Toll-like receptor-7 signaling in Kupffer cells exacerbates concanavalin A-induced liver injury in mice.

Concanavalin A (ConA) is a plant lectin that can induce immune-mediated liver damage. ConA induced liver damage animal model is a widely accepted model that can mimic clinical acute hepatitis and immune-mediated liver injury in humans. Toll-like receptor-7 (TLR7), a member of the TLR family, plays a key role in pathogen recognition and innate immune activation. The aim of this study was to examine the role of TLR7 in the pathogenesis of ConA-induced liver injury. Acute liver injury was induced by intravenous injection with ConA in WT (wild-type) and TLR7 knockout (KO) mice. Results showed that attenuated liver injury in TLR7-deficient mice, as indicated by increased survival rate, decreased aminotransferase levels, and reduced pathological lesions, was associated with decreased release of pro-inflammatory cytokines in livers. Consistently, significantly decreased proliferation of CD4+ T cell was detected in ConA-stimulated TLR7-deficient splenocytes, but not in CD3/CD28 stimulated TLR7-deficient CD4+ T cells. Moreover, TLR7 deficiency in KCs specifically suppressed the expression of TNF-α (tumor necrosis factor-α). Depletion of KCs abolished the detrimental role of TLR7 in ConA-induced liver injury. Taken together, these results demonstrate that TLR7 can regulate the expression of TNF-α in KCs, which is necessary for the full progression of ConA-induced liver injury.

A novel model of image acquisition and processing for holistic quantification of angiogenesis disrupted by application of mainstream and sidestream cigarette smoke solutions.

Angiogenesis is a vital process in the growth of new blood vessels from pre-existing vasculature. Among several approaches being used for studies related to angiogenesis, chicken chorioallantoic membrane assay (CAM) is an excellent model system. However, its utility has been limited due to difficulty in quantifying putative angiogenic and anti-angiogenic response to an experimental compound in an objective and quantifiable manner. Herein, we report a novel approach of image acquisition and processing for better evaluation of neovascularization. The effects of mainstream cigarette smoke solutions (MSCSS) and sidestream cigarette smoke solutions (SSCSS) from different commercially available cigarettes on angiogenesis were quantified, using CAM assay. Different gross and nanometer scale topographies of CAMs were quantified, which are vital for 3D image scrutiny and can precisely enumerate angiogenesis. Pattern formation of blood vessels, diameter, area and 3D surface roughness of CAMs were substantially disrupted by application of cigarette-smoke extracts. An important point revealed in our study that SSCSS appeared to be significantly more toxic than MSCSS with respect to their effects on angiogenesis. This new imaging technique combined with other modalities, will provide a robust platform to optimize trial design and more patent studies in angiogenesis.

Diminished embryonic movements of developing embryo by direct exposure of sidestream whole smoke solutions.

Embryonic movements (EM) are considered to be the first sign of life and cigarette smoking during pregnancy has been linked to affect EM. Exposure to sidestream smoke, produced from the emissions of a smoldering cigarette, may result in poor pregnancy outcome and increased risk of serious perinatal morbidity and mortality. In this study, the chicken embryo bioassay was used to systematically assess the effects of short-term exposure to sidestream whole smoke solutions (SSWSS) on EM, recorded in real time by a video camera for 60 min and each EM was counted for every 3-min interval. Application of different types of SSWSS to the embryos caused significant changes in all types of EM from 15 to 18 min of recording time. Extensive reduction (P<0.001) and some time complete stoppage of swing-like movements and whole-body movements were observed in almost all treated embryos. Our data clearly link between exposure of SSWSS and substantial decrease in EM. It is unclear whether nicotine and/or other ingredients present in sidestream smoke are responsible for these alterations in EM. This article provides an outline of the relevance of SSWSS on EM for evolutionary developmental biology and this assay can be used to investigate the complex mixtures with regard to their effects on EM.

Toxicological screening for the effects of short-term exposure of sidestream cigarette smoke on angiogenesis.

Angiogenesis is a fundamental process that ensures adequate metabolic supply to tissues in numerous biological and pathological states, including tumorigenesis. Cigarette smoking causes numerous adverse effects, some of which are associated with disruption of the normal process of angiogenesis. It is believed that sidestream cigarette smoke severely affects different components that help in the normal process of angiogenesis. Sidestream cigarette smoke is composed primarily of smoke that emanates from the burning end of a cigarette, and contaminants that diffuse through the cigarette paper. The aim of the current study was to ascertain the toxicological effects of different sidestream cigarette smoke solutions (SSCSS) on angiogenesis by using chicken chorioallantoic membrane (CAM) assay. Decrease in total vascular area, diameter of secondary and tertiary blood vessels, cell proliferation, migration of blood vessels toward ectoderm, and number of capillary plexuses formation was observed by application of SSCSS. Scanning microscopy also revealed deviation in pattern formed by the major capillary plexuses and the fibrillar elements of the mesoderm SSCSS in treated CAMs. It is concluded that SSCSS may hinder the normal process of angiogenesis resulting in abnormal blood supply to tissues and decreased repair and remodeling, which are common problems among smoke-exposed individuals. Further study is required to delineate the effects of different chemicals in SSCSS on angiogenesis.

Toxicological effects of mainstream whole smoke solutions on embryonic movements of the developing embryo.

Cigarette smoking is unrivaled among developmental toxicants in terms of total adverse impact on the human population. Maternal tobacco use during pregnancy adversely affects prenatal and postnatal growth and increases the risk of behavioral and developmental defects in children and adolescents. In the current study, the effects of different preparations of nicotine and mainstream whole smoke solutions (MSWSS) on embryonic movements during neonatal development were examined in vivo, using the chicken embryo model, recorded in real-time by a video camera. It was observed that low doses of nicotine induced hyperactivity and higher doses induced hypoactivity. Accordingly, a significant (p < 0.01) decrease in movements was observed by application of 10 microg of nicotine and different preparations of MSWSS. A dose-dependent decrease in embryonic movements was observed, which did not recover by the end of experiment. It was concluded that nicotine could alter embryonic movements, which are important during embryogenesis for differentiation and maturation of the body systems.

Disruption of normal embryonic angiogenesis by direct exposure of mainstream whole smoke solutions of commercial cigarettes.

Angiogenesis is activated in the female reproductive system during embryogenesis and embryo implantation. Smoking during pregnancy has been linked to interfere with normal process of angiogenesis resulting an increased incidence of ectopic pregnancy, spontaneous abortion, preterm delivery and sudden infant death syndrome. Chorioallantoic membrane (CAM) assay was used as an alternative in vivo approach to evaluate the toxicological effects of different mainstream whole smoke solutions (MSWSS) of commercial cigarettes on embryonic angiogenesis. Seventy 5-day-old CAMs, divided in seven groups were exposed to MSWSS with different nicotine concentration: 0.2mg (group B), 0.3mg (group C), 0.5mg (group D), 0.6mg (group E), 0.7mg (group F) and 1mg (group G). All smoke solutions caused varying levels of disruption on the normal process of angiogenesis and have shown to adversely affect the diameters of blood vessels, capillary plexus formation and organization of the fibrillar materials of CAMs. Abbot curve, angular spectrum and 3D surface roughness of CAMs were also measured for precise quantification of angiogenesis. Moderate to dramatic changes were observed in all treated groups with a very highly significant (P<0.001) disruption observed on CAMs of group G. No significant change was observed in different groups treated with pure nicotine. Current observations demonstrated that MSWSS of different commercial cigarettes have toxic effects on the process of angiogenesis and smoking during pregnancy may lead to an increased risk of spontaneous abortion and preterm delivery.

A novel image probing system for precise quantification of angiogenesis.

The rapid development of clinical diagnostic imaging technology, in tandem with medical and angiogenesis research, has led to some major advances in healthcare. The chorioallantoic membrane assay is commonly used for studying normal angiogenesis as well as putative angiogenic and antiangiogenic substances. Despite the progress, it is generally recognized that a major problem is the lack of a suitable quantitative bioassay for angiogenesis. Image probing is a novel solution to this problem, which, together with its associated discipline of evaluating angiogenesis, is showing great potential not only for accurate measurement of even very small blood vessels but also for detailed three-dimensional quantification of blood vessels and surface characterization. This technique could be a helpful tool for quantification in angiogenesis research.