Actin Antibodies Research Articles

Evaluation of the Role of Myofibroblast and Fibronectin in the Aetiopathogenesis of Cholesteatoma.

Primary acquired Cholesteatoma is a complex issue for otolaryngologists, with its development mechanisms still unclear due to the intricate anatomy of this region. It's aetiopathogenesis remains poorly understood and this aggressive clinical condition often leads to various complications. Recent research explores myofibroblast and fibronectin's potential roles in pathomechanisms of Cholesteatoma. To determine and analyze the role of myofibroblast and fibronectin in the aetiopathogenesis of Cholesteatoma. In a cross-sectional study at a tertiary care hospital, 30 patients with chronic suppurative otitis media with cholesteatoma were surgically treated, and intraoperative biopsy specimens were collected. These specimens were processed and subjected to histopathological examination, including immunohistochemical staining with Alpha-smooth muscle actin and anti-fibronectin antibody to identify myofibroblast and fibronectin presence. The data were then analyzed to investigate the aetiopathogenesis of cholesteatoma in this cohort. On immunostaining, 25 blocks (83.33%) were positively stained for Alpha-SMA (p-value-0.0007), whereas 29 blocks (96.67%) were positively stained for fibronectin (p-value < 0.0001), suggesting a statistically significant association between the presence of both myofibroblast and fibronectin with cholesteatoma perimatrix. Additionally, a statistically significant association was noted between complications and positive staining for myofibroblast (p-value - 0.0415) and positive staining for fibronectin (p-value-0.0254). Our study indicates that Cholesteatoma retraction and progression are driven by myofibroblast and fibronectin mechanisms, and also links them to disease severity. This understanding opens avenues for innovative diagnostics and treatments targeting these biomarkers.

P148 CCN3-derived peptides as anti-fibrotic therapies for systemic sclerosis

Abstract Background/Aims Effective therapy for skin fibrosis in scleroderma (systemic sclerosis: SSc) remains an unmet clinical challenge. Persistently activated M2 macrophages are believed to stimulate myofibroblasts in the disease microenvironment creating a pro-fibrotic niche in the skin. Expression of the anti-fibrotic matricellular protein CCN3 is reduced in animal models of fibrosis. We have identified a small peptide based on an amino acid sequence in CCN3, BLR-200, which mimics the anti-fibrotic activity of CCN3, and propose that BLR-200 could be used to treat patients in whom lack of CCN3 activity is permissive for fibrosis. However, whether there is lack of CCN3 activity in SSc and whether BLR-200 has effective anti-fibrotic activity in SSc are unknown. Methods We used ELISAs (R&amp;D Systems) to detect CCN3 and the M2 macrophage marker CD206 in the serum of scleroderma patients with early stage diffuse subset disease (within two years of disease onset), those with late-stage diffuse subset disease (over five years duration) as well as healthy controls (all n = 20). The bleomycin-induced mouse model of skin scleroderma, in which bleomycin is injected subcutaneously every day for 21 days, was used to assess the antifibrotic ability of BLR200 in vivo. Results In scleroderma serum, CCN3 was significantly reduced, relative to matched healthy controls and patients with inactive disease, in early onset scleroderma patients with active disease that show elevated CD206 levels (p &amp;lt; 0.01). In the mouse model of human SSc, compared to injection with a scrambled control peptide and relative to control mice treated with phosphate buffered saline, injection with BLR-200 prevented bleomycin-induced changes in collagen deposition, myofibroblast differentiation and skin thickness as measured by Trichrome stain, indirect immunofluorescence analysis with an anti-a-smooth muscle actin antibody, and morphometric analysis, respectively (N = 8 mice per group, p &amp;lt; 0.05). Similarly, RNA sequencing and real-time polymerase chain reaction analysis revealed that BLR-200 significantly impaired the ability of bleomycin to induce expression of fibrogenic genes such as: integrin alpha 11, CCN2, CCN1, Smad3, wnt4, YAP1 and tenascin-C (all N = 5, p &amp;lt; 0.05). Spatial transcriptomics analysis revealed that BLR-200 impaired bleomycin-induced alterations in skin cell populations, including the activation and expansion of epithelial and reticular fibroblast niches and the induction of Wnt, hippo, focal adhesion and actin cytoskeleton gene expression cluster, all of which are known to be activators of fibrosis and myofibroblast activation and persistence. Conclusion CCN3 expression is reduced in serum of scleroderma patients with active skin disease that show M2 macrophage activation as visualized by elevated CD206 levels. Since BLR-200 has antifibrotic activity in the bleomycin model of skin scleroderma, our data are consistent with the hypothesis that BLR-200 could be used to block fibrosis progression in early onset diffuse scleroderma patients who possess both low CCN3 and high CD206 in serum. Disclosure J. Nguyen: None. K. Zestranjyan: None. S. Xu: None. B.L. Riser: Corporate appointments; CEO of BLR Bio. R.J. Stratton: None. A. Leask: None.

Abstract 2791: Establishment of immortalized endometrial cell lines using Sendai virus

Abstract Objective: To establish endometrial immortalized cell lines using the Sendai virus and analyze their characteristics. Methods: The study included patients with benign ovarian tumors and endometrioid carcinoma who had regular menstrual cycles. Endometrial cells were collected and separated into epithelial and non-epithelial cells using flowcytometry (FCM) (CD326 (EpCAM) antibody). Furthermore, the isolated cells were infected with Sendai virus carrying three immortalization genes (Bmi-1, hTERT, and SV40T) to create immortalized cell lines. The cells were characterized as epithelial cells by immunocytochemistry (ICC), and receptor expression was assessed by reverse transcription PCR (RT-PCR) and ICC. Non-epithelial cells from the patients with endometrial carcinoma were evaluated using ICC. Results: The immortalized cell lines were capable of more than 20 passages. Immortalized endometrial epithelial cells tested positive for anti-Keratin antibodies and negative for anti-Vimentin antibodies by ICC. Additionally, FCM detected these cells as positive for anti-EpCAM antibody, confirming that they were epithelial cells. RT-PCR confirmed mRNA expression of estrogen α-receptor and progesterone receptor after 20 passages and ICC confirmed protein expression of them after 10 passages, establishing an endometrial epithelial immortalized cell line. Non-epithelial cells isolated from patients with endometrial carcinoma tested positive for anti-α-smooth muscle actin and anti-Vimentin antibodies by ICC, indicating their characterization as cancer-associated fibroblasts. Conclusion: We successfully established an immortalized cell line of endometrial epithelial cells using Sendai virus and generated cancer-associated fibroblasts. Citation Format: Kohei Hikino, Hiroaki Komatsu, Genki Hichiwa, Kanako Kazuki, Yasuhiro Kazuki, Hiroyuki Kugoh, Masayo Okawa, Yuki Ida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Shinya Sato, Fuminori Taniguchi. Establishment of immortalized endometrial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2791.

Biosensing using antibody-modulated motility of actin filaments on myosin-coated surfaces

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport within living cells. The characteristics of molecular motors, i.e., their motility over long distances, their capacity of transporting cargoes, and their very efficient energy consumption, recommend them as potential operational elements of a new class of dynamic nano-devices, with potential applications in biosensing, analyte concentrators, and biocomputation. A possible design of a biosensor based on protein molecular motor comprises a surface with immobilized motors propelling cytoskeletal filaments, which are decorated with antibodies, presented as side-branches. Upon biomolecular recognition of these branches by secondary antibodies, the ‘extensions’ on the cytoskeletal filaments can achieve considerable lengths (longer than several diameters of the cytoskeletal filament carrier), thus geometrically impairing or halting motility. Because the filaments are several micrometers long, this sensing mechanism converts an event in the nanometer range, i.e., antibody-antigen sizes, into an event in the micrometer range: the visualization of the halting of motility of microns-long cytoskeletal filaments. Here we demonstrate the proof of concept of a sensing system comprising heavy-mero-myosin immobilized on surfaces propelling actin filaments decorated with actin antibodies, whose movement is halted upon the recognition with secondary anti-actin antibodies. Because antibodies to the actin-myosin system are involved in several rare diseases, the first possible application for such a device may be their prognosis and diagnosis. The results also provide insights into guidelines for designing highly sensitive and very fast biosensors powered by motor proteins.

Changes of biliary cilia, smooth muscle tissue distribution, innervation and extracellular matrices during morphological evolution of hepatic architectures in vertebrates

BackgroundThe liver architecture of vertebrates can be classified into two types, the portal triad type (having periportal bile ducts) and the non-portal triad type (having bile ducts independent of the course of portal veins). The former is typically detectable in livers of tetrapods and cartilaginous fish, and its ancestral state is found in the hagfish, an earliest diverged lineage among vertebrates. Teleosts other than osteoglossomorphs have the latter. The aim of the present study is to reveal the changes of the hepatic innervation, biliary cilia and smooth muscle distribution, and extracellular matrices along vertebrate evolution with attention to the two types of liver architectures. MethodsThe hepatic innervation, biliary cilia and smooth muscle distribution, and collagen deposition were immunohistochemically and histochemically compared among livers of various vertebrates, using anti-acetylated tubulin and anti-α-smooth muscle actin antibodies, and Sirius red staining. These were also ultrastructurally examined. ResultsAlthough the hagfish liver had periportal intrahepatic bile ducts and ductules as detected in mammalian livers, it lacked smooth muscles around bile ducts and portal veins. Extracellular matrices in their connective tissues had thick collagen fibers. Its innervation was restricted to intrahepatic bile ducts and portal veins in the hilum. In livers of other vertebrates, including teleosts, the innervation was broadly detectable, especially around bile ducts, hepatic arteries and portal veins (afferent vessels), but not around central veins (efferent vessels). The chondrichthyans ultrastructurally had smooth muscle tissue around bile ducts. Cilia distribution was confirmed in intrahepatic bile ducts of tetrapods and basal actinopterygians. Teleosts other than osteoglossomorphs lacked cilia in their intrahepatic bile ducts. ConclusionsThe liver architecture of the hagfish may be unique for innervation and extracellular matrices. Hepatic innervation may not have occurred in vertebrate ancestors. Hepatic innervation in bile ducts, hepatic arteries and portal veins may have been conserved among the extant jawed vertebrates. Cilia distribution in bile ducts may have changed during evolution of actinopterygians.

Role of myofibroblasts in oral plasma cell granuloma: Immunohistochemical evaluation of α-SMA and ALK in a retrospective study of 30 cases

IntroductionOral plasma cell granuloma (OPCG) is a rare reactive lesion with generally benign yet occasionally aggressive behavior. Myofibroblasts are important in many physiologic, and pathologic conditions. The role of myofibroblasts in the clinical behavior of OPCG was assessed as well as its usefulness in differentiating this lesion from the inflammatory myofibroblastic tumor mimicking plasma cell granuloma. Material and MethodsThis retrospective study included 30 paraffin blocks of OPCG. Immunohistochemical evaluation of alpha-smooth muscle actin (α-SMA) and Anaplastic lymphoma kinase (ALK) antibodies was performed. The mean area of positive expression was calculated and scored semiquantitatively with clinicopathologic correlations. ResultsMost of the cases were clinically non-aggressive. Alveolar bone resorption was observed in nine cases, two of them showed severe resorption and stromal fibrosis. Negative α-SMA was observed in 70% of cases showing a predominance of plasma cells in the stroma. All cases of stromal fibrosis revealed positive α-SMA of a weak percentage. A statistically significant difference was observed between α-SMA expression and the clinicopathologic variables. Negative ALK expression was noted in all cases. DiscussionMyofibroblasts were infrequently found in OPCG. Remarkably, the aggressive behavior in cases with intense fibrosis was related to the existence of myofibroblasts even of non-neoplastic nature and minimal amount. The number of myofibroblasts and their nature assessed via α-SMA and ALK immunohistochemical expression respectively might be valuable in predicting the biological behavior of OPCG and may hold diagnostic significance in challenging OPCG cases that might mimic inflammatory myofibroblastic tumor.

The Predictive Value of Autoantibodies in Determining Autoimmune Hepatitis (AIH) Severity

Background: The presence of autoantibodies is a prerequisite for the diagnosis of autoimmune hepatitis (AIH). However, most autoantibodies are not disease-specific, and serological overlap between AIH and other chronic liver diseases is common. Since the prognostic parameters of AIH are limited, this study aimed to investigate the relationship between histopathological findings on liver biopsy with different types of autoantibodies associated with AIH and how autoantibodies can predict the severity and extent of disease. Methods: The present study was performed on 30 patients with a definite diagnosis of AIH according to the International Autoimmune Hepatitis Group (IAIHG) criteria. Pediatric AIH patients underwent liver tissue examinations at the time of diagnosis at accession, which confirmed characteristic histological changes. AIH-related serologic major and minor autoantibodies were measured using indirect immunofluorescence assays and ELISA kit (EUROIMMUN, Germany), respectively, and were compared within all patients, and the results were recorded. Finally, the obtained data were analyzed using SPSS V25 software. Results: Out of 30 patients, 17 (56.66%) were female, and the age range of patients was 17-11 years (8.46±6.95). Anti-nuclear antibody (ANA) (73.3%), smooth muscle antibody (SMA)-anti-smooth muscle actin antibody (ASMA) (70%), perinuclear anti-neutrophilic cytoplasmic antibodies (p-ANCA) (63%), and liver kidney microsomal (LKM) (43.3%) were the most common autoantibodies found in children with AIH. There was a significant relation between the severity of histological findings and the presence of LKM antibodies (P&lt;0.05). The highest sensitivity for predicting severe AIH based on histopathological findings was ANA autoantibody positivity and the presence of at least two primary autoantibodies (LKM and SMA-ASMA). On the other hand, positive LKM antibodies had the highest specificity and positive predictive value (PPV) in AIH severity prediction. Conclusion: The results of the present study suggested that there might be a significant correlation between the presence of primary LKM autoantibodies and biopsy results, so it can possibly act as an accurate autoantibody for predicting the severity of AIH, while other AIH-related autoantibodies did not seem to have a significant correlation with biochemical and histological findings.

Anatomical and Histological Study of the Upper Tarsus in Asian.

The aim of this study was to observe and analyze the anatomical and histological characteristics of the upper tarsus in Asian. A total of 15 Asians (14 adults, 1 child) were used. The sagittal sections with thickness of 3μm in the middle of the upper eyelid were prepared and stained with hematoxylin-eosin, Masson trichrome and anti-smooth muscle actin antibody staining, and then were observed and photographed under light microscopy. On the sagittal section, the upper end of the tarsus was connected with the Müller muscle and the part of the posterior layer of the levator aponeurosis; the lower end was the inflated part of the palpebral margin; the superficial surface was connected with the pretarsal fascia; and the deep surface was connected with Müller muscle aponeurosis and palpebral conjunctiva. Histologically, the tarsus was similar to the structure between dense fibrous connective tissue and cartilage, and its main structures include meibomian glands and its ducts, accessory lacrimal glands, glands of Moll, glands of Zeis, eyelash hair follicles, Riolan muscle, blood vessels, and collagen fibers. Through the observation of the tarsus of a child, compared with the adult specimen, the meibomian gland tissue was more abundant; the collagen fibers density was significantly lower and arranged orderly; the blood vessel density was significantly higher; α-SMA positive smooth muscle cells could be seen in the upper end of the tarsus and its superficial and deep surfaces. In this study, the internal and adjacent anatomical structures of the upper tarsus in Asians were observed under a microscope, and according to the histological characteristics, the tarsus was divided into 3 parts and 3 types. Through the preliminary analysis, the tarsus was likely to show different histological characteristics in different individuals and age groups of Asians. There was a certain correlation between the degree of meibomian gland atrophy, the degree of collagen fibrosis and the density of blood vessel in the tarsus.

P150 The Triptryium wilfordii derivative celastrol has anti-fibrotic effects in systemic sclerosis

Abstract Background/Aims Scleroderma (systemic sclerosis, SSc) is an autoimmune inflammatory-fibrosis syndrome that damages the skin and internal organs and is considered a prototypic complex fibrotic disease. In SSc, persistently activated myofibroblasts are maintained by an excessive, autocrine mechanotranductive/pro-adhesive signaling loop. Drugs targeting this pathway are therefore of likely therapeutic benefit in SSc. The mechanosensitive transcriptional co-activator, yes activated protein-1 (YAP1), is activated in SSc fibroblasts. The terpenoid plant-derivative celastrol has recently been identified as a YAP inhibitor; however, if celastrol can alleviate SSc fibrosis, and its underlying mechanism of action, is as yet unclear. Methods We cultured age-, sex-, and site-matched human dermal fibroblasts sampled from healthy individuals and patients with early onset (&amp;lt;18 month after first diagnosis) diffuse cutaneous scleroderma (systemic sclerosis, dcSSc) and treated with or without transforming growth factor β1 (TGFβ1, 4ng/ml) in the presence or absence of celastrol (500nM). We also subjected C57BL6J mice to the inflammatory-driven bleomycin-induced model of skin SSc, in the presence or absence of celastrol (28days, every day, 0.1U bleomycin, 1mg/kg celastrol). RNA expression was assessed using RNAseq and real-time polymerase chain reaction analysis. Protein expression was determined using Western blot and enzyme-linked immunosorbent assay. Fibrosis was monitored by hematoxylin and eosin and trichrome staining of tissue sections, and by using indirect immunofluorescence analysis with anti-alpha-smooth muscle actin (SMA) antibodies to detect myofibroblasts. Results In dermal fibroblasts, celastrol impaired the ability of TGFβ1 to induce an SSc-like pattern of gene expression, including the induction of cellular communication network factor 2 (CCN2), collagen I and TGFβ1 protein (N = 6, all p &amp;lt; 0.01). Celastrol also alleviated the persistent fibrotic phenotype of dermal fibroblasts cultured from lesions of SSc patients, including the overproduction of CCN2 and collagen (N = 3, p &amp;lt; 0.05). Finally, in the bleomycin-induced model of SSc dermatopathology, celastrol inhibited skin thickness/collagen deposition in vivo and blocked nuclear localization of YAP in SMA-positive myofibroblasts (all N = 6, p &amp;lt; 0.01). Conclusion Our data are consistent with the hypothesis that compounds, such as celastrol, that antagonize the YAP pathway warrant further consideration as potential treatments for SSc skin fibrosis. Disclosure A. Leask: Grants/research support; CIHR, NSERC, Arthritis Society of Canada. R.J. Stratton: None. S. Xu: None. P. Chitturi: None.

An Immunohistochemical Analysis for Evaluating the Diagnostic Role of Myofibroblasts in Oral Squamous Cell Carcinoma using α-Smooth Muscle Actin Antibody.

One of the most common types of malignancies affecting the head and neck region is oral squamous cell carcinoma (OSCC). Little less is known about the role of myofibroblasts in the pathogenetic process of OSCC. Hence, we assessed the involvement of myofibroblasts in the invasive process of OSCC using α-SMA (α-smooth muscle actin) antibody. Four study groups in total were organized as follows: 40 cases each of well-differentiated OSCC (WDOSCC), moderately differentiated OSCC (MDOSCC), poorly differentiated OSCC (PDOSCC), and controls make up Group 1, Group 2, Group 3, and Group 4, respectively. The percentage of α-SMA immunopositive cells and staining intensity (A) multiplied together to determine the final staining score (B). The final staining index was produced by multiplying staining intensity (A) by the proportion of immunopositive cells that were stained with α-SMA (B) (FSI). Score Zero was graded as Index Zero by FSI while scores One and Two received an Index Low rating, scores Three and Four an Index Moderate rating, and scores Six and Nine an Index High rating. Significantly higher expression of myofibroblast was observed in OSCC group in comparison with the control group. However; no significant difference in myofibroblast expression was observed while comparing different grades of OSCC. We recommend using myofibroblasts as a stromal marker to track the severity and development of OSCC.

EFFECT OF BEVACIZUMAB ON α-SMOOTH MUSCLE ACTIN EXPRESSION AND FIBROBLAST COUNT IN TRABECULECTOMY AREA TOWARDS PREVENTION OF FIBROSIS

Objective to analyze the effect of bevacizumab on α-smooth muscle actin expression and fibroblast count in trabeculectomy area of rabbit models in order to find a safer modulator of wound healing to improve surgical outcome. Material and methods 16 New Zealand white rabbits aged 4-6 months and weigh between 2,5-3,5 kg were performed trabeculectomy in their right eyes with postoperative subconjunctival injection of BSS and Bevacizumab. Rabbits were put into control and bevacizumab group using simple random sampling. Examination were done postoperative day 1, 7, and 14 and subjects were terminated and performed enucleation on postoperative day 14. The samples were histologically stained with Haematoxyline-Eosin to count the fibroblast and immunohistochemistry using α-smooth muscle actin antibody to differ the myofibroblast from fibroblast and the expression of α-SMA were collected using immunoreactive score. Result Mann Whitney u test and independent t-test were used to analyze the data, and we found both less expression of α-smooth muscle actin and fibroblast count on bevacizumab group compared to control group which indicates less myofibroblast, fibroblast, and less scarring potential in trabeculectomy area. Conclusion bevacizumab inhibits fibroblast proliferation and its differentiation to myofibroblast that lead to less collagen production and fibrosis.

Post synaptic density 95 (PSD95)–Actin interaction: A critical regulator of synaptic strength and plasticity in Alzheimer’s disease pathology

AbstractBackgroundPSD95 serves as a scaffolding protein to several neurotransmitter receptors, signal transduction components, and adhesion molecules at the synapse. It is a potential regulator of synaptic strength and plasticity by regulating glutamatergic receptor trafficking, which is impaired in AD. PSD95 interacts directly with NMDA receptors while the interactions with AMPA receptors are through an auxiliary subunit TARPs. Actin cytoskeleton is highly enriched at the synapse. However, the interaction of PSD‐95 – actin and its consequences in synaptic transmission is not understood. Therefore, we investigated the critical role of PSD‐95 association at the synapse with actin and its impact on AMPA and NMDA receptors using AD mice.MethodsSynaptosomes were isolated from adolescent and middle aged wildtype and APP/PS1 mice. Immunoprecipitation was performed using the synaptosomes and F‐actin fractions with anti‐PSD95 antibody followed immunoblotting against glutamatergic receptor subunit antibodies and actin antibody. Statistical comparisons between two groups were performed with the two‐tailed unpaired Mann–Whitney U test.ResultsPSD95 association with actin is detected in synaptosomes isolated from adolescent and middle aged WT and APP/PS1 mouse brain cortex. We found that PSD95‐actin association is significantly decreased in middle aged APP/PS1 mice compared with WT while it remains unaffected in the F‐actin fraction of synaptosomes in APP/PS1 mice. Then, we examined the levels of AMPA and NMDA receptor subunits in APP/PS1 mice. GluA1 and GluA2 subunits of AMPAR are significantly decreased in middle aged but not in adolescent APP/PS1 mice in comparison to age matched WT mice. Protein levels of GluN1 and GluN2B subunits are significantly decreased in both adolescent and middle aged APP/PS1 mice, while GluN2A levels are reduced only in adolescent mice. We detected that PSD95 is physically associated with both AMPAR and NMDAR subunits and this association with GluA1 subunit is affected only in middle aged APP/PS1 mice. Interestingly, interaction of NMDAR subunits (GluN1 and GluN2B) with PSD‐95 is greatly enhanced at adolescent APP/PS1 mice even though these subunit protein levels are significantly diminished.ConclusionOur findings indicate that perturbation of PSD95‐actin association and glutamatergic receptors in AD leads to synaptic dysfunction. It may have critical implications in defective glutamatergic neurotransmission.

Newly raised anti-c-Kit antibody visualizes morphology of interstitial cells of Cajal in the developing gut of chicken embryos.

The gut peristaltic movement, a wave-like propagation of a local contraction, is important for the transportation and digestion of ingested materials. Among three types of cells, the enteric nervous system (ENS), smooth muscle cells, and interstitial cells of Cajal (ICCs), the ICCs have been thought to act as a pacemaker, and therefore it is important to decipher the cellular functions of ICCs to further our understanding of gut peristalsis. c-Kit, a tyrosine kinase receptor, has widely been used as a marker for ICCs. Most studies with ICCs have been conducted in mammals using commercially available anti-c-Kit antibody. Recently, the chicken embryonic gut has emerged as a powerful model to study gut peristalsis. However, since the anti-c-Kit antibody for mammals does not work for chickens, cellular mechanisms by which ICCs are regulated have largely been unexplored. Here, we report a newly raised polyclonal antibody against the chicken c-Kit protein. The specificity of the antibody was validated by both western blotting analyses and immunocytochemistry. Co-immunostaining with the new antibody and anti-α smooth muscle actin (αSMA) antibody successfully visualized ICCs in the chicken developing hindgut in the circular muscle and longitudinal muscle layers. As previously shown in mice, common progenitors of ICCs and smooth muscle cells at early stages were double positive for αSMA and c-Kit, and at later stages, differentiated ICCs and smooth muscle cells exhibited only c-Kit and αSMA, respectively. A novel ICC population was also found that radially extended from the submucosal layer to the circular muscle layer. Furthermore, the new antibody delineated individual ICCs in a cleared hindgut. The antibody newly developed in this study will facilitate the study of peristaltic movement in chicken embryos.

High Actin Expression in Thrombus of Acute Ischemic Stroke Can Be a Biomarker of Atherothrombotic Origin Stroke.

BackgroundAs the treatment target, the imaging information and histologic characteristics of the thrombus may differ according to the stroke subtype. This study aimed to provide the correlative study of stroke etiology with the non-contrast CT, and histological composition of retrieved clots in acute ischemic stroke (AIS).Materials and MethodsA total of 94 patients with AIS who underwent the endovascular treatment with successfully retrieved clots from January 2017 to October 2020 were enrolled in the present study. Histological analysis was performed using hematoxylin and eosin (H&E) staining and immunostaining with CD3, CD20, CD105, and actin antibodies. CT obtained at the patients' admission was to measure the attenuation and volume of all thrombus.ResultsA total of 94 subjects were included in this study. Fifty-six patients were classified as cardioembolic (CE), and 38 were classified with large-artery atherosclerosis (LAA). The subjects with LAA tend to exhibit higher actin and CD105 levels, and lower Hounsfield Unit (HU) values than subjects with CE. After adjusting for confounders, the actin was positively correlated with CD105 but not with HU values. Logistics regression shows actin was valuable for the prediction of LAA (OR, 1.148; 95% CI, 1.075–1.227; p < 0.001), even adjusted for age, sex, and intervention type (OR, 1.129; 95% CI, 1.048–1.216; p = 0.001), CT density and CD105 (OR, 1.161; 95% CI, 1.056–1.277; p = 0.002). Actin levels have a strong accuracy in differentiating LAA from CE, especially combined with CT density and CD105, which yielded a sensitivity of 63.2%, a specificity of 89.3%, with the area under the curve (AUC) at 0.821 (95% CI, 0.731–0.912).ConclusionOur findings suggest that actin's level was a major factor differentiating atherothrombotic origin strokes from the cardioembolic stroke.Clinical Trial RegistrationChiCTR2100051173.

Rapamycin-filgrastim combination therapy ameliorates portal hypertension-induced splenomegaly: Role of β actin and S100A9 proteins modulation.

Objective(s):Thioacetamide (TAA) was administered to induce an animal model of liver disease with secondary splenomegaly to assess the mechanisms underlying the effects of rapamycin and filgrastim when taken separately or in combination on the biochemical and histopathological aspects of the liver and spleen. Materials and Methods:Thirty adult male albino rats were divided into five groups (control, TAA-treated group, TAA+rapamycin, TAA+filgrastim, and TAA+rapamycin+filgrastim group). We measured relative liver and spleen weights, serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and albumin. Molecular docking modeling and histopathological examination of liver and spleen sections with hematoxylin and eosin and Masson trichrome staining with immunohistochemical detection of splenic CD3 and CD20 lymphocytes, S100A9 and β actin antibodies were detected. Morphometric and statistical analyses of the results were performed. Results:TAA administration altered the histological structure of the liver and spleen and impaired liver function. It increased the expression of splenic CD3, CD20 lymphocytes, and S100A9 while diminishing the expression of β actin. Each of rapamycin and filgrastim, when administered separately, improved liver and spleen indices and liver function, but rapamycin did not affect the albumin level. They lowered splenic B and T lymphocyte levels. Expression levels of S100A9 showed down-regulation while β actin levels were up-regulated when compared with TAA. Combination therapy improved liver and spleen tissue pathology and significantly ameliorated the expression of splenic lymphocytes through regulation of S100A9 and β actin expression. Conclusion:The synergistic effect of combination therapy was dependent on the regulation of splenic S100A9 and β actin levels.

Myofibroblasts as important diagnostic and prognostic indicators of oral squamous cell carcinoma: An immunohistochemical study using alpha-smooth muscle actin antibody.

Background:Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity, with multifactorial etiopathogenesis. Data from the past literature suggest that myofibroblasts (MFs) can also contribute significantly to the pathogenesis of the disease. Hence, the present study was undertaken for assessing the expression of MF in well-differentiated OSCC (WDOSCC), moderately differentiated OSCC (MDOSCC), poorly differentiated OSCC (PDOSCC) and healthy controls by immunohistochemistry using alpha-smooth muscle actin (α-SMA) antibody.Methodology:Forty cases each of WDOSCC, MDOSCC, PDOSCC and healthy controls were included. 4-μm thick sections from each tissue sample were stained with routine hematoxylin and eosin as well as immunohistochemically using α-SMA. Among different grades of OSCC, expression of MFs was compared. All the results were subjected to statistical analysis.Results:While comparing the expression of MFs in between different grades of OSCC, nonsignificant results were obtained. While comparing the expression of MF in between OSCC cases and normal controls, significant results were obtained.Conclusion:MFs are one of the vital pathogenetic components in OSCC cases in predicting their invasive behaviors. We advocate the use of MFs as a stromal marker for visualizing invasion and progression in OSCC patients.

Cholesterol Induces Oxidative Stress, Mitochondrial Damage and Death in Hepatic Stellate Cells to Mitigate Liver Fibrosis in Mice Model of NASH.

Liver fibrosis and its end-stage disease cirrhosis are major world health problems arising from chronic injury of the liver. In recent years, the hypothesis that hepatic stellate cells’ (HSCs’) activation and fibrosis can be mitigated by HSC apoptosis and cell death has become of interest. In the current study, we evaluated the effect of cholesterol and bile acids on HSC apoptosis and liver fibrosis. Male C57BL/6J mice (wild type), aged four to five weeks, were fed an AIN-93G based diet (normal diet, ND), ND diet + 1% (w/w) cholesterol (CHOL group), ND diet + 0.5% (w/w) cholic acid (CA group) or ND diet + 1% (w/w) cholesterol + 0.5% (w/w) cholic acid (CHOL + CA group). Female Mdr2(-/-) mice were also treated with ND with and without 1% cholesterol. The effect of cholesterol on liver fibrosis and HSC clearance was evaluated. In addition, we studied the mechanism of cholesterol-induced apoptosis in HSC-T6 and AML-12 hepatocyte cell lines. In animals treated with cholic acids, increased lipid peroxidation and fibrosis were observed after six weeks of treatment. However, addition of cholesterol to the diet of C57BL/6J mice led to HSC-specific apoptosis and resolution of liver fibrosis, verified by double-staining with active caspase and α smooth muscle actin antibodies. In Mdr2 (-/-) mice, a diet supplemented with cholesterol corrected fibrosis and induced active hepatic stellate cells’ clearance. HSC-T6 were found to be much more sensitive to cholesterol-induced oxidative stress, mitochondrial damage and apoptosis compared to hepatocytes. These results indicate that cholesterol may be a trigger of HSC lipid peroxidation and death in the liver in a model of non-alcoholic steatohepatitis. A high cholesterol-to-bile acid ratio may determine the trajectory of the liver disease toward mitigation of fibrosis.

Histological Study of Dysplastic Dermal Tissue in Newborn with Myelomeningocele: A Comparative Analysis of the Population of Myofibroblasts

Objectives: Lumbar myelomeningocele is the most common type of spina bifida, a defect of embryonic development, in which an incomplete closure of the vertebral arches is observed in addition to dysplastic alterations of the nervous, meningeal, bone and cutaneous tissue. The tissue response to postoperative closure will depend, among other factors, on the population of pluripotent cells such as myofibroblasts present in the tissue at the edges of the lesion. The purpose of the following study was to determine the population of myofibroblasts in the dysplasic dermis associated with lumbar myelomeningocele, to compare the population of myofibroblasts of the edges of the lesion with those of the normal skin of the defect, and to assess the usefulness of the Ammoniac Silver Nitrate technique, in the identification of myofibroblasts. Methods: It was randomly selected 6 newborn patients who went to intervention for the surgical closure of lumbar myelomeningocele, 10 millimeters of both dysplastic and normal eutrophic skin were extracted from each patient, the histological sections were stained with hematoxylin/eosin and ammoniac silver nitrate to detect myofibroblasts. Immunohistochemistry: Anti-alpha-actin Smooth Muscle Actin (SMA) antibody was used to detect alpha actin-positive intracytoplasmic bodies and subplasmalemal pla¬cas. Results: the presence of myofibroblasts, presence of dense subplasmalemal plaques and positive immunostaining for alpha-actin in the area of dysplastic dermal tissue were identified. The amount of myofibroblasts was higher in the dysplasic zone, Ammoniac silver nitrate highlighted the dense subplasmalemal plaques of myofibroblasts. A 5:1 ratio was found when comparing the amount of myofibroblasts of the dysplastic dermis with respect to the healthy one, student's T-test showed a standard deviation between samples 1.87 and p&lt;0.05. The differences between the two cell populations studied are statistically significant. Conclusions: The presence of myofibroblasts demonstrates the possibility of generating new post-surgical tissue in the cases studied. Ammoniac silver nitrate is a novel and reliable method in the histological determination of subplaslemal plaques of myofibroblasts.

Immunoreactivity and a new staining method of monocarboxylate transporter 1 located in endothelial cells of cerebral vessels of human brain in distinguishing cerebral venules from arterioles.

Distinguishing brain venules from arterioles with arteriolosclerosis is less reliable using traditional staining methods. We aimed to immunohistochemically assess the monocarboxylate transporter 1 (MCT1), a specific marker of venous endothelium found in rodent studies, in different caliber vessels in human brains. Both largeand small-caliber cerebral vessels were dissected from four autopsy donors. Immunoreactivity for MCT1 was examined in all autopsied human brain tissues, and then each vessel was identified by neuropathologists using hematoxylin and eosin stain, the Verhoeff’s Van Gieson stain, immunohistochemical stain with antibodies for α-smooth muscle actin and MCT1 in sequence. A total of 61 cerebral vessels, including 29 arteries and 32 veins were assessed. Immunoreactivity for MCT1 was observed in the endothelial cells of various caliber veins as well as the capillaries, whereas that was immunenegative in the endothelium of arteries. The different labeling patterns for MCT1 could aid in distinguishing various caliber veins from arteries, whereas assessment using the vessel shape, the internal elastic lamina, and the pattern of smooth muscle fibers failed to make the distinction between small-caliber veins and sclerotic arterioles. In conclusion, MCT1 immunohistochemical staining is a sensitive and reliable method to distinguish cerebral veins from arteries.

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death