Tissue Survival Research Articles

94. Efficacy of SCY-247, a Second-generation Triterpenoid Antifungal, in Three Murine Models of Invasive Fungal Infections

Abstract Background There are limited treatment options for invasive fungal infections (IFIs) with broad-spectrums of activity that can be delivered both orally and intravenously. SCY-247 is a second-generation novel triterpenoid antifungal in development. It has broad-spectrum activity covering yeasts, molds and dimorphic fungi, including azole- and echinocandin-resistant strains and can be given orally or intravenously. Recent in vivo murine efficacy studies were performed with SCY-247 and results from 3 studies demonstrating activity against Candida and Mucorales are presented.Table 1:Study Methodology Details Methods The in vivo activity of SCY-247 was assessed in murine models of invasive candidiasis (IC) caused by C. albicans and C. glabrata and a murine model of pulmonary mucormycosis (PM) caused by R. delemar. In each model, SCY-247 was administered by oral gavage, twice daily (BID), for seven days with doses ranging from 5 to 48 mg/kg. Efficacy endpoints included changes in tissue fungal burden (CFU) and/or survival. Details are shown in Table 1. Results SCY-247 demonstrated potent in vivo activity in all three murine IFI models. In each dose-dependent responses based on the primary endpoints of fungal burden and/or survival were observed. Significant reductions in kidney fungal burden vs placebo (P < 0.01) were observed at doses ≥10 mg/kg against C. albicans and at doses ≥16 mg/kg against C. glabrata; significant reductions in lung fungal burden (P < 0.01) were also observed in the C. glabrata model at doses ≥32 mg/kg. Bioanalysis demonstrated SCY-247 preferentially distributed to kidney and lung tissues versus plasma and reductions in C. glabrata fungal burden correlated with dose and exposure. Against pulmonary mucormycosis, treatment with SCY-247 at doses ≥32 mg/kg resulted in prolonged survival (P < 0.05) and reductions in both lung and brain fungal burden vs placebo (P < 0.05). Conclusion SCY-247 demonstrated significant in vivo activity in murine models of invasive candidiasis and pulmonary mucormycosis, and the antifungal responses correlated with dose and exposure. Disclosures Steve Wring, PhD, SCYNEXIS, Inc.: Advisor/Consultant Katyna Borroto-Esoda, MS, Scynexis Inc: Advisor/Consultant Nathan P. Wiederhold, PharmD, BioMerieux: Grant/Research Support|F2G: Advisor/Consultant|F2G: Grant/Research Support|Mycovia: Grant/Research Support|Scynexis: Grant/Research Support|Sfunga: Grant/Research Support Ashraf S. Ibrahim, PhD, SCYNEXIS, Inc.: Advisor/Consultant|SCYNEXIS, Inc.: Grant/Research Support David A. Angulo, MD, SCYNEXIS, Inc.: Board Member|SCYNEXIS, Inc.: SCYNEXIS, INC Officer and Board Member, SCYNEXIS Patents|SCYNEXIS, Inc.: Stocks/Bonds (Public Company)

HMGB2 knockdown ameliorates retinal ganglion cell injury by inhibiting NLRP3 inflammasome activation after retinal ischemia.

To explore the neuroprotective effects of high mobility group box 2 (HMGB2) knockdown on retinal ganglion cells (RGCs) in the retinal ischemia-reperfusion injury (RIRI). Oxygen-glucose deprivation (OGD)-injured RGCs from postnatal three-day C57BL/6 mice pups and high intraocular pressure (IOP)-induced RIRI mice were used as cellular and animal models of RIRI. The expression of HMGB2 in the retina of RIRI mice and OGD-injured RGCs was detected through reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting. The effects of HMGB2 silencing on the morphological changes, RGCs survival, and cell apoptosis in mouse retinal tissues were observed through H&E staining, immunofluorescence staining with RNA-binding protein with multiple splicing (RBPMS) antibody, and TUNEL staining, respectively. RGC viability and apoptosis were examined by CCK-8 and flow cytometry assays. The levels of proteins associated with NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated pyroptosis [NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-18] in vivo and in vitro were measured by Western blotting. HMGB2 protein and NLRP3 were upregulated in the retina of RIRI mice and OGD-injured RGCs (P<0.001). The retina was edematous, accompanied by disorganized cell arrangement and decreased thickness of all layers, and obvious vacuoles in ganglion cell layer. HMGB2 silencing alleviated the reduction in total retinal thickness and the severity of retinal tissue damage as well as suppressed RGC loss and retinal cell apoptosis in RIRI mice. OGD-induced RGC apoptosis was ameliorated after downregulation of HMGB2 in vitro. Intravitreal injection of the AAV-sh-HMGB2 and si-HMGB2 resulted in significantly decrease of NLRP3, Caspase-1, GSDMD-N, IL-1β, and IL-18 protein levels in the retinal tissues of RIRI mice and OGD-injured RGCs, respectively (all P<0.001). HMGB2 knockdown protects against RGC apoptosis and pyroptosis after RIRI through suppressing NLRP3 inflammasome activation.

∆9-Tetrahydrocannabinol Increases Growth Factor Release by Cultured Adipose Stem Cells and Adipose Tissue in vivo.

Because of its biocompatibility and its soft and dynamic nature, the grafting of adipose tissue is regarded an ideal technique for soft-tissue repair. The adipose stem cells (ASCs) contribute significantly to the regenerative potential of adipose tissue, because they can differentiate into adipocytes and release growth factors for tissue repair and neovascularization to facilitate tissue survival. The present study tested the effect of administering a chronic low dose of ∆9-tetrahydrocannabinol (THC) on these regenerative properties, in vitro and in vivo. Human ASCs were exposed to increasing concentrations of THC. Resazurin conversion was applied to investigate the effect on metabolic activity, cell number was assessed by crystal violet staining, tri-linear differentiation was evaluated by specific colorimetric approaches, and the release of growth factors was analyzed by ELISA. Two groups of mice were treated daily either with a low dose of THC (3mg/kg) or a vehicle solution. After 3weeks, adipose tissue was obtained from excised fat deposits, homogenized and tested for growth factor contents. THC decreased ASC proliferation but increased metabolic activity as well as adipogenic and chondrogenic differentiation. A low concentration of THC (1µM) enhanced the growth factor release by ASCs. The concentration of these cytokines was also increased in adipose tissue of mice treated with THC. Our results indicate that chronic activation of the endocannabinoid system promoted differentiation and growth factor release of ASCs, which could be of specific value for enhancing the regenerative potential of adipose tissue.

Rooting depth and specific leaf area modify the impact of experimental drought duration on temperate grassland species

Abstract Due to climate change, temperate grasslands are being exposed to increasingly severe droughts. Concurrently, land‐use intensification is altering grasslands' functional composition by promoting fast‐growing, resource‐acquisitive species with high specific leaf area (SLA). How SLA affects the ability of grassland species to resist and recover from increasingly severe droughts and if deep roots improve their drought performance remains unclear. To investigate this, we established a common‐garden field experiment including temperate grassland species with SLAs of 17.9–39.3 mm2 g−1 and maximal rooting depths of 16–252 cm. After 1.5 years, we simulated droughts for 0, 79, 134, 177 and 220 days. Drought effects on plant performance increased with drought length, reducing the survival of green tissue and annual biomass by up to ~50% across all 32 species considered. As plant‐available water remained in deep soil layers by the end of all treatments, deep roots mitigated the negative effect of increasing drought length on productivity in the later stage of drought and favoured productivity after a longer drought. The low‐to‐high SLA trait gradient among the 16 graminoid species seemed to represent alternative survival strategies ranging from dehydration tolerance to dehydration avoidance, rather than drought sensitivity. Variable drought responses along the SLA gradient of forbs imply that multiple other traits are related to drought resistance across evolutionarily distant species. Synthesis. Our results suggest that deep roots are beneficial for temperate grassland species subjected to longer periods without rainfall when plant‐available water is lacking in shallow soil layers but remaining in deep soil layers. In the face of increasing drought severity, we thus recommend (1) fostering deep‐rooted species in intensive grasslands on deep, productive soil and (2) directing further studies towards identifying management practices that support deep rooting in semi‐natural grasslands.

Endothelial-specific CXCL12 regulates neovascularization during tissue repair and tumor progression.

C-X-C motif chemokine ligand 12 (CXCL12; Stromal Cell-Derived Factor 1 [SDF-1]), most notably known for its role in embryogenesis and hematopoiesis, has been implicated in tumor pathophysiology and neovascularization. However, its cell-specific role and mechanism of action have not been well characterized. Previous work by our group has demonstrated that hypoxia-inducible factor (HIF)-1 modulates downstream CXCL12 expression following ischemic tissue injury. By utilizing a conditional CXCL12 knockout murine model, we demonstrate that endothelial-specific deletion of CXCL12 (eKO) modulates ischemic tissue survival, altering tissue repair and tumor progression without affecting embryogenesis and morphogenesis. Loss of endothelial-specific CXCL12 disrupts critical endothelial-fibroblast crosstalk necessary for stromal growth and vascularization. Using murine parabiosis with novel transcriptomic technologies, we demonstrate that endothelial-specific CXCL12 signaling results in downstream recruitment of non-inflammatory circulating cells, defined by neovascularization modulating genes. These findings indicate an essential role for endothelial-specific CXCL12 expression during the neovascular response in tissue injury and tumor progression.

Enhanced cell survival in prepubertal testicular tissue cryopreserved with membrane lipids and antioxidants rich cryopreservation medium

The present study explores the advantages of enriching the freezing medium with membrane lipids and antioxidants in improving the outcome of prepubertal testicular tissue cryopreservation. For the study, testicular tissue from Swiss albino mice of prepubertal age group (2 weeks) was cryopreserved by slow freezing method either in control freezing medium (CFM; containing DMSO and FBS in DMEM/F12) or test freezing medium (TFM; containing soy lecithin, phosphatidylserine, phosphatidylethanolamine, cholesterol, vitamin C, sodium selenite, DMSO and FBS in DMEM/F12 medium) and stored in liquid nitrogen for at least one week. The tissues were thawed and enzymatically digested to assess viability, DNA damage, and oxidative stress in the testicular cells. The results indicate that TFM significantly mitigated freeze–thaw-induced cell death, DNA damage, and lipid peroxidation compared to tissue cryopreserved in CFM. Further, a decrease in Cyt C, Caspase-3, and an increase in Gpx4 mRNA transcripts were observed in tissues frozen with TFM. Spermatogonial germ cells (SGCs) collected from tissues frozen with TFM exhibited higher cell survival and superior DNA integrity compared to those frozen in CFM. Proteomic analysis revealed that SGCs experienced a lower degree of freeze–thaw-induced damage when cryopreserved in TFM, as evident from an increase in the level of proteins involved in mitigating the heat stress response, transcriptional and translational machinery. These results emphasize the beneficial role of membrane lipids and antioxidants in enhancing the cryosurvival of prepubertal testicular tissue offering a significant stride towards improving the clinical outcome of prepubertal testicular tissue cryopreservation.

Genome-Wide CRISPR Screen Identifies Genes Involved in Metastasis of Pancreatic Ductal Adenocarcinoma.

Background/Objectives: Early and aggressive metastasis is a major feature of pancreatic ductal adenocarcinoma. Understanding the processes underlying metastasis is crucial for making a difference to disease outcome. Towards these ends, we looked in a comprehensive manner for genes that are metastasis-specific. Methods: A genome-wide CRISPR-Cas9 gene knockout screen with 259,900 single guide RNA constructs was performed on pancreatic cancer cell lines with very high or very low metastatic capacity, respectively. Functional aspects of some of the identified genes were analysed in vitro. The injection of tumour cells with or without a gene knockout into mice was used to confirm the effect on metastasis. Results: The knockout of 590 genes-and, with higher analysis stringency, 67 genes-affected the viability of metastatic cells substantially, while these genes were not vital to non-metastasizing cells. Further evaluations identified different molecular processes related to this observation. One of the genes was MYBL2, encoding for a well-known transcription factor involved in the regulation of cell survival, proliferation, and differentiation in cancer tissues. In our metastasis-focussed study, no novel functional activity was detected for MYBL2, however. Instead, a metastasis-specific transformation of its genetic interaction with FOXM1 was observed. The interaction was synergistic in cells of low metastatic capacity, while there was a strong switch to a buffering mode in metastatic cells. In vivo analyses confirmed the strong effect of MYBL2 on metastasis. Conclusions: The genes found to be critical for the viability of metastatic cells form a basis for further investigations of the processes responsible for triggering and driving metastasis. As shown for MYBL2, unexpected processes of regulating metastasis might also be involved.

Endothelial NRG-1 knock-out causes left ventricle dilation in the presence and absence of angiotensin II administration

Abstract Introduction Neuregulin-1 (NRG-1) is a growth factor derived from endothelial cells and belongs to the epidermal growth factor (EGF) family. While NRG-1 is expressed by various cell types, including epithelial cells, glial cells, neurons, and myocytes, the primary source of NRG-1 is considered to be the endothelium. NRG-1 exerts its effects through the EGF receptors ERBB2, ERBB3, and ERBB4. Upon ligand binding, these receptors dimerize and become phosphorylated, leading to downstream signaling that impacts tissue proliferation, differentiation, and survival. The biological role of NRG-1 is intricate due to its isoforms and differential expression across distinct tissue types. In our study, we investigated the impact of endothelial NRG-1 knock-out (KO) on the heart, both in the presence and absence of angiotensin II (ANGII) treatment. ANGII serves as a trigger for NRG-1 upregulation. Methods C57BL/6N-VE-cad-cre-ERT2 mice were crossbred with C57BL/6N-NRG F/F mice, enabling endothelial-specific NRG-1 knockout (KO) using a tamoxifen-inducible promoter. At eight weeks of age, C57BL/6N-VE-cad-cre-ERT2-NRG F/F mice received tamoxifen (1 mg) or vehicle injections for nine consecutive days intraperitoneally (i.p.) to induce the KO. Two days after injection, osmotic minipumps were subcutaneously implanted, releasing a stable dose of angiotensin II (ANGII) at a rate of 400 ng·kg·min⁻¹ for a four-week period. Sham operations served as controls (SHAM). In the final week of treatment, high-frequency ultrasound imaging was performed to assess cardiac function and dimensions. After four weeks of ANGII treatment, mice were sacrificed, and organs were weighed and harvested for further molecular analysis. Results High-frequency ultrasound revealed left ventricle dilation in the absence of endothelial NRG-1, both in ANGII-treated and SHAM-treated mice. This was demonstrated by a significant increase in systolic and diastolic left ventricle internal diameter (LVID) in the endothelial NRG-1 KO groups compared to their respective controls. Additionally, systolic and diastolic LV volume (LV-vol) was significantly increased in the endothelial NRG-1 KO groups, regardless of ANGII treatment. Notably, there was no difference in LVID and LV-vol between SHAM and ANGII-treated groups in the presence of endothelial NRG-1, indicating that LV dilation is primarily due to the absence of endothelial NRG-1 rather than ANGII. Furthermore, heart weights of the endothelial NRG-1 KO mice were significantly increased compared to the control groups, as shown in Figure 1. Conclusion Our study demonstrates that endothelial NRG-1 KO leads to LV dilation, suggesting a crucial role for endothelial-secreted NRG-1 in maintaining normal LV function in adulthood. Further research is needed to fully understand the precise role of NRG-1 in LV homeostasis.

Removal-Free and Multicellular Suspension Bath-Based 3D Bioprinting.

Suspension bath-based 3D bioprinting (SUB3BP) is effective in creating engineered vascular structures. The transfer of oxygen and nutrients via engineered vascular networks is necessary for tissue or organ survival and integration following transplantation. Existing SUB3BP techniques face challenges in fabricating hierarchical structures with multicellular organization, including issues related to suspension bath removal, restricted material choices, and low accuracy. A next-generation SUB3BP technique that is removal-free and multicellular is presented. A simple, storable, stable, and scalable starch hydrogel design leverages the diverse spectrum of hydrogels available for use in SUB3BP. Starch granules (8.1µm) create vascular structures with minimal surface roughness (2.5µm) that simulate more natural vessel walls compared to prior research. The development of cells and organoids, as well as the bioprinting of multicellular skin models with vasculature, demonstrates that starch suspension baths eliminate the removal process and have the potential for fabricating artificial tissue with a hierarchical structure and multicellular distribution.

Selective, Intrinsically Fluorescent Trk Modulating Probes.

Neurotrophins (NTs) elicit the growth, survival, and differentiation of neurons and other neuroectoderm tissues via activation of Trk receptors. Hot spots for NT·Trk interactions involve three neurotrophin loops. Mimicry of these using "cyclo-organopeptides" comprising loop sequences cyclized onto endocyclic organic fragments accounts for a few of the low molecular mass Trk agonists or modulators reported so far; the majority are nonpeptidic small molecules accessed without molecular design and identified in random screens. It has proven difficult to verify activities induced by low molecular mass substances are due to Trk activation (rather than via other receptors), enhanced Trk expression, enhanced NT expression, or other pathways. Consequently, identification of selective probes for the various Trk receptors (e.g., A, B, and C) has been very challenging. Further, a key feature of probes for early stage assays is that they should be easily detectable, and none of the compounds reported to date are. In this work, we designed novel cyclo-organopeptide derivatives where the organic fragment is a BODIPY fluor and found ones that selectively, though not specifically, activate TrkA, B, or C. One of the assays used to reach this conclusion (binding to live Trk-expressing cells) relied on intrinsic fluorescence in the tested materials. Consequently, this work established low molecular mass Trk-selective probes exhibiting neuroprotective effects.

A Novel Human Amniotic Membrane Suspension Improves the Therapeutic Effect of Mesenchymal Stem Cells on Myocardial Infarction in Rats

Mesenchymal stem cell (MSC) therapy aids cardiac repair and regeneration, but the low rate of MSC survival and engulfment in the infarcted heart remains a major obstacle for routine clinical application. Here, an injectable suspension of human acellular amniotic membrane (HAAM) that may serve as synergistic cell delivery vehicle for the treatment of myocardial infarction (MI) by improving MSC homing and survival is developed. The results demonstrate that compared with MSC transplantation alone, HAAM‐loaded MSCs have higher survival and engraftment rates in infarcted tissue, alleviated hypoxia‐induced myocardial damage, achieved higher improvements in cardiac function, promoted angiogenesis, and reduced myocardial fibrosis. In addition, HAAM‐loaded MSCs increase N‐cadherin levels and thereby enhance the efficacy of MSCs in treating MI. This study provides a new approach for MSC‐based cardiac repair and regeneration.

Bridging the Gap: Advances and Challenges in Heart Regeneration from In Vitro to In Vivo Applications.

Cardiovascular diseases, particularly ischemic heart disease, area leading cause of morbidity and mortality worldwide. Myocardial infarction (MI) results in extensive cardiomyocyte loss, inflammation, extracellular matrix (ECM) degradation, fibrosis, and ultimately, adverse ventricular remodeling associated with impaired heart function. While heart transplantation is the only definitive treatment for end-stage heart failure, donor organ scarcity necessitates the development of alternative therapies. In such cases, methods to promote endogenous tissue regeneration by stimulating growth factor secretion and vascular formation alone are insufficient. Techniques for the creation and transplantation of viable tissues are therefore highly sought after. Approaches to cardiac regeneration range from stem cell injections to epicardial patches and interposition grafts. While numerous preclinical trials have demonstrated the positive effects of tissue transplantation on vasculogenesis and functional recovery, long-term graft survival in large animal models is rare. Adequate vascularization is essential for the survival of transplanted tissues, yet pre-formed microvasculature often fails to achieve sufficient engraftment. Recent studies report success in enhancing cell survival rates in vitro via tissue perfusion. However, the transition of these techniques to in vivo models remains challenging, especially in large animals. This review aims to highlight the evolution of cardiac patch and stem cell therapies for the treatment of cardiovascular disease, identify discrepancies between in vitro and in vivo studies, and discuss critical factors for establishing effective myocardial tissue regeneration in vivo.

Osteochondral Tissue-On-a-Chip: A Novel Model for Osteoarthritis Research.

The existing in vitro and in vivo models for studying osteoarthritis have significant limitations in replicating the complexity of joint tissues. This research aims to validate a Tissue-On-a-Chip system for osteoarthritis research. Osteochondral tissues obtained from knee replacement surgeries of patients with osteoarthritis were cultured in an Organ-On-a-Chip system. This system was designed to supply oxygen and glucose to the cartilage from the bone. The distribution of oxygen and glucose was evaluated by fluorescence using Image-iT Green Hypoxia and 2-NBDG, respectively. Cytotoxicity was measured using lactate dehydrogenase (LDH) levels in chip cultures compared to plate cultures (12 tissues per method). Glycosaminoglycans (GAGs), alkaline phosphatase (ALP), Coll2-1, and procollagen type II N-terminal propeptide (PIINP) were measured in the perfused medium of the Tissue-On-a-Chip over a period of 70 days. Fluorescence of Image-iT Green Hypoxia was observed only in the cartilage area, while 2-NBDG was distributed throughout the tissue. An increase in LDH levels was noted in the plate cultures on day 24 and in the Tissue-On-a-Chip cultures on day 63. Compared to the start of the culture, GAG content increased on day 52, while ALP showed variations. A notable increase in GAG, ALP, and Coll2-1 levels was observed on day 59. PIINP levels remained stable throughout the experiment. The validated osteochondral Tissue-On-a-Chip system can replicate the joint microenvironment, with hypoxic conditions in cartilage and normoxic conditions in bone. Tissue survival and component stability were maintained for approximately two months. This platform is a useful tool for evaluating new drugs and represents a viable alternative to animal models.

Choke Anastomosis: A Key Element Acting as a Shunt Converter Between Adjacent Angiosomes.

Choke anastomosis is commonly recognized as a resistance factor that detrimentally affects the hemodynamics of the skin flap; however, its additional potential physiological roles in normal skin function are currently not fully understood. Ten cadaveric forehead flap specimens pedicled with unilateral STAs were perfused with lead oxide-gelatin mixture, and then dissected into 3 layers, including the super temporal fascia-frontalis-galea aponeurotica layer, the subcutaneous adipose tissue layer, and the "super-thin flap" layer. The forehead flap and stratified specimens underwent molybdenum target x-ray and subsequent transparent processing to effectively visualize the microscopic spatial architecture of arterial vessels across all levels. Based on the different anastomoses near the midline area of the flap, 2 types of arterial perfusion were identified: choke anastomosis type (8/10) and true anastomosis type (2/10). The former formed multiple choke anastomoses near the midline. In the "super-thin flap" layer, arterial perfusion density on the ipsilateral side was significantly higher compared to that on the contralateral side. The arterioles on the ipsilateral side exhibited a dense and uninterrupted distribution, whereas those on the contralateral side appeared sparse and dispersed. The latter exhibited an alternative perfusion pattern; the bilateral arterial vessels were connected with 3 to 5 true anastomoses near the midline. Furthermore, the microscopic architecture confirmed a uniform distribution of arterioles that remained continuous from ipsilateral to contralateral sides in the "super-thin flap" layer. This study demonstrated that choke anastomosis not only impairs blood perfusion in the adjacent angiosomes but also acts as a shunt converter to impact the blood supply of distal skin flaps at different levels through the "trans-territory diversion phenomenon." This results in necrosis of the superficial dermis while preserving survival of the deep subcutaneous adipose tissue.

The Potential of Transforming Growth Factor-Beta1 (TGFβ1) in Fibrotic Tissue of Leiomyoma Specimens

Background: Fibrosis can define as the development of excessive fibrous tissue in an organ or tissue. In the situation of the uterus, leiomyoma or scar tissue may involve fibrosis state. The main feature of fibrosis in myoma is the existence of disordered smooth muscle cells and collagen that can be recognized via histological examinations in addition to extracellular component deposition. Leiomyoma is the most public uterine disorder that is mainly consists of smooth muscle cells, myo-fibroblast, and an obvious content of extracellular matrix. The myofibroblast in myoma lesion produced an elevated content of ECM. This paper displays the fibrotic circumstantial in leiomyoma and the mechanisms related to deposition of ECM components. Methods: this study involved (50) patients of 18-80 years old women diagnosed with benign uterine fibroid. Tumor samples were attained by consent from women undergoing hysterectomy or myomectomy at the Al Karama Teaching Hospitals in Kut, Wasit Province, Iraq between October 2022 to March 2023. samples from normal and fibroid were subjected to routein histological processing and subsequently stained with routein stains for general description, Masson Trichrome stain, Van Gieson's stain to visualize collagen content and histochemical stain (Alcian Blue-Periodic Acid Schiff) to evaluate the profile of mucins secreted via tumor cells.Normal myometrium and myoma tissue expression of TGF β1 growth factor was compared using immunohistochemical staining. Qupath and Image J programs were used to analysis the content of fibrous tissue within normal and myoma tissue. Results: Microscopical results revealed that myoma have a proliferation of overlap fusiform cells organized in bundles alienated by variable amounts of connective tissue fibers, enclosed peripherally by a pseudo capsule. Special stained sections analysis revealed deposition of extracellular martix components specially collagen fibers that vary within each nodular fibroid (intrafascicular, interfascicular) enclosed myocytes that reflect the state of fibrosis that appears as blue – green colors enclosed in Masson's trichrome stain, while in Van Gieson's stain appears as red color. Proliferative tumor cells were revealed via Alcian Blue-PAS stain in dark blue color. results showed distribution of perivascular fibers in myoma as compared with normal adjacent myometrium, which appears in red color in Van Gieson's stain. Immunostaining recorded the significantly higher intensity of TGF-β in the leiomyoma tissue than in the normal myometrium (P &lt; 0.05). The effects of the TGF-β1 inhibitor significantly differed between normal myometrium and leiomyoma tissue, with a greater decrease in cell survival in the leiomyoma tissue (P &lt; 0.05). Conclusion: highly fibrous tissue contents showed within myoma and perivascular than adjacent myometrium, these fibrous tissues were detected via special colours in both Masson Trichrome and Van Gieson's stains. The fibrous tissue within leiomyoma showed higher intensity to TGF than normal myometrium.

Effects of Three Retrograde Filling Materials on Production of Inflammatory Cytokines and Resorbing Mediators

: Root-end filling materials are typically used following endodontic surgeries, and due to their different chemical compositions, they have varying effects on the survival and differentiation of tissues around the root apex. The purpose of this in vitro study was to evaluate the effects of three retrograde filling materials—ProRoot MTA, Super ethoxy benzoic acid (EBA), and Geristore—on the production of pro-inflammatory and resorptive cytokines (RANK, RANK-L, OPG, IL-1β, and TNF-α). After mixing and setting, the experimental materials were exposed to UV rays and then applied to the prepared cells. This study used osteoblast (MG-63) and THP-1 monocyte cell lines, as well as peripheral blood monocytes (PBM). The evaluation times for RANK-L and OPG were 24 and 48 hours, while IL-1β and tumor necrosis factor (TNF-α) were evaluated at 24 hours. The RANK levels were measured using flow cytometry, and monocyte survival was assessed at different times using the MTT assay. The results were analyzed using Minitab statistical software, which indicated that the type of material significantly affected cytokine production. Exposure of monocytes to EBA resulted in a decrease in TNF-α and IL-1β secretion compared to the other groups. However, in the THP-1 cell line, there was no significant difference in the release of pro-inflammatory cytokines between the groups. Conversely, Geristore induced the highest level of RANK-L and the lowest level of OPG in cultures with the MG-63 cell line. According to the MTT assay, the viability order of the materials from early to late was Geristore &lt; EBA &lt; MTA.

The paradoxical role of transforming growth factor-β in controlling oral squamous cell carcinoma development.

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that plays a vital role in regulating cell growth, differentiation and survival in various tissues. It participates in a variety of cellular processes, including cell apoptosis, cell migration and evasion, and plays a paradoxical role in tumor genesis and development. In the early stage of tumor, TGF-β inhibits the occurrence of tumor by inhibiting cell proliferation and regulating cell apoptosis. In the advanced stage of tumor, TGF-β promotes tumor development and affects prognosis by promoting cell survival and proliferation, cell migration and invasion, participates in immune escape, etc. In this article, we will review the paradoxical role of TGF-β on the occurrence and development of oral squamous cell carcinoma.

Adipose-derived stem cell-based anti-inflammatory paracrine factor regulation for the treatment of inflammatory bowel disease

Stem cell-based therapies offer promising avenues for treating inflammatory diseases owing to their immunomodulatory properties. However, challenges persist regarding their survival and efficacy in inflamed tissues. Our study introduces a novel approach by engineering adipose-derived stem cells (ADSCs) to enhance their viability in inflammatory environments and boost the secretion of paracrine factors for treating inflammatory bowel disease (IBD). An arginine-glycine-aspartate peptide-poly (ethylene glycol)-chlorin e6 conjugate (RPC) was synthesized and coupled with ADSCs, resulting in RPC-labeled ADSCs (ARPC). This conjugation strategy employed RGD-integrin interaction to shield stem cells and allowed visualization and tracking using chlorin e6. The engineered ARPC demonstrated enhanced viability and secretion of paracrine factors upon light irradiation, regulating the inflammatory microenvironment. RNA-sequencing analysis unveiled pathways favoring angiogenesis, DNA repair, and exosome secretion in ARPC(+) while downregulating inflammatory pathways. In in vivo models of acute and chronic IBD, ARPC(+) treatment led to reduced inflammation, preserved colon structure, and increased populations of regulatory T cells, highlighting its therapeutic potential. ARPC(+) selectively homed to inflammatory sites, demonstrating its targeted effect. Overall, ARPC(+) exhibits promise as an effective and safe therapeutic strategy for managing inflammatory diseases like IBD by modulating immune responses and creating an anti-inflammatory microenvironment.

Systemic allopurinol administration reduces malondialdehyde, interleukin 6, tumor necrosis factor α, and increases vascular endothelial growth factor in random flap Wistar rats exposed to nicotine.

Smoking poses a risk to flap viability, with nicotine being a major contributor to the formation of free radicals. Allopurinol, known for its antioxidant properties, has been shown to enhance tissue survival in ischemic conditions by reducing the production of reactive oxygen species (ROS). This study aims to assess the impact of allopurinol on the viability and success of skin flaps in Wistar rats exposed to nicotine. This study examined skin flap survival in nicotine-exposed rats treated with allopurinol. Twenty-eight rats were separated into two groups. During 1 month of nicotine exposure, the treatment group received systemic allopurinol 7 days before and 2 days after the flap procedure, while the control group received no allopurinol. Pro-angiogenic factors, proinflammatory factors, anti-inflammatory factors, and oxidative markers were assessed on the 7th day after the flap procedure using enzyme-linked immunosorbent assay method. Macroscopic flap viability was evaluated on the 7th day using Image J photos. As an oxidative marker, malondialdehyde levels were significantly lower in rats given allopurinol than in controls (P &lt; 0.001). The levels of interleukin 6 and tumor necrosis factor α, as markers of inflammatory factors, were significantly lower in the group of rats given allopurinol compared to controls (P &lt; 0.001). The level of angiogenesis in rats given allopurinol, measured by vascular endothelial growth factor levels, was also higher in the treatment group compared to controls (P &lt; 0.001). Macroscopically, the percentage of distal flap necrosis in Wistar rats given allopurinol was lower and statistically significant compared to controls (P &lt; 0.001). Xanthine oxidoreductase is part of a group of enzymes involved in reactions that produce ROS. Allopurinol, as an effective inhibitor of the xanthine oxidase enzyme, can reduce oxidative stress by decreasing the formation of ROS. This reduction in oxidative stress mitigates the risk of ischemic-reperfusion injury effects and significantly increases the viability of Wistar rat flaps exposed to nicotine.

Pembrolizumab and Cabozantinib in Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma: Long-term Survival Update with a Biomarker Analysis.

Anti-programmed cell death protein 1 (PD-1) therapy is a standard of care in recurrent and/or metastatic head and neck squamous cell carcinoma (RMHNSCC). Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKI) have immunomodulatory properties and improve clinical outcomes in combination with anti-PD-1 therapy in different malignancies. We report the long-term efficacy and safety of pembrolizumab and cabozantinib in patients with RMHNSCC and include a correlative biomarker analysis. This open-label, single-arm, multicenter, phase 2 study screened 50 patients with RMHNSCC, of whom 36 received pembrolizumab and cabozantinib. The primary endpoint was overall response rate (ORR), safety, and tolerability. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and correlative studies of tissue and blood. We report the long-term PFS, OS, and safety of treated patients and describe correlative biomarkers evaluating p-MET expression and tumor immune microenvironment (TIME) using multiplex immunohistochemistry. With median follow-up of 22.4 months, the median PFS was 12.8 months with a 2-year PFS of 32.6% (95% CI, 18.8%-56.3%) and the median OS was 27.7 months with a 2-year OS of 54.7% [95% confidence interval (CI), 38.9%-76.8%]. The median duration of response was 12.6 months with a 2-year rate of 38.5% (95% CI, 30.8%-81.8%). Long-term treatment-related adverse events included manageable hypothyroidism (5.5%) and grade 1 elevated aspartate aminotransferase and alanine aminotransferase (2.8%). Baseline tumor p-MET expression correlated with ORR (P = 0.0055). Higher density of CD8+, CD103+, and CSF1-R+ cells at baseline correlated with improved OS [hazard ratio (HR) = 5.27, P = 0.030; HR = 8.79, P = 0.017; HR = 6.87, P = 0.040, respectively]. Pembrolizumab and cabozantinib provided prolonged encouraging long-term disease control and survival with a maintained favorable safety profile. The prognostic significance of higher density of CD8+, CD103+, and CSF1-R+ cells in TIME deserve further evaluation in similar clinical settings.