxicity was induced in Wistar albino rats by oral administration of 1.25 ml/kg CCl4 once every day for 7 consecutive days. A candidate kalka formulation (fine paste) was prepared and administered to rats at different dose rates of 0.54 g/kg, 1.08 g/kg and 2.16 g/kg daily. At the end of the study-period, the serum levels of aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total bilirubin, total protein, albumin and total cholesterol were monitored. Further, the hepatic pathology was evaluated for assessing the extent of hepatotoxicity in the control and hepatoprotective effect in treatment groups. Meanwhile in-vitro antioxidant activity of kalka was evaluated by hydroxy radical, nitric oxide and DPPH (2, 2 diphenyl-1-picrylhydrazil) radical scavenging assays. Further, a 'limit test' was done in accordance with OECD Guidelines 425 (acute toxicity). RESULTS: The animals treated with the fine paste of L. alsinoides did not show an elevation in the biochemical values compared to CCl4 treated rats and during histomorphologic evaluation, hepatoprotective effect was evident with scattered mitotic figures in the parenchyma. Acute toxicity evaluation indicated that doses up to 2500 mg/kg are not toxic to rats. It has a good anti-oxidant activity also. CONCLUSIONS: From the study, it was obvious that L. alsinoides had significant hepatoprotective effect in CCl4 induced liver toxicity in rats. This ethno medicinal plant is certainly a promising hepatoprotective drug in liver disorders.

Sirtuin 6 deficiency transcriptionally up-regulates TGF-ß signaling and induces fibrosis in mice.

Caloric restriction has been associated with increased life span and reduced aging-related disorders and reduces fibrosis in several diseases. Fibrosis is characterized by deposition of excess fibrous material in tissues and organs and is caused by aging, chronic stress, injury, or disease. Myofibroblasts are fibroblast-like cells that secrete high levels of extracellular matrix proteins, resulting in fibrosis. Histological studies have identified many-fold increases of myofibroblasts in aged organs where myofibroblasts are constantly generated from resident tissue fibroblasts and other cell types. However, it remains unclear how aging increases the generation of myofibroblasts. Here, using mouse models and biochemical assays, we show that sirtuin 6 (SIRT6) deficiency plays a major role in aging-associated transformation of fibroblasts to myofibroblasts, resulting in tissue fibrosis. Our findings suggest that SIRT6-deficient fibroblasts transform spontaneously to myofibroblasts through hyperactivation of transforming growth factor ß (TGF-ß) signaling in a cell-autonomous manner. Importantly, we noted that SIRT6 haploinsufficiency is sufficient for enhancing myofibroblast generation, leading to multiorgan fibrosis and cardiac dysfunction in mice during aging. Mechanistically, SIRT6 bound to and repressed the expression of key TGF-ß signaling genes by deacetylating SMAD family member 3 (SMAD3) and Lys-9 and Lys-56 in histone 3. SIRT6 binding to the promoters of genes in the TGF-ß signaling pathway decreased significantly with age and was accompanied by increased binding of SMAD3 to these promoters. Our findings reveal that SIRT6 may be a potential candidate for modulating TGF-ß signaling to reduce multiorgan fibrosis during aging and fibrosis-associated diseases.

Acute oral toxicity of pesticide combination (acephate 50% and imidacloprid 1.8% as active ingredients) in Sprague-Dawley rats.

AIM: The aim of this study was to assess the acute toxic interaction and lethal dose (LD50) of pesticide combination product (acephate 50% and imidacloprid 1.8% as active ingredients) available in the market in Sprague-Dawley female rats by oral route. MATERIALS AND METHODS: A total of 10 Sprague-Dawley female rats were divided into two groups, comprising five rats in each dose group. Both groups were identified as control and test groups, respectively. Control group received sterile water as vehicle and test group received pesticide combination (acephate 50% and imidacloprid 1.8% as active ingredients) at a dose of 0 and 2000 mg/kg body weight. As per the Organization for Economic Cooperation and Development Guideline 420, initially one animal each from both the control and test groups were dosed with 0 and 2000 mg/kg, respectively, as sighting study. Based on the results of sighting study, additionally, four animals each from both groups were dosed with the same dose to make a total of five animals in each group. Dose volume was constant as 10 mL/kg. All animals were observed daily twice for clinical signs and mortality. Body weight was recorded on day 0 and weekly thereafter during 14 days' observation period; last body weight (fasted) was recorded on day 15. All the rats of both the groups were humanely sacrificed on day 15 for gross pathology, collection of organs for histopathology, organ weighing, and morphometry. Organ weights were taken as absolute values, and relative organ weights to last fasted body weights were calculated. RESULTS: Pesticide combination (acephate 50% and imidacloprid 1.8% as active ingredients) treated rats showed cholinergic signs with one mortality in the test group. No significant difference was observed in body weight, relative organ weights, and organ morphometry between pesticide combination exposed and non-exposed groups. Gross pathology of the treated rats was also comparable with respect to control group. Histopathological changes in the liver, kidneys, heart, lung, adrenaline, spleen, and ovaries of test group rats were found to be comparable with control group rats. CONCLUSION: The present study demonstrated the LD50 of one of the combination products available in the market having acephate 50% and imidacloprid 1.8% as active ingredients in Sprague-Dawley female rats which is >2000 mg/kg body weight. Furthermore, gross, histopathology and histoarchitectural alterations of all the vital organs of the test group were comparable to the control.

Diabetes induces fibrotic changes in the lung through the activation of TGF-ß signaling pathways.

In the long term, diabetes profoundly affects multiple organs, such as the kidney, heart, brain, liver, and eyes. The gradual loss of function in these vital organs contributes to mortality. Nonetheless, the effects of diabetes on the lung tissue are not well understood. Clinical and experimental data from our studies revealed that diabetes induces inflammatory and fibrotic changes in the lung. These changes were mediated by TGF-ß-activated epithelial-to-mesenchymal transition (EMT) signaling pathways. Our studies also found that glucose restriction promoted mesenchymal-to-epithelial transition (MET) and substantially reversed inflammatory and fibrotic changes, suggesting that diabetes-induced EMT was mediated in part by the effects of hyperglycemia. Additionally, the persistent exposure of diabetic cells to high glucose concentrations (25 mM) promoted the upregulation of caveolin-1, N-cadherin, SIRT3, SIRT7 and lactate levels, suggesting that long-term diabetes may promote cell proliferation. Taken together, our results demonstrate for the first time that diabetes induces fibrotic changes in the lung via TGF-ß1-activated EMT pathways and that elevated SMAD7 partially protects the lung during the initial stages of diabetes. These findings have implications for the management of patients with diabetes.

A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces.

OBJECTIVE: We aim to develop a simple, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which can be used for studying cardiac hypertrophy in vitro. METHODS: Keratin was extracted from human hair and applied as nanoscale coating onto the culture dishes. Physical parameters such as surface morphology and roughness of the coating were studied by SEM and AFM. Cardiomyocyte specific markers were assessed by immunofluorescence. Signaling pathways activated in hypertrophy were analyzed by western blotting and changes in the expression of fetal genes were analyzed by qPCR. The changes in the calcium fluxes were observed microscopically using Fluo-4. RESULTS: Keratin coated surfaces displayed a uniform coating and comparable roughness across dishes. Our optimized protocol for isolating cardiomyocytes yielded up to ~106 cells per heart. Characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contractions on keratin-coated substrates similar to fibronectin-coated surfaces. Phenylephrine (PE) treated cardiomyocytes grown on keratin-coated substrates exhibited increased cell size, sarcomere organization and perinuclear ANP expression indicating the development of cardiac hypertrophy. In addition, we observed increased activation of Akt and ERK pathways, induction of the fetal genes and increased protein synthesis upon PE treatment, which are characteristics of cardiomyocyte hypertrophy. The protocol was extended to mouse cardiomyocytes and found to show similar results upon examination. CONCLUSION: We demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.

Comparative profiling of extractable proteins in extracellular matrices of porcine cholecyst and jejunum intended for preparation of tissue engineering scaffolds.

Scaffolds prepared from cholecyst and jejunum have differential immunological potential, despite similar biocompatibility, when used as subcutaneous grafts. The reason for differential immunogenicity is probably due to differences in the nature of protein composition and biomolecules in the extracellular matrices (ECMs) of source organs that are used for preparation of the scaffolds. Against this background, the present study aims to identify the extractable proteins of ECMs derived from porcine cholecyst and jejunum. The proteins were extracted and identified through a conventional database search following sodium dodecyl sulfate-polyacrylamide gel-electrophoresis separation and mass spectroscopy. The resultant protein profile was analyzed and at least 154 proteins in cholecyst-derived extracellular matrix (CDE) and 186 proteins in jejunum-derived extracellular matrix (JDE) were identified. Both the matrices contained several extracelluar proteins including fibronectin, nidogen, decorin, and lumican that are known to participate in wound healing responses. However, the CDE had fewer cellular proteins than JDE, especially the latter contained class-I and class-II histocompatibility antigens which are incriminated as potent immunogens responsible for graft rejection. The results of the study suggested that the ECMs used for the scaffold preparation need not be "acellular" and differences in the protein composition of the ECMs might have caused the differential wound healing responses. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 489-496, 2017.

Wound healing potential of scaffolds prepared from porcine jejunum and urinary bladder by a non-detergent/enzymatic method.

Scaffolds prepared using extracellular matrices of mammalian organs/tissues, when used as grafts, have wound healing potential. This paper evaluated the physical properties and in vivo wound healing potential of jejunum-derived scaffold (JDS) and urinary bladder-derived scaffold (UDS) of porcine origin prepared by a non-detergent/enzymatic method. The former had higher flexural rigidity and suture retention strength compared to the latter, but both of them had the essential flexural rigidity and suture retention strength required for skin grafts. Full thickness skin-wounds on rabbit dorsum were treated with these scaffolds and the wound healing ability was compared by studying histomorphology parameters such as re-epithelialisation, collagen deposition, angiogenesis, proliferation of cells, mesenchymal cell infiltration and myofibroblast response. The extent of these reactions was assessed using histomorphometry. The results indicated that both grafts initiated healing faster than those wounds without any graft, as evidenced by the extent of cell proliferation and mesenchymal cell infiltration. The myofibroblast response persisted longer in the non-graft assisted wound healing reaction compared to the healing in the graft assisted wounds. Moreover, the JDS induced higher cell proliferation and greater angiogenesis than UDS probably indicating better healing by the former. The results suggested that JDS and UDS prepared by non-detergent/enzymatic method have potential clinical applications.

Biocompatibility and Immunophenotypic Characterization of a Porcine Cholecyst-derived Scaffold Implanted in Rats.

Comparative histomorphological assessment of local response to implanted reference biomaterial, also called biocompatibility testing/evaluation, in an appropriate animal model is a widely practiced safety evaluation procedure performed on biomaterials before clinical use. Standardized protocols and procedures, originally designed for testing synthetic materials, available for the testing/evaluation do not account for the immunogenic potential of a candidate biomaterial. Therefore, it is appropriate to supplement the routine biocompatibility test reports with adjunct data that may provide insight into the immunogenic potential of candidate biomaterials, especially when testing biomaterials that are derived from mammalian sources. This article presents expanded safety evaluation data of a porcine cholecyst-derived scaffold (CDS) intended as a xenogeneic graft. The biocompatibility was tested in rat subcutaneous model in comparison with a reference material and the CDS was found biocompatible. However, when studied by immunohistochemistry and real-time reverse transcription polymerase chain reaction for the number and/or polarization of M1 macrophage, M2 macrophage, cytotoxic T-cell, helper T cell, TH1 cell, and TH2 cell, the CDS appeared to induce a differential local immunopathological tissue reaction despite the similarity in biocompatibility with the reference material. The adjunct data collected were useful for objectively assessing the safety of CDS as a xenograft.

Comparative local immunogenic potential of scaffolds prepared from porcine cholecyst, jejunum, and urinary bladder in rat subcutaneous model.

Extracellular matrices isolated from several mammalian organs/tissues have found several clinical uses as xenografts or implants. However, they may cause complications because of adverse immunologic reactions. Scaffolds that promote favorable graft-acceptance reaction are preferred for fabricating xenografts. The objective of this study was to evaluate the immunogenic potential of a porcine cholecyst-derived scaffold (CDS), prepared by a non-detergent/enzymatic method, in comparison with jejunum and urinary bladder-derived scaffolds in a rat subcutaneous model. Key graft-rejection/acceptance reaction was evaluated at the site of implantation by studying the occurrence and/or function of immunocompetent cells in the tissue reaction. There was differential occurrence of M1-macrophage, M2-macrophage, T-helper cells, T-cytotoxic cells, B-cells, and mast cells in the tissue reaction and the CDS attracted few cells compared with other scaffolds. Real-time polymerase chain reaction for evaluating mRNA of functional markers like inducible nitric oxide synthase (M1 macrophage), arginase 1 (M2 macrophage), interferon gamma (TH1 lymphocytes), and interlukin-4 (TH2 lymphocytes) suggested that the CDS, compared with the scaffolds prepared from small intestine and urinary bladder, elicited M2 macrophage and TH2 lymphocyte polarization that are congenial graft-acceptance reactions. The results indicated that CDS has less immunogenic potential compared with the scaffolds prepared from jejunum and urinary bladder when used as subcutaneous graft in rats. It was concluded that CDS is a promising animal-derived xenograft for biomedical application.

Biomaterial properties of cholecyst-derived scaffold recovered by a non-detergent/enzymatic method.

Isolation procedures for the recovery of extracellular matrices (ECMs) from animal organs/tissues that are useful in regenerative medicine involve multiple sequential steps/stages including collection of the source organ at slaughter, their transportation to laboratory, decellularization, decontamination, stabilization, and sterilization. Most of these steps require extensive use of chemicals/reagents/enzymes which may also adversely affect the quality of the scaffold. With an effort to minimize the use of chemicals/reagents/enzymes, while extracting biomaterial-grade ECM from porcine cholecyst (gall bladder), we performed preisolation ex situ incubation of the organ in a stabilizing agent that also caused in situ crosslinking of tissue-components and delaminated the collagen-rich ECM from the tissue-layer beneath the mucosa. The physical, chemical, and biological properties of the isolated scaffolds were similar to that of a commercially available porcine small intestinal submucosa. The cholecyst-derived scaffold not only satisfied preclinical safety-test procedures such as cytotoxicity, local response, and endotoxin load but also showed the potential to promote healing of full-thickness skin wound in a rabbit model. The procedure was also suitable for isolating scaffolds from other hollow organs such as jejunum and urinary bladder. It was concluded that enzyme/detergent treatment may be an avoidable step while isolating biomaterial-grade scaffolds from hollow organs.

Phenotypic modulation of cell types around implanted polyethylene terephthalate fabric in rabbit muscle.

Whereas the nature of healing reaction in skeletal muscle following implantation of biomaterial has been extensively studied, the extent of variation in cell phenotypes is poorly known. Here, we studied the phenotypic alteration of cell types following injury in skeletal muscle of rabbits implanted with a commonly used biomaterial, polyethylene terephthalate (PET) fabric. Following implantation, histomorphological features were studied after 1, 4, and 12 weeks. Routine objective histomorphological evaluation was supplemented with histochemistry for collagen and immunohistochemistry for proliferating cell nuclear antigen (PCNA), CD34, vimentin, and alpha smooth muscle actin (α-SMA). The extent of reaction was quantified. The foreign body giant cells were found to comprise subpopulations, based on the variation in vimentin detectability or the presence of differentially capable proliferating nuclei (PCNA positive). Many rhabdomyocytes adjacent to the implant were PCNA-positive and some of them showed CD34 positivity. Often, the rhabdomyocytes very near to implanted PET fabric assumed a myofibroblast phenotype as evidenced by vimentin and/or α-SMA positivity at immunohistochemistry. Overall, the results suggested a phenotypic alteration of native cell types following implantation of PET fabric in rabbit skeletal muscle. Quantification of such cell types at the tissue-material interphase in relation to the deposition of collagen may be desirable during safety evaluation of biomaterials by histomorphology.

Porcine cholecyst-derived scaffold promotes full-thickness wound healing in rabbit.

Graft-assisted healing is an important strategy for treating full-thickness skin wounds. This study evaluated the properties of porcine cholecyst-derived scaffold and its use for treating full-thickness skin wound in rabbit. The physical properties of cholecyst-derived scaffold were congenial for skin-graft application. Compared to a commercially available skin-graft substitute made of porcine small intestinal submucosa, the cholecyst-derived scaffold was rich in natural biomolecules like elastin and glycosaminoglycans. When used as a xenograft, it promoted healing with excess cell proliferation at early phases and acceptable collagen deposition in the later remodelling phases.

Advantages of hyaluronic acid as a component of fibrin sheet for care of acute wound.

Skin injury is followed by accumulation of a fibrin based provisional matrix which normally drives the process of wound repair. Exogenous fibrin with extra cellular matrix (ECM) components can also favor the wound healing process. In a preliminary study we found that lyophilized fibrin sheet (FS) arrest bleeding from rabbit skin wound but it remained dry during the repair period and did not accelerate the healing process better than untreated control. In the current study, hyaluronic acid (HA) was incorporated into FS and the resultant HA-FS promoted water retention and improved wound healing process. Gross-morphology, histopathology and histomorphometry were employed to compare qualitative and quantitative difference of wound healing in treated group against controls. In experimental sites (HA-FS), re-epithelialization was completed by 14 days with neo-vascularization and deposition of wavy bundles of collagen in the treated sites. Rate of healing process was different in treated and untreated wounds and most striking difference was the appearance of appendages, sebaceous gland and hair follicle at some locations in HA-FS treated sites. Therefore, HA with fibrin can create an effective wound care matrix which promotes water retention and wound healing potential.