Detachment Of Epithelial Cells Research Articles

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

Research Note: The effect of photoperiod on the NLRP3 inflammasome and gut microbiota in broiler chickens

The present study aimed to investigate the effect of photoperiod on the intestinal inflammation and gut microbiota. A total of 96 broiler chickens were divided into 2 groups and fed separately under 2 different photoperiods (12L:12D group and 23L:1D group) for 21 d. The results showed that the photoperiod of 23L:1D damaged duodenal tissue structure (intestinal villus erosion, mucosal epithelial cell detachment, and inflammatory cell infiltration), significantly increased the concentration of inflammatory cytokines (IL-1β, IL-18, IL-6, and TNF-α) and significantly increased the mRNA expression levels and protein expression levels of NOD-, LRR-, pyrin domain-containing protein 3 (NLRP3) and caspase1 (P <0.05) compared with 12L:12D, which indicating that extended photoperiod induced intestinal injury and activated NLRP3 inflammasome. 16S rRNA sequencing analysis revealed that Bacteroides was significantly decreased, Ruminococcus_torques_group, norank_f_Desulfovibrionaceae, GCA-900066575, Defluviitaleaceae_UCG-011, Lachnospiraceae_FCS020_group, norank_f_UCG-010 and norank_f_norank_o_Clostridia_vadinBB60_group and were significantly increased in the 23L:1D group, compared with the 12L:12D group (P < 0.05). The correlation analysis between differential microbial communities and intestinal inflammation showed that the relative abundance of Bacteroides was negatively correlated with the mRNA expression level of NLRP3 (P < 0.05) and the relative abundance of Ruminococcus_torques_group was positively correlated with the mRNA expression level of NLRP3 (P < 0.05). linear discriminant analysis (LDA) effect size (LEfSe) results (LDA > 4) showed that the relative abundance of Bacteroides was dramatically higher (P < 0.05) in the 12L:12D group, whereas the relative abundance of Ruminococcus_torques_group was noticeably higher (P < 0.05) in the 23L:1D group. By the comprehensive analysis of the gut microbiota, the interaction of gut microbiota (Bacteroides and Ruminococcus_torques_group) and NLRP3 inflammasome may contribute to the intestinal injury under the condition of extended photoperiod.

Online monitoring of epithelial barrier kinetics and cell detachment during cisplatin-induced toxicity of renal proximal tubule cells.

Real-time and non-invasive assessment of tissue health is crucial for maximizing the potential of microphysiological systems (MPS) for drug-induced nephrotoxicity screening. Although impedance has been widely considered as a measure of the barrier function, it has not been incorporated to detect cell detachment in MPS with top and bottom microfluidic channels separated by a porous membrane. During cell delamination from the porous membrane, the resistance between both channels decreases, while capacitance increases, allowing the detection of such detachment. Previously reported concepts have solely attributed the decrease in the resistance to the distortion of the barrier function, ignoring the resistance and capacitance changes due to cell detachment. Here, we report a two-channel MPS with integrated indium tin oxide (ITO) electrodes capable of measuring impedance in real time. The trans-epithelial electrical resistance (TEER) and tissue reactance (capacitance) were extracted from the impedance profiles. We attributed the anomalous initial increase observed in TEER, upon cisplatin administration, to the distortion of tight junctions. Cell detachment was captured by sudden jumps in capacitance. TEER profiles illuminated the effects of cisplatin and cimetidine treatments in a dose-dependent and polarity-dependent manner. The correspondence between TEER and barrier function was validated for a continuous tissue using the capacitance profiles. These results demonstrate that capacitance can be used as a real-time and non-invasive indicator of confluence and will support the accuracy of the drug-induced cytotoxicity assessed by TEER profiles in the two-channel MPS for the barrier function of a cell monolayer.

Comparative histomorphologic study of basement membrane side staining and additional epithelial side staining of the anterior lens capsule with Trypan Blue.

To compare the histomorphologic changes on the anterior lens capsule by both epithelial and basement membrane side staining to those of only basement membrane side staining of the anterior lens capsule with Trypan Blue (TB). A cross-sectional study was done on 72 samples from patients who underwent cataract surgery between April 2021 and September 2022. After capsulorhexis of the TB-stained capsule, it was made into two halves externally and one half labeled as controls (sample A). The other half was immediately stained further with TB on the epithelial side and was taken as cases (sample B). Samples were analyzed for lens epithelial cells and basement membrane changes. The loss of intactness of lens epithelial cells, partial or complete detachment of lens epithelial cells, degeneration of lens epithelial cells, and basement edema were significantly higher in cases compared to controls, whereas intactness of the basement membrane did not show any statistical significance between the two groups. There was a statistically significant decrease in cell density in cases compared to controls. Staining the epithelial side of the capsular bag with TB is more detrimental to lens epithelial cells and paves the way for a further study of staining the capsular bag before intra-ocular lens implantation to reduce the incidence of posterior capsule opacification.

Atmospheric pollutant black carbon induces ocular surface damage in mice

The threats of air pollution to human health have been gradually discovered, including its effects on eyes. The purpose of the study is to investigate the potential correlation between ocular surface exposure to black carbon and ocular surface structural damage as well as tear film dysfunction. To achieve this goal, 60 6-8-week-aged male BALB/C mice were randomly divided into 4 groups (n = 15). 0.5 mg/ml (group A), 1 mg/ml (group B), 5 mg/ml (group C) black carbon suspension droplets and PBS solution (group D) were used in the right eyes, 4 μl per time of three times per day. Tear break-up time, corneal fluorescein staining scores, and tear volume were assessed before treatment (day 0) and on days 4, 7, 10, and 14 after treatment. On day 14, the mice were sacrificed, and corneal and conjunctival tissues were collected for histological analysis. As the exposure time increased, there were no significant changes in the measured parameters from PBS-treated group of mice (P > 0.05). However, in the black carbon-treated group, there were significant decreases in tear film break-up time, significant increases in corneal fluorescein staining scores, and significant reductions in tear secretion (all P < 0.05). After 14 days, H&E staining of the corneal epithelium showed that in the PBS-treated group of mice, the corneal epithelial cells were neatly arranged, with no inflammatory cell infiltration, while in the black carbon-treated group, the corneal epithelium was significantly thickened, the basal cell arrangement was disrupted, the number of cell layers increased, and there was evidence of inflammatory cell infiltration. In the ultrastructure of the corneal epithelium, it could be observed that the black carbon-treated group had an increased amount of corneal epithelial cell detachment compared to the PBS-treated group, at the same time, the intercellular connections were looser, and there was a decrease in the number of microvilli and desmosomes in the black carbon-treated group. The results indicate that the ocular surface exposure to black carbon can result in a decrease in tear film stability and tear secretion in mice. Moreover, it can induce alterations in the corneal structure.

An enhanced model for environmental dry eye: Exploring pathological features and underlying factors

This study aimed to develop an enhanced environmental dry eye (EDE) model that accurately reproduces the etiology of prolonged visual fatigue and investigates the underlying pathological features. A total of 40 adult SPF-grade Wistar rats were randomly assigned to control (n = 20) and model (n = 20) groups. Rats in the control group were maintained under normal conditions, while rats in the model group were exposed to a controlled frontal airflow of 2–4 m/s from a fan for 7.5 h daily while placed on a suspended cylindrical wire mesh frame. Various assessments were performed at different time points during the 14-day experiment, including blink frequency, tear secretion (phenol red thread test), tear film breakup time (BUT), fluorescein staining (FL), corneal epithelial status (light microscopy), ultrastructure of corneal epithelial cells (electron microscopy), and expression levels of inflammatory cytokines (IL-1β, TNF-α) in tears (enzyme-linked immunosorbent assay). Additionally, mRNA and protein expression levels of MMP-9, IL1β, IL6, TNF-α, IFN-γ, and caspase-3 in corneal tissues were quantified (real-time quantitative PCR and Western blotting). Compared to the control group, the model group rats exhibited significant decreases in blink frequency (P < 0.001), tear secretion (Schirmer I test) values (P < 0.001), and tear film breakup time levels (P < 0.001). There was also a significant increase in fluorescein staining scores (P < 0.001) in the model group. Histological examination revealed distinct differences of the corneal epithelium between groups. The corneal epithelium of the model group appeared thicker, with disorganized cell arrangement in the superficial and basal layers, partial defects or detachment of superficial epithelial cells, and a rough, uneven surface. Scanning electron microscopy observations showed a rough corneal epithelial surface with numerous cracks and scattered vesicular-like structures in the model group. Furthermore, the model group rats exhibited a significant increase in expression of IL-1β and TNF-α in tears (P < 0.001), and upregulated expression levels of MMP-9, TNF-α, IL-1β, caspase-3, IL-6, and IFN-γ at both the mRNA and protein levels in corneal tissues (P < 0.001). In conclusion, the modified “wire-meshing cylindrical board” model effectively overcomes the limitations of the traditional “jogging board " dry eye model and successfully simulates the etiology of prolonged visual fatigue. This innovative EDE model demonstrates a high degree of relevance to dry eye conditions resulting from prolonged visual tasks, with a high success rate of model induction. Moreover, it proves to be a simple, practical, and easily replicable model, making it highly suitable for further studies on prolonged visual fatigue and facilitating its widespread adoption in research and clinical applications.

Evaluation of Imidacloprid Induced Changes in Thyroid Gland and its Amelioration with Withania somnifera in Wistar Rats

The study was conducted on a total of 48 female Wistar rats divided into four groups, each containing 12 rats. Group 1 was control, group 2 was imidacloprid control, group 3 was Withania somnifera control and group 4 was administered with both imidacloprid and Withania somnifera. The rats of group 3 and 4 were subjected to oral treatment of Withania somnifera (1 gm/kg feed) for a period of 30 days to measure the protective effect against thyroid toxicity induced by imidacloprid (30 mg/kg body weight/day for 30 days). Hormonal assays showed a significant (p&lt;0.05) rise in thyroid stimulating hormone and fall in triidothyronine and thyroxine in imidacloprid administered rats when compared to control. No gross lesions were noticed in thyroid gland. Group 2 thyroid gland sections revealed congestion, degenerated thyroid follicles with loss of colloid substance, increase in inter-follicular space, atropic follicles, fibrosis, pyknotic nuclei, detachment of follicular epithelial cells, increase in number of micro-follicles and proliferation of follicular epithelium towards lumen. Electron microscopy of thyroid gland from group 2 rats revealed significant pathological changes. The degree of lesions was minimal in group 4 rats when compared to group 2 rats. These results indicate that Withania somnifera is beneficial in ameliorating the imidacloprid induced thyroid toxicity in rats.

Deciphering the Role of p60AmotL2 in Epithelial Extrusion and Cell Detachment.

Preserving an accurate cell count is crucial for maintaining homeostasis. Apical extrusion, a process in which redundant cells are eliminated by neighboring cells, plays a key role in this regard. Recent studies have revealed that apical extrusion can also be triggered in cells transformed by oncogenes, suggesting it may be a mechanism through which tumor cells escape their microenvironment. In previous work, we demonstrated that p60AmotL2 modulates the E-cadherin function by inhibiting its connection to radial actin filaments. This isoform of AmotL2 is expressed in invasive breast and colon tumors and promotes invasion in vitro and in vivo. Transcriptionally regulated by c-Fos, p60AmotL2 is induced by local stress signals such as severe hypoxia. In this study, we investigated the normal role of p60AmotL2 in epithelial tissues. We found that this isoform is predominantly expressed in the gut, where cells experience rapid turnover. Through time-lapse imaging, we present evidence that cells expressing p60AmotL2 are extruded by their normal neighboring cells. Based on these findings, we hypothesize that tumor cells exploit this pathway to detach from normal epithelia and invade surrounding tissues.

Assessment of the water quality and toxicity effects on zebrafish (Danio rerio) of a stream near a phosphorus chemical plant in Guizhou Province, southwestern China.

To reveal the influence of the phosphorus chemical industry (PCI) on regional water environmental quality and safety, the water quality and ecotoxicological effects of a stream near a phosphorus chemical plant (PCP) in Guizhou Province, southwestern China, were investigated based on water samples collected from the stream. The results showed that the average concentrations of NH3-N, TN, P, F-, Hg, Mn, and Ni were 3.14mg/L, 30.09mg/L, 3.34mg/L, 1.18mg/L, 1.06μg/L, 45.82μg/L, and 11.30μg/L, respectively. The overall water quality of the stream was in the heavily polluted category, and NH3-N, TN, P, F-, and Hg were the main pollution factors. The degree of pollution was in the order of rainy period > transitional period > dry period, and the most polluted sample site was 1100m from the PCP. After 28 days of exposure to stream water, there was no significant change in the growth parameters of zebrafish. The gills of zebrafish showed a small amount of epithelial cell detachment and a small amount of inflammatory cell infiltration, and the liver tissue displayed a large amount of hepatocyte degeneration with loose and lightly stained cytoplasm. Compared with the control group, the%DNA intail, tail length, tail moment, and olive tail moment were significantly increased (p < 0.05), indicating that the water sample caused DNA damage in the peripheral blood erythrocytes of zebrafish. The stream water in the PCI area was found to be polluted and exhibited significant toxicity to zebrafish, which could pose a threat to regional ecological security.

Plasmin-Induced Lens Epithelial Cells Detachment for the Reduction of Posterior Capsular Opacification.

We sought to evaluate the efficacy and safety of plasmin injection in the capsular bag during the cataract operation for the prevention of posterior capsule opacification. Thirty-seven anterior capsular flaps taken from phacoemulsification surgery were immersed in either 1 µg/mL plasmin (plasmin group, n = 27) or phosphate-buffered saline (control group, n = 10) for 2 minutes and photographed after fixation and nuclear staining to compare the numbers of residual lens epithelial cells. In the animal experiments, the plasmin solution was injected into the capsular bag and remained for 5 minutes during hydrodissection or after lens extraction. The degree of posterior capsular opacity of the rabbits at 2 months were photographed by slit lamp biomicroscopy. In HLE-B3 cell culture, the cell detachment rate, proliferation, and apoptosis after the plasmin digestion were analyzed. The residual lens epithelial cell numbers on the capsule after plasmin treatment were 168 ± 190.7/mm2 in the 1 µg/mL plasmin group, which was significantly lower than that of the control (1012 ± 798.8/mm2; P < 0.0001). In a rabbit model, the treatment of plasmin resulted in a significantly clearer posterior capsule compared with that of the control group at 2 months postoperatively. This study suggested that plasmin injection can induce effective lens epithelial cell detachment, which could be a promising adjunctive treatment to further improve the success rate in posterior capsule opacification prevention. Plasmin injection for lens epithelial cell detachment could significantly decrease the number of residual lens epithelial cells. This approach could be a promising treatment incorporating the current treatment approach to further improve the success rate in posterior capsule opacification prevention.

Rosiglitazone attenuates hypoxia-induced renal cell apoptosis by inhibiting NF-κB signaling pathway in a PPARγ-dependent manner

Background In recent years, peroxisome proliferator-activated receptor γ (PPARγ) has been found to be closely associated with hypoxia renal disease. The aim of this study was to investigate the relationship between rosiglitazone and mitochondrial apoptosis in renal tissue and its associated mechanisms. Methods Twenty-four male Sprague-Dawley rats were randomly divided into three groups (n = 8 in each): normal control group, hypoxia injury group (equal volume of 0.9% saline), and PPARγ agonist group (Rosiglitazone, 10 mg/kg · d, intraperitoneally). The hypoxia injury group and PPARγ agonist group were placed in a hypoxia chamber and the simulated altitude was set at 7,000 m for 7 days. Blood and kidney samples were collected after 7 days. The quantitative real-time polymerase chain reaction and Western blot methods were used to determine the expression of PPARγ, nuclear factor kappa-B (NF-κB), B-cell lymphoma-2 (Bcl-2), and Bax. Results The results showed that compared with the normal control group, the renal tissue of rats after hypoxia was severely damaged, as shown by massive renal tubular epithelial cell degeneration and detachment, and renal tubular dilation. The NF-κB protein expression significantly increased, the Bcl-2 protein and mRNA expression significantly decreased, and Bax protein and mRNA expression significantly increased (p < .05 for all). Renal injury was much less severe in the PPARγ agonist group compared to the hypoxia injury group. Conclusions Rosiglitazone can alleviate hypoxia renal injury, with the possible mechanism involving attenuation of apoptosis by inhibiting the activation of the NF-κB signaling pathway in a PPARγ-dependent manner and increasing Bcl-2 and decreasing Bax expression.

ErbB2/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.

Distinct Cohorts of Aspergillus fumigatus Transcription Factors Are Required for Epithelial Damage Occurring via Contact- or Soluble Effector-Mediated Mechanisms.

Damage to the lung epithelium is a unifying feature of disease caused by the saprophytic fungus Aspergillus fumigatus. However, the mechanistic basis and the regulatory control of such damage is poorly characterized. Previous studies have identified A. fumigatus mediated pathogenesis as occurring at early (≤ 16 hours) or late (>16 hours) phases of the fungal interaction with epithelial cells, and respectively involve direct contact with the host cell or the action of soluble factors produced by mature fungal hyphae. Both early and late phases of epithelial damage have been shown to be subject to genetic regulation by the pH-responsive transcription factor PacC. This study sought to determine whether other transcriptional regulators play a role in modulating epithelial damage. In particular, whether the early and late phases of epithelial damage are governed by same or distinct regulators. Furthermore, whether processes such as spore uptake and hyphal adhesion, that have previously been documented to promote epithelial damage, are governed by the same cohorts of epithelial regulators. Using 479 strains from the recently constructed library of A. fumigatus transcription factor null mutants, two high-throughput screens assessing epithelial cell detachment and epithelial cell lysis were conducted. A total of 17 transcription factor mutants were found to exhibit reproducible deficits in epithelial damage causation. Of these, 10 mutants were defective in causing early phase damage via epithelial detachment and 8 mutants were defective in causing late phase damage via epithelial lysis. Remarkably only one transcription factor, PacC, was required for causation of both phases of epithelial damage. The 17 mutants exhibited varied and often unique phenotypic profiles with respect to fitness, epithelial adhesion, cell wall defects, and rates of spore uptake by epithelial cells. Strikingly, 9 out of 10 mutants deficient in causing early phase damage also exhibited reduced rates of hyphal extension, and culture supernatants of 7 out of 8 mutants deficient in late phase damage were significantly less cytotoxic. Our study delivers the first high-level overview of A. fumigatus regulatory genes governing lung epithelial damage, suggesting highly coordinated genetic orchestration of host-damaging activities that govern epithelial damage in both space and time.

Effects of hypoxia and acidification stress on ion regulation and gill structure of juvenile Larimichthys crocea.

To investigate the ion regulation of large yellow croaker (Larimichthys crocea) under hypoxia and acidification stresses, we investigated the effects of hypoxia (dissolved oxygen DO 3.5 mg·L-1, pH 8.1), acidification (DO 7.0 mg·L-1, pH 7.35) and combined stresses of hypoxia and acidification (DO 3.5 mg·L-1, pH 7.35) on gill tissue structure and physiological indices related to ion regulation of juvenile L. croaker. The results showed that, under hypoxia stress, gill Na+/K+-ATPase activity, serum Na+, Ca2+ and Cl- contents of juvenile L. croaker decreased first and then increased. Under acidification stress, gill Ca2+-ATPase activity, serum Na+ and Ca2+ contents of juvenile L. croaker increased first and then decreased. Under the combined stresses of hypoxia and acidification, Na+/K+-ATPase activity and Na+, K+ and Ca2+ contents increased first and then decreased, while Ca2+-ATPase activity and Cl- content decreased first and then increased. The results of gill histology showed that hypoxia and acidification stresses led to the detachment of gill epithelial cells, and the combined stresses of hypoxia and acidification led to proliferation, hypertrophy and swelling of gill epithelial cells. Comprehensive analysis showed that hypoxia and acidification stress affected the activities of major ion regulatory enzymes in juvenile L. croaker and caused different degrees of damage to gill tissue, resulting in imbalanced ion regulation in juvenile L. croaker.

Bacterial Toxins from Staphylococcus aureus and Bordetella bronchiseptica Predispose the Horse's Respiratory Tract to Equine Herpesvirus Type 1 Infection.

Respiratory disease in horses is caused by a multifactorial complex of infectious agents and environmental factors. An important pathogen in horses is equine herpesvirus type 1 (EHV-1). During co-evolution with this ancient alphaherpesvirus, the horse’s respiratory tract has developed multiple antiviral barriers. However, these barriers can become compromised by environmental threats. Pollens and mycotoxins enhance mucosal susceptibility to EHV-1 by interrupting cell junctions, allowing the virus to reach its basolateral receptor. Whether bacterial toxins also play a role in this impairment has not been studied yet. Here, we evaluated the role of α-hemolysin (Hla) and adenylate cyclase (ACT), toxins derived from the facultative pathogenic bacterium Staphylococcus aureus (S. aureus) and the primary pathogen Bordetella bronchiseptica (B. bronchiseptica), respectively. Equine respiratory mucosal explants were cultured at an air–liquid interface and pretreated with these toxins, prior to EHV-1 inoculation. Morphological analysis of hematoxylin–eosin (HE)-stained sections of the explants revealed a decreased epithelial thickness upon treatment with both toxins. Additionally, the Hla toxin induced detachment of epithelial cells and a partial loss of cilia. These morphological changes were correlated with increased EHV-1 replication in the epithelium, as assessed by immunofluorescent stainings and confocal microscopy. In view of these results, we argue that the ACT and Hla toxins increase the susceptibility of the epithelium to EHV-1 by disrupting the epithelial barrier function. In conclusion, this study is the first to report that bacterial exotoxins increase the horse’s sensitivity to EHV-1 infection. Therefore, we propose that horses suffering from infection by S. aureus or B. bronchiseptica may be more susceptible to EHV-1 infection.

Escherichia coli Alpha-Hemolysin HlyA Induces Host Cell Polarity Changes, Epithelial Barrier Dysfunction and Cell Detachment in Human Colon Carcinoma Caco-2 Cell Model via PTEN-Dependent Dysregulation of Cell Junctions.

Escherichia coli (E. coli) of the B2 phylotype reside in human and animal intestines. The bacteria possess pathogenicity factors such as α-hemolysin (HlyA) that can induce intestinal epithelial leaks. We addressed the questions which host cell processes were dysregulated by E. coli HlyA that can potentiate intestinal diseases. The colon carcinoma cell line Caco-2 was infected by HlyA+ E. coli. Cell polarity regulation was analyzed by live cell imaging for the phosphatidylinositol-4,5-bisphosphate (PIP2) abundance. In Caco-2 monolayers, transepithelial electrical resistance was measured for characterization of barrier function. Cell proliferation and separation were assessed microscopically. Epithelial regulation and cell signaling were analyzed by RNA-Seq and Ingenuity Pathway Analysis (IPA). Our main findings from E. coli HlyA toxinogenicity in the colon carcinoma cell line are that (i) PIP2 at the membrane decrease, (ii) PTEN (phosphatase and tensin homolog) inhibition leads to cell polarity changes, (iii) epithelial leakiness follows these polarity changes by disruption of cell junctions and (iv) epithelial cell detachment increases. HlyA affected pathways, e.g., the PTEN and metastasis signaling, were identified by RNA-Seq bioinformatics calculations in IPA. In conclusion, HlyA affects cell polarity, thereby inducing epithelial barrier dysfunction due to defective tight junctions and focal leak induction as an exemplary mechanism for leaky gut.

Automated Quantitative Analysis of Airway Epithelial Cell Detachment Upon Fungal Challenge.

Host-pathogen interactions involve a complex interplay between host and pathogen factors, resulting in either host protective immunity or establishment of disease. One of the hallmarks for disease progression is host tissue destruction. The first host surface to interact with the opportunistic respiratory fungal pathogen, Aspergillus fumigatus, is the airway epithelium. Unravelling the mechanisms involved in airway epithelial cell damage by A. fumigatus is essential to understanding the establishment and progression of infection in the host. Although host cell damage can be measured in vitro by indirect cell lysis assays, here, we describe an automated, simple, and low-cost assay to directly visualize and quantify epithelial cell line damage after challenge with A. fumigatus. We employ the previously characterized tissue noninvasive A. fumigatus ΔpacC mutant to demonstrate the quantitative difference in cell damage relative to its parental tissue invasive strain. This assay is easily scaled up for high-throughput screening of multiple Aspergillus mutants and can be adapted to suit diverse host cell lines, different time points of infection, challenge with other microbes, and drugs or novel compounds.

Characterization and pathogenicity of Aeromonas veronii associated with mortality in cage farmed grass carp, Ctenopharyngodon idella (Valenciennes, 1844) from the Central Himalayan region of India.

In the study, Aeromonas strains (n = 12) were isolated from moribund grass carp fry reared in the cage culture unit from the Central Himalayan region of India. They were identified as Aeromonas veronii, by biochemically and 16S rRNA analysis. The experimental bath infection of grass carp fry was performed using A. veronii GCAFBLC 228, one of the 12 isolates at cell concentrations 106 and 108CFUmL-1. The infected fry showed varied behavioural characteristics followed by tail rot, black pigmentation and hemorrhage in the body 48-96h post infection. The post bath challenged demonstrated maximum mortality (23%) at cell concentration 108CFUmL-1 during 10th and 12th day. Histopathology revealed hypertrophy, hyperplasia, fusion of gill lamellae, detachment and epithelial cell detachment in gill, swelling of hepatocytes, granular deposition in liver and tubular degeneration and yellow pigmented macrophage aggregates in the kidney. The in vitro assays for virulence traits recorded that A. veronii GCAFBLC 228 was β-haemolytic having strong cell surface hydrophobicity (CHS) characteristic (> 50%), precipitated after boiling, produced slime, non-suicidal and bound to crystal violet. The antibiogram showed that the strain was susceptible to ciprofloxacin (5μg), cefotaxime (30μg), ceftazidime (30μg), cefoxitin (30μg), ceftriaxone (30μg), chloramphenicol (30μg) and tetracycline (30μg). Negative staining transmission electron microscopy revealed presence of the lateral flagellum-like structure and cell adherence possibly could be correlated with the pathogenicity of A. veronii GCAFBLC 228. The further investigation is warranted to study the transmission, pathogenesis and epidemiology of A. veronii GCAFBLC 228 to develop the best health management practice for cage farmed fish.

Influence of Punica granatum peel on sperm parameters of domestic rabbit (Oryctolagus cuniculus) in zinc chloride -induced toxicity

Zinc is an essential component of biological function, play a significant role in male reproductive functioning and deficiency of this trace metal has negative impact on spermatogenesis and semen quality. The purpose of the present study was to find out if high concentrations of zinc lead to the opposite and evaluate the protective effect of natural products such as the ethanolic extract of P. granatum peel against the toxicity and oxidative stress induced by zinc chloride in male rabbitsTwenty adult rabbits were divided into equally four groups; the control, the ZnCl2 (5 mg/kg bw), the P.granatum peel(4 mg/kg bw) and the combined group (ZnCl2- PGP). The ZnCl2 and the PGP were administrated daily by gavage for a period of 6 weeks, and then the reproductive organs’ weight, the plasma testosterone, and the biology of sperms were evaluated.The results showed that the number of sperms in the epididymis and the concentration of testosterone in serum were decreased with an increased dose of ZnCl2 .Testicular and epididymis histopathological lesions like detachment, atrophy and vacuolization of germ, Leydig and epithelial cells were observed. On the other hand, the study shows improvement in all parameters when treated with P. granatum peel extract. In conclusion, High concentrations of zinc chloride may lead to sterility, which can be treated with natural product such as P. granatum peel.

A novel gingipain regulatory gene in Porphyromonas gingivalis mediates host cell detachment and inhibition of wound closure.

The black pigmentation‐related genes in Porphyromonas gingivalis are primarily involved in regulating gingipain functions. In this study, we identified a pigmentation‐related gene, designated as pgn_0361. To characterize the role of pgn_0361 in regulating P. gingivalis‐mediated epithelial cell detachment and inhibition of wound closure, PgΔ0361, an isogenic pgn_0361‐defective mutant strain, and PgΔ0361C, a complementation strain, were constructed using P. gingivalis ATCC 33277. The gingipain and hemagglutination activities, as well as biofilm formation, were examined in all three strains. The effect of P. gingivalis strains on epithelial cell detachment was investigated using the HO‐1‐N‐1 and Ca9‐22 epithelial cell lines. The inhibition of wound closure by heat‐killed P. gingivalis cells and culture supernatant was analyzed using an in vitro wound closure assay. Compared to the wild‐type strain, the PgΔ0361 strain did not exhibit gingipain or hemagglutination activity but exhibited enhanced biofilm formation. Additionally, the PgΔ0361 strain exhibited attenuated ability to detach the epithelial cells and to inhibit wound closure in vitro. Contrastingly, the culture supernatant of PgΔ0361 exhibited high gingipain activity and strong inhibition of wound closure. The characteristics of PgΔ0361C and wild‐type strains were comparable. In conclusion, the pgn_0361 gene is involved in regulating gingipains. The PGN_0361‐defective strain exhibited reduced virulence in terms of epithelial cell detachment and inhibition of wound closure. The culture supernatant of the mutant strain highly inhibited wound closure, which may be due to high gingipain activity.