TNFalpha increases in vitro migration of human HPV18-positive SW756 cervical carcinoma cells.

TNFalpha has been associated with both, tumor survival and apoptosis. This cytokine is also involved in promoting cell migration during wound healing and tumorigenesis. SW756 is a HPV18-positive cervical carcinoma cell line, which has been used to study different mechanisms of cervical cancer progression. An in vitro assay of scratch wound healing onto monolayers of SW756 cells was used to assess the effect of TNFalpha on cell migration into a wound space. It was found that SW756 cells have the ability to migrate, but not proliferate in response to scratch wounding in a serum-free medium supplemented with TNFalpha. RT-PCR analysis showed that SW756 cells express TNFalpha mRNA when incubated in medium with and without serum. Wound closure and migration rate of SW756 cells were significantly increased in the presence of serum-free media supplemented with TNFalpha (10 ng/mL) as compared to serum-free media, and media supplemented with either anti-TNFalpha antibody or both TNFalpha and anti-TNFalpha antibody (p < 0.05). The results showed a stimulatory effect of TNFalpha on the migration of SW756 cervical carcinoma cells, suggesting a novel and important role for TNFalpha in cervical cancer progression.     

Probiotics enhance the recovery of gut atrophy in experimental malnutrition.

AIM: The aim of this study was to evaluate the effect of probiotics on the recovery of the bowel atrophy induced by malnutrition in rats. METHODS: Twenty-and-six Wistar rats (200-250g) were fed with either a normoproteic (sham group, n=6) or a free-protein diet (n=20) during 12 days. Twelve malnourished rats were randomized to recover during 15 days with either a hydrolyzed diet (control group, n=6) or the same diet enriched with probiotics (Streptococcus thermophilus and Lactobacillus helveticus; probiotic group, n=6). RESULTS: Probiotic group showed similar gain of body, liver and bowel weight than controls. At the jejunum, both the villus height (383+/-49 vs. 321+/-46mm; p=0.04) and crypt depth (157+/-31 vs. 125+/-10mm; p=0.04) were greater in probiotic group than in controls. The crypt depth at the cecum (214+/-22 vs. 169+/-43 mm; p=0.05) and the wall width at both the cecum (410+/-18 vs. 340+/-61 mm; p=0.02) and sigmoid (479+/-130 vs. 330+/-62 mm; p=0.03) were higher in probiotic than in control group. CONCLUSION: Streptococcus thermophilus and Lactobacillus helveticus enhance the recovery of gut atrophy induced by malnutrition. Probiotics can be useful as oral adjuvants during the recovery of malnutrition.     

Morphological and molecular pathology of CCL4‐induced hepatic fibrosis in connexin43‐deficient mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl₄)‐induced hepatic fibrosis in mice. We administered CCl₄ to both Cx43‐deficient (Cx43^+/?) and wild‐type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real‐time PCR. We demonstrated that Cx43^+/? mice developed excessive liver fibrosis compared with wild‐type mice after CCl₄‐induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43^+/? mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43^+/? mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl₄‐treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

An electron microscopic analysis on the ultra structural abnormalities in sperm of the common carp Cyprinus carpio L. inhabiting a polluted lake, Umiam (Meghalaya, India)

The present communication reports the ultra structural abnormalities in sperm of a fish species Cyprinus carpio inhabiting a polluted lake, Umiam in North‐East India. Transmission electron microscopy (TEM) revealed absence of differentiation between head and midpiece (neck) of some sperm while scanning electron microscopy (SEM) showed some sperm tails with highly reduced length and some sperm with folded tail. Abnormal shape of some sperm head was also revealed by Scanning electron microscopy. Detachment of membrane from some parts of the sperm head and an outward expansion of the same was observed from Transmission electron micrographs of transverse section of sperm head. The well developed mitochondria surrounding the cytoplasmic channel in the sperm tail, as observed in control were found to be drastically disorganized in fish inhabiting the polluted lake. The study suggests that the fish C. carpio inhabiting the polluted lake Umiam is under severe stress as far as its male reproductive system is concerned. The study further suggests that Electron microscopic approach is extremely important in the assessment of adverse effects of environmental pollution on fish tissue. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.     

The hepatocytes of the brown trout (Salmo trutta fario): A stereological study of some cytoplasmic components with the breeding cycle

Sex differences exist in fish hepatocytes, but studies for characterizing their cytology throughout the breeding cycle are still scarce; suggesting changes, but most lacking quantitative data. To address this limitation, to complement baseline data generated from the brown trout model, and to prove that sex‐specific seasonal changes exist, we made an unbiased stereological evaluation of the hepatocytic cytoplasm. Unprecedentedly for fish liver, the stereological design was exempt from model (biased) assumptions. Five (3 years old) animals per sex were studied in endogenous vitellogenesis, exogenous vitellogenesis, and spawning season end. Liver pieces for analysis were systematically sampled. Stereology was done in transmission electron microscopy (TEM) micrographs. Primary data generated relative volume estimates of the major cytoplasmic components. Such values were used for deriving absolute volumes (per cell and per liver). Lipid droplets did not show changes. As to other targets, trends at cell and liver levels were not always equal. If the hepatocyte was the reference space, the contents in mitochondria, dense bodies, glycogen, and cytosol changed seasonally, in both sexes. If taking the liver as the reference, changes attained the Golgi apparatus and rough endoplasmic reticulum (RER), besides dense bodies, glycogen (in females), and cytosol. The components volumes (namely per liver) were often positively (negatively for glycogen) correlated with the ovary weight, disclosing new associations and implications in fish. While also offering gold‐standard data for backing morphofunctional correlations and pathology, we revealed a new process by which females increase the amount of RER and Golgi throughout vitellogenesis, breaking from the idea on how this event happens in fish. Microsc. Res. Tech. 73:766–778, 2010. © 2010 Wiley‐Liss, Inc.     

Bacterial endotoxins in periodontal health and diseases

Bacterial endotoxins are a major concern in periodontal health and diseases owing to their structure and biological activity. With up-to-date knowledge of endotoxins and the recent findings about the influence of endotoxins in dental health, their probable mode of pathogenesis, and standard detection methods, this review analyzes the potential efficacy and benefits of probiotics in combination with conventional and contemporary treatment measures. In the oral cavity, Gram-negative bacteria are documented to predominate in the pulpal lesions with radiolucent areas and in the root canal with pulp necrosis, where they pose an absolute threat by promoting a series of inflammatory reactions. Endotoxin, a constituent of Gram-negative bacteria establishes a nexus between cytokine stimulation and proinflammatory reactions, therefore plays a critical role in decaying dental pulp and modulating periodontal diseases. Currently, the treatment regimen involves several biochemical preparations. In addition, probiotics have been reported to control endotoxin in gingivitis and contribute to the overall improvement of dental health. A potential benefit of a combination of probiotics as a complementary treatment along with the conventional treatment warrant more empirical evidence to elucidate its role and mechanism in resolving the clinical manifestations associated with endotoxins in the periodontal region.     

The influence of probiotics of different microbiological composition on histology of the gastrointestinal tract of juvenile Oncorhynchus mykiss

This article presents results of the influence of three probiotic feed additive of various microbiological composition: Bacillus subtilis (VKPM B-2335); B. subtilis (OZ-2 VKPM-11966) + Bacillus amyloliquefaciens (OZ-3 VKPM-11967); Lactobacillus acidophilus (VKPM B-3235) on the growth and histology of the organs of the gastrointestinal tract of juvenile Oncorhynchus mykiss by morphometric parameters. These probiotic bacteria are the most commonly used in aquaculture. The effect of the probiotic feed additive led to the increase in fish growth and influenced different sections of the gastrointestinal tract. The biggest change was found in the mid intestine and the reliable difference compared with the control diet was obtained at the following parameters: lamina propria width, intraepithelial lymphocytes number of prismatic epithelium and goblet cells area. The changes in the pyloric appendages were less obvious but reported as playing an important functional role in digestion. The liver preserved normal functional structure in all series of the experiment except for the group with L. acidophilus, where hepatocyte small-drop vacuolization was observed. That might be connected with the change of the digest activity resulting from a decrease in secretory activity of the intestinal exocrinocytes. The use of all probiotic feed additives led to a similar change in morphometric parameters in all groups, which suggests a decrease in the immune response.     

Immunocytochemical localization of apolipoprotein A-I using polyclonal antibodies raised against the formaldehyde-modified antigen

The preparation of cells for electron microscopy, in particular the fixation and embedding routine, influences the antigenicity, often resulting in a markedly reduced labelling intensity. To overcome the difficulties associated with fixation-induced changes in antigenicity, we produced antibodies against pre-fixed human apolipoprotein (apo) A-I. Purified apo A-I was fixed with 4% formaldehyde and was used to raise polyclonal antibodies in rabbits. The antiserum was purified by protein-A-Sepharose followed by affinity chromatography with the fixed antigen coupled to vinylsulphone-activated agarose. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed lipoproteins. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, indicating that the antibodies were directed against epitopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells. When HepG2 cells were exposed to anti-apo A-I antibodies followed by a secondary FITC-labelled antibody, fluorescence was found intracellularly in distinct regions. Electron microscopy revealed that the endoplasmic reticulum, and in particular the trans elements of the Golgi complexes, were the main compartments stained for apo A-I both in HepG2 cells, as shown by the immunoperoxidase technique, and in human hepatocytes, as shown by the protein-A-gold technique on ultrathin cryosections. The findings demonstrate the potential of using antibodies to fixed antigens as a strategy to overcome impaired localization due to fixation in cytochemistry at the light microscopic and electron microscopic levels.     

Sinusoidal endothelial cells of the liver: Fine structure and function in relation to age

Liver endothelial cells form a continuous lining of the liver capillaries, or sinusoids, separating parenchymal cells and fat-storing cells from sinusoidal blood. Liver sinusoidal endothelial cells differ in fine structure from endothelial cells lining larger blood vessels and from other capillary endothelia in that they lack a distinct basement membrane and also contain open pores, or fenestrae, in the thin cytoplasmic projections which constitute the sinusoidal wall. This distinctive morphology supports the protective role played by liver endothelium, the cells forming a general barrier against pathogenic agents and serving as a selective sieve for substances passing from the blood to parenchymal and fat-storing cells, and vice versa. Sinusoidal endothelial cells, furthermore, significantly participate in the metabolic and clearance functions of the liver. They have been shown to be involved in the endocytosis and metabolism of a wide range of macromolecules, including glycoproteins, lipoproteins, extracellular matrix components, and inert colloids, establishing endothelial cells as a vital link in the complex network of cellular interactions and cooperation in the liver. Fine structural studies in combination with the development of cell isolation and culture techniques from both experimental animal and human liver have greatly contributed to the elucidation of these endothelial cell functions. Morphological and biochemical investigations have both revealed little changes with age except for an accumulation of iron ferritin and a decrease in the activities of glucose-6-phosphatase, Mg-ATPase, and in glucagon-stimulated adenylcyclase. Future studies are likely to disclose more fully the role of sinusoidal endothelial cells in the regulation of liver hemodynamics, in liver metabolism and blood clearance, in the maintenance of hepatic structure, in the pathogenesis of various liver diseases, and in the aging process in the liver.     

Scanning electron microscopic and histological studies of the buccal cavity of a phytoplanktivorous small freshwater fish,Amblypharyngodon mola

The electron microscopic and histological studies of the buccal‐cavity of herbivorous fish Mola (Amblypharyngodon mola) were performed. The studies revealed that the architectures of the buccal cavity of A. mola support the herbivory nature of the fish. Both the upper and lower jaws of the fish are rich in mucus glands, unculi, and microridges. The presence of papillae like taste buds in the lower jaw of A. mola indicates the mechanosensory role of the lower jaw during gustation. These features directly support a gustatory feeding behavior associated with filter feeding in this small freshwater fish.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

Melanization stimulating factor (MSF) and melanization inhibiting factor (MIF) in the integument of fish

The present studies were directed to demonstrate that adult fish skin contains putative factors that affect chromatophore and/or chromatoblast function. This hypothesis is based upon the possibility that hypo and/or hyperpigmented areas of the skin are so pigmented because of the localized expression of intrinsic factors that are either stimulatory or inhibitory to the differentiation of specific pigment cell types. In all the morphological and biochemical experiments carried out, we used culture media conditioned by dorsal (DCM) or ventral (VCM) skin from different species of fish. Both DCM and VCM were capable of stimulating differentiation of melanophores in neural crest explants. While the stimulation of melanization is an activity present in both dorsal and ventral skin, an inhibitory activity is also present in ventral skin at such a concentration that it overrides the stimulatory activity afforded by DCM. With biochemical assays, we demonstrated that the three important sequential enzymatic steps in melanogenesis are all stimulated by the conditioned media in a dose-dependent manner and this results in an increase in the amount of melanin present in cultured cells. The results of our investigations provide strong evidence that there are intrinsic pigment cell regulatory factors in the integument of fish, the inhibitory activity being stronger in the ventrum, and that those factors strongly influence, perhaps even determine, the pigment patterns of fish.     

Calcium hypochlorite as a dentin deproteinization agent: Microleakage,scanning electron microscopy and elemental analysis

Purpose: This study investigated the influence of collagen removal with calcium hypochlorite on the surface morphology of acid‐etched dentin and on the microleakage of composite restorations. In addition, the elemental composition (EC) of dentin after removal of the collagen fibrils was analyzed. Materials and Methods: Forty third molars received two cavities and were divided into four groups according to dentin treatment: CTRL—no pre‐treatment; Na10—10% NaOCl for 30 s; Ca10—10% CaOCl for 30 s, and Ca15—15% CaOCl for 30 s. The cavities were filled using an acetone‐based adhesive system and a resin composite; they were then subjected to thermal cycling for 5,000 cycles, immersed in methylene blue for 4 h and sectioned into 1‐mm thick slabs. Two examiners evaluated two slices per tooth using a stereomicroscope and assigned the degree of infiltration (scores 0–3). The data were analyzed using the Kruskal–Wallis (α = 0.05). Four teeth received surface treatment according to the groups and were submitted to SEM and EDS to carry at the EC. Results: There was no significant difference between the experimental groups (P = 0.533). CaOCl alters the morphology and surface composition of the dentin, resulting in an increase in the amount of calcium in the interface. Conclusions: When used prior to an acetone‐based adhesive system, CaOCl did not produce any differences in microleakage when compared to the CTRL group or to the Na10 group. Microsc. Res. Tech. 78:676–681, 2015. © 2015 Wiley Periodicals, Inc.     

Estimating volumes from common carp hepatocytes using design‐based stereology and examining correlations with profile areas: Revisiting a nutritional assay and unveiling guidelines to microscopists

Assessing fish liver status is common in aquaculture nutrition assays. This often implies determining hepatocytes profile areas in routine thin (5–7 μm) histological sections. However, there are theoretical problems using planar morphometry in thin sections: inherent sampling cells biases, too small numbers of sampled cells, under/overestimation of size, measuring size as areas when cells are three‐dimensional (3D) entities. The gold standard for assessing/validate cell size is stereology using thick sections (20–40 μm). Here, we estimated the volume of hepatocytes and their nuclei by the nucleator and optical disector stereological probes (in thick sections), and, innovatively, in thin sections too (using single‐section disectors). The liver of common carp eating feed containing either low or high level of lipids was targeted. Results were compared with prior profile areas from planar morphometry using thin sections, and with profile areas estimated here with the two‐dimensional (2D) nucleator. Ratios between nucleus and cell/cytoplasm (N/C) areas and volumes were calculated and compared. There was high positive correlation between volumes in thin and thick sections (r = .85 to .89; p < .001), empirically validating the single‐section disector. Strong correlations existed between profile‐derived versus 2D‐nucleator areas (r = .74 to .83; p < .001). There was systematic underestimation of cells and nucleus size using planar morphometry. The N/C ratios derived from the 2D‐nucleator data were higher than those from planar morphometry. Despite theoretical premises for using simple planar morphometry in thin sections are flawed, our results support that such morphometry on carp/fish hepatocytes may offer some valid biological conclusions. Anyway, we advanced guidelines for implementing proper methods.     

Wear damage to spur gears

Electron microscopic studies of damage to spur gears in an experimental gearbox have shown that rolling contact fatigue plays a major role supplemented by abrasive wear. It is noteworthy that the most voluminous part of the wear debris consisted of extremely fine particles of thicknesses below 0.2 μm.     

Knockdown of lncRNA XIST prevents the epithelial-mesenchymal transition of TGF-β2-induced human lens epithelial cells via miR-124/Slug axis

Posterior capsular opacification (PCO) is linked to the pathological process of lens epithelial cells, which includes proliferation, migration, and epithelial-mesenchymal transition (EMT). Our goal was to investigate whether long noncoding RNA (lncRNA) XIST contributes to EMT via targeting miR-124/Slug axis in TGF-β2-induced SRA01/04 cells. EMT was induced by stimulating SRA01/04 cells with 10 ng/mL TGF-β2 for 24 h, and PCO microenvironment was simulated. The expressions levels of lncRNA XIST, miR-124, and Slug were measured by real-time polymerase chain reaction (RT-PCR) and western blot. The role and mechanism of lncRNA XIST in promoting EMT of TGF-β2-treated SRA01/04 cells were investigated by using cell transfection, cell counting kit-8 (CCK-8), immunofluorescence staining, transwell assay, wound-healing assay, RT-PCR, western blot and dual-luciferase reporter assay. The expression of Slug and lncRNA XIST was markedly increased, while miR-124 was downregulated in TGF-β2-treated SRA01/04 cells compared with the control group. Knockdown of lncRNA XIST reduced EMT, migration, and cell viability in TGF-β2-induced SRA01/04 cells; moreover, it adversely modulated miR-124 and adjusted the expression of Slug in SRA01/04 cells, while these effects were diminished by co-transfection with AMO-miR-124. Our data demonstrated that lncRNA XIST functioned as a competitive endogenous RNA (ceRNA) of miR-124 to modulate the expression level of Slug, thereby modulating EMT, migration, and cell viability in SRA01/04 cells. In conclusion, lncRNA XIST has a crucial role in promoting TGF-β2-induced EMT via modulating the miR-124/Slug axis in SRA01/04 cells, and it may provide a novel therapeutic option for PCO treatment.     

Effects of exposition to polluted environments on blood cells of the fish Prochilodus lineatus

In this study, we investigated the effects of contaminated water on the blood parameters of the fish Prochilodus lineatus exposed during 7 and 20 days. Blood was collected with heparinized syringes, and blood smears were prepared and stained with Leishman stain. Slides were examined and photographed using a Leica light microscope. Total white blood cell counts and differential counts of thrombocytes and leukocytes were obtained for statistical analysis. The group exposed to water samples from Lago Azul exhibited an increase in the number of leukocytes and in the total number of white blood cells, suggesting that the chemical contaminants in this environment were acting similarly to antigens in this fish species, causing the proliferation of defense cells. In the group exposed to detergent during 20 days, the number of thrombocytes decreased. These results suggest that the variations in the number of leukocytes were indicators of environmental pollution and that biodegradable detergents may, after a certain time of exposure, affect vital functions in fish, such as coagulation and prevention of infections, which directly involves thrombocytes.     

Qualitative study of young,adult, and aged wistar rats temporomandibular synovial membrane employing light,scanning, and transmission electron microscopy

The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

Uncommon hepatic macrophagic foamy‐cell nodules in Iberian barbel (Luciobarbus bocagei Steindachner, 1864) from the Vizela River (Portugal)

The main intent of this work (after the by chance finding, in archived histological slides) is to characterize one previously non‐described liver lesion of the Iberian barbel from the Vizela River (Portugal). This ran through a textile and dyeing industrial region. The lesion type was made of groups of foamy cells (presumptive macrophages), which appear either as a “smaller non‐nodular form,” without a connective tissue capsule and displaying an irregular profile, or as a “bigger nodular form,” presenting a thin capsule and a circular profile. The nodular forms could appear multi‐layered, resembling “cross‐sectioned onions”. The lesions number, dimension, and structural complexity were greater in bigger fish, appearing only after a history of poor water quality. In extreme cases, the lesions slightly protruded the liver surface. Special histological staining proved the connective tissue nature of the capsule (and its eventual septa), the presence of proteins, glycoproteins, lipofuscin, melanin, iron (putative hemosiderin), and copper, in a variable number of foamy cells within the lesions. At times, degenerating hepatocytes appeared at the border of the lesions. It is proposed that this lesion type incorporates both macrophages and degenerating hepatocytes, looking as one (unpublished) form of a macrophage aggregate. The term “foamy‐cell nodules” was advanced for this abnormality. The lesion could have been induced by pollution, because: no parasites were ever associated with the lesion; there was co‐existence of the lesion with a water quality status scored as “bad”; such lesion neither existed in fish sampled after mitigation and remediation measures nor in reference fish.