The influence of a biofilm-dispersing wound gel on the wound healing process.

Topical antimicrobials that reduce the bacterial bioburden within a chronically-infected wound may have helpful or harmful effects on the healing process. We used murine models of full-thickness skin wounds to determine the effects of the novel biofilm-dispersing wound gel (BDWG) and its gel base on the healing of uninfected wounds. The rate of wound closure over 19 days was comparable among the BDWG-treated (BT) wounds and the controls. Compared with the controls, histology of the BT wounds showed formation of a stable blood clot at day 1, more neovascularisation and reepithelialisation at day 3, and more organised healing at day 7. Fluorescence-activated cell sorting analysis showed a lower percentage of neutrophils in wounded tissues of the BT group at days 1 and 3, and significantly more M2 macrophages at day 3. Levels of proinflammatory cytokines and chemokines were increased over the uninjured baseline within the wounds of all treatment groups but the levels were significantly lower in the BT group at day 1, modulating the inflammatory response. Our results suggest that BDWG does not interfere with the wound healing process and may enhance it by lowering inflammation and allowing transition to the proliferative stage of wound healing by day 3.

MORPHOLOGICAL FEATURES OF CELLULAR INFILTRATION IN THE MUCOSA OF LARGE INTESTINE IN ULCERATIVE COLITIS AND IRRITABLE BOWEL SYNDROME.

OBJECTIVE: The aim is to improve morphological diagnostics, including differential, of UC and IBS, identifying morphological features of cellular infiltration in the mucous membrane ofthe large intestine. PATIENTS AND METHODS: Material and methods: Autopsy and biopsy material - fragments of the mucous membrane of the large intestine was used in this study. All the material was divided into 5 groups. Group 1 included autopsy material from 6 cases, in which no general pathological processes in the gastrointestinal tract were detected during autopsy and microscopic examination. Group 2 included biopsy material from 34 patients with diagnosed UC of the 1st activity degree. Group 3 included the biopsy material of 27 patients with UC of the 2nd degree of activity. Group 4 included biopsy material from 19 patients, diagnosed with UC of the 3rd degree of activity. Group 5 included biopsy material from 82 patients with clinically diagnosed IBS. Histological, morphometrical, immunohistochemical and statistical methods of investigation were used. RESULTS: Results: The mucous membrane of the large intestine in patients with ulcerative colitis of varying degrees of activity, compared with the physiological norm, has pronounced infiltration by plasma cells, T-lymphocytes, B-lymphocytes, macrophages, mast cells, eosinophilic and neutrophilic leukocytes in the superficial parts of the epithelium, crypts,lamina propria. There is also an increase in the number and size of lymphoid follicles in the lamina propria. Predominant cellular elements in the infiltrate are plasma cells, T-lymphocytes, eosinophilic and neutrophilic leukocytes. The growth of ulcerative colitis activity leads to an increase the inflammatory cell infiltration in the mucous membrane of the colon, as evidenced an increase the density of cellular infiltrate; the severity of inflammatory changes in crypts and an increase in the number of crypt abscesses; a decrease the number of cases with focal infiltration in the lamina propria and an increase the number of cases with diffuse infiltration; the spread of inflammatory cell infiltration from the superficial parts of the lamina propria to its deep parts with the subsequent involvement of its entire thickness; an increase the central trends of the indexes of the severity of all cellular infiltration, infiltration by plasma cells, T-lymphocytes, macrophages, neutrophilic leukocytes. The mucous membrane of the large intestine in patients with irritable bowel syndrome has moderately pronounced cellular infiltration in the superficial epithelium andlamina propria, in comparison with the physiological norm. The number and size of lymphoid follicles increase. Inflammatory cell infiltration often spreads to the upper onethird or two thirds of the thickness of the lamina propria, characterized by the presence of plasma cells, T-lymphocytes, B-lymphocytes, macrophages, mast cells, eosinophilic and neutrophilic leukocytes. In this case, plasma cells, T-lymphocytes, mast cells and macrophages dominate. The indexes of the severity of all cellular infiltration, as well as infiltration by plasma cells, T-lymphocytes, B-lymphocytes, macrophages, mast cells, eosinophilic and neutrophilic leukocytes, increases in the mucous membrane of the large intestine in irritable bowel syndrome in comparison with the norm. In the mucous membrane of the large intestine in irritable bowel syndrome compared with ulcerative colitis of varying degrees of activity inflammatory cell infiltration is less pronounced. It often extends to one third or two thirds of the thickness of the lamina propria. There are fewer lymphoid follicles, cryptitis and crypt abscesses are not determined. The indexes of the severity of all cellular infiltration, as well as infiltration by plasma cells, T-lymphocytes, eosinophilic and neutrophilic leukocytes are lower. CONCLUSION: Conclusions: The revealed features of cellular infiltration in the mucous membrane of the large intestine make it possible to improve morphological diagnostics, including differential, of ulcerative colitis of varying degrees of activity and irritable bowel syndrome.

Cellular infiltration in traumatic brain injury.

Traumatic brain injury leads to cellular damage which in turn results in the rapid release of damage-associated molecular patterns (DAMPs) that prompt resident cells to release cytokines and chemokines. These in turn rapidly recruit neutrophils, which assist in limiting the spread of injury and removing cellular debris. Microglia continuously survey the CNS (central nervous system) compartment and identify structural abnormalities in neurons contributing to the response. After some days, when neutrophil numbers start to decline, activated microglia and astrocytes assemble at the injury site-segregating injured tissue from healthy tissue and facilitating restorative processes. Monocytes infiltrate the injury site to produce chemokines that recruit astrocytes which successively extend their processes towards monocytes during the recovery phase. In this fashion, monocytes infiltration serves to help repair the injured brain. Neurons and astrocytes also moderate brain inflammation via downregulation of cytotoxic inflammation. Depending on the severity of the brain injury, T and B cells can also be recruited to the brain pathology sites at later time points.

Transglutaminase 2-specific coeliac disease autoantibodies induce morphological changes and signs of inflammation in the small-bowel mucosa of mice.

Coeliac disease is hallmarked by an abnormal immune reaction against ingested wheat-, rye- and barley-derived gluten and the presence of transglutaminase 2 (TG2)-targeted autoantibodies. The small-bowel mucosal damage characteristic of the disorder develops gradually from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early-stage coeliac disease have TG2-autoantibodies present in serum and small-intestinal mucosa and they may already suffer from abdominal symptoms before the development of villus atrophy. Previously, we have shown that intraperitoneal injections of coeliac patient-derived sera or purified immunoglobulin fraction into mice induce a condition mimicking early-stage coeliac disease. In the current study, we sought to establish whether recombinantly produced patient-derived TG2-targeted autoantibodies are by themselves sufficient for the development of such an experimentally induced condition in immune-compromised mice. Interestingly, mice injected with coeliac patient TG2-antibodies had altered small-intestinal mucosal morphology, increased lamina propria cellular infiltration and disease-specific autoantibodies deposited in the small bowel, but did not evince clinical features of the disease. Thus, coeliac patient-derived TG2-specific autoantibodies seem to be sufficient for the induction of subtle small-bowel mucosal alterations in mice, but the development of clinical features probably requires additional factors such as other antibody populations relevant in coeliac disease.

[Investigation of the structure of a functionally intact xenopericardial bioconduit after long-term implantation]. / Issledovanie struktury funktsional'no sokhrannogo ksenoperikardial'nogo bioproteza posle prodolzhitel'nogo perioda implantatsii.

AIM: to investigate the cellular composition of a functionally intact xenopericardial valve in a recipient with acquired mitral defect after long-term implantation. MATERIAL AND METHODS: A Uniline bioconduit (BC) ('Neocor', Kemerovo) removed from the heart in the mitral position at 7.2 years after implantation was investigated. Heart valve leaflets were fixed in a buffered 4% paraformaldehyde solution and imbedded in paraffin or epoxy resin. Slices made from the paraffin samples were stained with hematoxylin and eosin or underwent immunohistochemical (IHC) examination for typing endothelial cells, smooth muscle cells, macrophages, fibroblasts, and T and B lymphocytes. The epoxy resin-embedded samples were examined using light and scanning electron microscopy according to the original procedure. For this, the samples were ground and polished, then stained with toluidine blue and basic fuchsin or contrasted with uranyl acetate and lead citrate. RESULTS: Different cell types were found in the outer layers of heart valve leaflets. IHC showed that endothelial cells, macrophages, smooth muscle cells, and fibroblasts were present in the samples. A relationship was found between the degree of degenerative changes in the BC surface and the magnitude of cellular infiltration in xenotissue. This paper debates whether impaired integrity of the surface leaflet layers plays a trigger role in structural dysfunctions of the implanted valves and whether BC endothelialization has a protective effect, which can considerably reduce the immunogenicity of xenotussie and prevent the penetration of recipient cells. CONCLUSION: The paper shows that it is expedient to modify the surface of the heart valve leaflets in order to create favorable conditions for the attachment and function of endothelial progenitor cells.

An in vivo analysis of Miromesh--a novel porcine liver prosthetic created by perfusion decellularization.

BACKGROUND: Bioprosthetics derived from human or porcine dermis and intestinal submucosa have dense, homogenous, aporous collagen structures that potentially limit cellular penetration, undermining the theoretical benefit of a "natural" collagen scaffold. We hypothesized that Miromesh-a novel prosthetic derived from porcine liver by perfusion decellularization-provides a more optimal matrix for tissue ingrowth. METHODS: Thirty rats underwent survival surgery that constituted the creation of a 4 × 1 cm abdominal defect and simultaneous bridged repair. Twenty rats were bridged with Miromesh, and 10 rats were bridged with non-cross-linked porcine dermis (Strattice). Ten Miromesh and all 10 Strattice were rinsed in vancomycin solution and inoculated with 10(4) colony-forming units of green fluorescent protein-labeled Staphylococcus aureus (GFP-SA) after implantation. Ten Miromesh controls were neither soaked nor inoculated. No animals received systemic antibiotics. All animals were euthanized at 90 d and underwent an examination of their gross appearance before being sectioned for quantitative bacterial culture and histologic grading. A pathologist scored specimens (0-4) for cellular infiltration, acute inflammation, chronic inflammation, granulation tissue, foreign body reaction, and fibrous capsule formation. RESULTS: All but one rat repaired with Strattice survived until the 90-d euthanization. All quantitative bacterial cultures for inoculated specimens were negative for GFP-SA. Of nine Strattice explants, none received a cellular infiltration score >0, consistent with a poor tissue-mesh interface observed grossly. Of 10 Miromesh explants also inoculated with GFP-SA, seven of 10 demonstrated cellular infiltration with an average score of +2.7 ± 0.8, whereas sterile Miromesh implants received an average score of 0.8 ± 1.0. Two inoculated Miromesh implants demonstrated acute inflammation and infection on histology. CONCLUSIONS: A prosthetic generated from porcine liver by perfusion decellularization provides a matrix for superior cellular infiltration compared with non-cross-linked porcine dermis.

The plasticity of inflammatory monocyte responses to the inflamed central nervous system.

Over the last three decades it has become increasingly clear that monocytes, originally thought to have fixed, stereotypic responses to foreign stimuli, mediate exquisitely balanced protective and pathogenic roles in disease and immunity. This balance is crucial in core functional organs, such as the central nervous system (CNS), where minor changes in neuronal microenvironments and the production of immune factors can result in significant disease with fatal consequences or permanent neurological sequelae. Viral encephalitis and multiple sclerosis are examples of important human diseases in which the pathogenic contribution of monocytes recruited from the bone marrow plays a critical role in the clinical expression of disease, as they differentiate into macrophage or dendritic cells in the CNS to carry out effector functions. While antigen-specific lymphocyte populations are central to the adaptive immune response in both cases, in viral encephalitis a prominent macrophage infiltration may mediate immunopathological damage, seizure induction, and death. However, the autoimmune response to non-replicating, non-infectious, but abundant, self antigen has a different disease progression, associated with differentiation of significant numbers of infiltrating monocytes into dendritic cells in the CNS. Whilst a predominant presence of macrophages or dendritic cells in the inflamed CNS in viral encephalitis or multiple sclerosis is well described, the way in which the inflamed CNS mobilizes monocytes in the bone marrow to migrate to the CNS and the key drivers that lead to these specific differentiation pathways in vivo are not well understood. Here we review the current understanding of factors facilitating inflammatory monocyte generation, migration and entry into the brain, as well as their differentiation towards macrophages or dendritic cells in viral and autoimmune disease in relation to their respective disease outcomes.

Truncated monocyte chemoattractant protein-1 can alleviate cardiac injury in mice with viral myocarditis via infiltration of mononuclear cells.

BALB/c mice inoculated intraperitoneally with coxsackievirus group B type 3 (CVB3) were allocated to five groups; namely, a viral myocarditis group infected with CVB3 alone (control group), an antibody intervention group that received intracardiac anti-MCP-1, an antibody intervention control group that received goat IgG, a tMCP-1 intervention group that received plasmid pVMt expressing tMCP-1, and a tMCP-1 intervention control group that received plasmid pVAX1. There was also a normal control group. The ratio of murine heart weight to body weight, pathological score of myocardial tissue, serum creatine kinase-MB titers and CVB3 loading of myocardial tissue were assessed. The cardiac lesions in mice that received 20, 40 or 60 µg pVMt (P < 0.05) were less severe than those in control mice with untreated viral myocarditis. In addition, fewer mononuclear cells had infiltrated the myocardium of mice who received 40 or 60 µg pVMt intramyocardially (P < 0.01), whereas there was no difference in mononuclear cell infiltration between mice with viral myocarditis and those that received 20 µg pVMt (P > 0.05). There was also no difference between mice that received anti-MCP-1 antibody and those that received 40 µg pVMt in ratio of HW/BW, serum CK-MB titers and pathological score (P > 0.05). This study showed that tMCP-1 can alleviate cardiac lesions and cardiac injury in mice with viral myocarditis via infiltration of mononuclear cells. Thus, tMCP-1 may be an alternative to anti-MCP-1 antibody treatment of viral myocarditis. Further research is required.

Lymphocytic infiltration as a marker of disease severity in patients with bullous pemphigoid-A case series.

Patients of Bullous Pemphigoid with predominant lymphocytic inflammatory infiltrate on histopathology have a severe form of the disease requiring high doses of steroids along with an adjuvant immunosuppressant. Thus, the histopathological evaluation would predict the severity of Bullous Pemphigoid, especially in countries where ELISA and immunofluorescence are not readily available.

Benefits of In Situ Foamed and Printed Porous Scaffolds in Wound Healing.

Macroporous hydrogels have shown significant promise in biomedical applications, particularly regenerative medicine, due to their enhanced nutrient and waste permeability, improved cell permissibility, and minimal immunogenicity. However, traditional methods of generating porous hydrogels require secondary post-processing steps or harmful reagents making simultaneous fabrication with bioactive factors and cells impossible. Therefore, a handheld printer is engineered for facile and continuous generation and deposition of hydrogel foams directly within the skin defect to form defect-specific macroporous scaffolds. Within the handheld system, a temperature-controlled microfluidic homogenizer is coupled with miniaturized liquid and air pumps to mix sterile air with gelatin methacryloyl (GelMA) at the desired ratio. An integrated photocrosslinking unit is then utilized to crosslink the printed foam in situ to form scaffolds with controlled porosity. The system is optimized to form reliable and uniform GelMA foams. The resulting foam scaffolds demonstrate mechanical properties with excellent flexibility making them suitable for wound healing applications. The results of in vitro cell culture on the scaffolds demonstrate significantly increased cellular activity compared to the solid hydrogel. The in vivo printed foam scaffolds enhanced the rate and quality of wound healing in mice with full-thickness wound without the use of biological materials.

Differential Gene Expression and Immune Cell Infiltration in Patients with Steroid-induced Necrosis of the Femoral Head.

OBJECTIVE: The study aimed to study the differential gene expression and immune cell infiltration in patients with steroid-induced necrosis of the femoral head (SANFH), identify the key genes and immune cells of SANFH, and explore the relationship between immune cells and SANFH. METHODS: The high-throughput gene chip dataset GSE123568 was downloaded from the GEO database, and the differential gene expression was analyzed with the R language. The STRING database and Cytoscape software were used to analyze the protein interaction network and screen key genes, and enrichment analysis was carried out on key genes. The infiltration of immune cells in SANFH patients was analyzed and verified by immunohistochemistry. RESULTS: EP300, TRAF6, STAT1, JAK1, CASP8, and JAK2 are key genes in the pathogenesis of SANFH, which mainly involve myeloid cell differentiation, cytokine-mediated signaling pathway, tumor necrosis factor-mediated signaling pathway, and cellular response to tumor necrosis factor through JAK-STAT, NOD-like receptor, toll-like receptor, and other signaling pathways, leading to the occurrence of diseases; immune infiltration and immunohistochemical results have shown the expression of memory B cells and activated dendritic cells as reduced in SANFH patients, while in the same SANFH samples, M1 macrophages have been positively correlated with monocytes, and neutrophils have been negatively correlated with monocytes expression. CONCLUSION: EP300, TRAF6, STAT1, JAK1, CASP8, and JAK2 have exhibited significant differences in SANFH (spontaneous osteonecrosis of the femoral head). Memory B cells, activated dendritic cells, M1 macrophages, monocytes, and neutrophils have shown abnormal expression in SANFH.

Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels.

Glioblastoma (GBM) is the deadliest brain tumor for which there is no cure. Bioengineered GBM models, such as hydrogel-encapsulated spheroids, that capture both cell-cell and cell-matrix interactions could facilitate testing of much needed therapies. Elucidation of specific microenvironment properties on spheroid responsiveness to therapeutics would enhance the usefulness of GBM models as predictive drug screening platforms. Here, GBM spheroids consisting of U87 or patient-derived GBM cells were encapsulated in soft (∼1 kPa), stiff (∼7 kPa), and dual-stiffness polyethylene glycol-based hydrogels, with GBM spheroids seeded at the stiffness interface. Spheroids were cultured for 7 days and examined for viability, size, invasion, laminin expression, hypoxia, proliferation, and response to the chemotherapeutic temozolomide (TMZ). We noted excellent cell viability in all hydrogels, and higher infiltration in soft compared to stiff hydrogels for U87 spheroids. In dual gels spheroids mostly infiltrated away from the stiffness interface with minimal crossing over it and some individual cell migration along the interface. U87 spheroids were equally responsive to TMZ in the soft and stiff hydrogels, but cell viability in the spheroid periphery was higher than the core for stiff hydrogels whereas the opposite was true for soft hydrogels. HIF1A expression was higher in the core of spheroids in the stiff hydrogels, while there was no difference in cell proliferation between spheroids in the stiff vs soft hydrogels. Patient-derived GBM spheroids did not show stiffness-dependent drug responses. U87 cells showed similar laminin expression in soft and stiff hydrogels with higher expression in the spheroid periphery compared to the core. Our results indicate that microenvironment stiffness needs to be considered in bioengineered GBM models including those designed for use in drug screening applications. STATEMENT OF SIGNIFICANCE: Recent work on tumor models engineered for use in drug screening has highlighted the potential of hydrogel-encapsulated spheroids as a simple, yet effective platform that show drug responses similar to native tumors. It has also been shown that substrate stiffness, in vivo and in vitro, affects cancer cell responses to drugs. This is particularly important for glioblastoma (GBM), the deadliest brain cancer, as GBM cells invade by following the stiffer brain structures such as white matter tracks and the perivascular niche. Invading cells have also been associated with higher resistance to chemotherapy. Here we developed GBM spheroid models using soft, stiff and dual-stiffness hydrogels to explore the connection between substrate stiffness, spheroid invasion and drug responsiveness in a controlled environment.

Cellular infiltration in an injectable sulfated cellulose nanocrystal hydrogel and efficient angiogenesis by VEGF loading.

BACKGROUND: Cellular infiltration and angiogenesis into implanted biomaterial scaffolds are crucial for successful host tissue integration and tissue regeneration. Cellulose nanocrystal (CNC) is a nano-sized cellulose derivative, which can form an injectable physical gel with salts. Sulfate groups of sulfated CNC (CNC-S) can act as a binding domain to various growth factors and cytokines with a heparin-binding domain for sustained release of them. Vascular endothelial growth factor (VEGF) can promote the proliferation of endothelial cells and angiogenesis. In this study, VEGF-loaded CNC-S hydrogel was evaluated as an injectable scaffold that can induce cellular infiltration and angiogenesis. METHODS: CNC-S was hydrolyzed to get desulfated CNC (CNC-DS), which was used as a negative control group against CNC-S. Both CNC-S and CNC-DS hydrogels were prepared and compared in terms of biocompatibility and VEGF release. The hydrogels with or without VEGF loading were subcutaneously injected into mice to evaluate the biocompatibility, cellular infiltration, and angiogenesis induction of the hydrogels. RESULTS: Both hydrogels possessed similar stability and shear-thinning behavior to be applicable as injectable hydrogels. However, CNC-S hydrogel showed sustained release (until 8 weeks) of VEGF whereas CNC-DS showed a very fast release of VEGF with a large burst. Subcutaneously injected CNC-S hydrogel showed much enhanced cellular infiltration as well as better biocompatibility with milder foreign body response than CNC-DS hydrogel. Furthermore, VEGF-loaded CNC-S hydrogel induced significant angiogenesis inside the hydrogel whereas VEGF-loaded CNC-DS did not. CONCLUSION: CNC-S possesses good properties as a biomaterial including injectability, biocompatibility, and allowing cellular infiltration and sustained release of growth factors. VEGF-loaded CNC-S hydrogel exhibited efficient angiogenesis inside the hydrogel. The sulfate group of CNC-S was a key for good biocompatibility and the biological activities of VEGF-loaded CNC hydrogel.

Retrospective Evaluation of the Most Frequently Observed Histological Changes in Duodenal and Rectal Mucosal Biopsies in Horses with Recurrent Colic.

Colic, a condition affecting the gastrointestinal tract of horses, manifests as severe pain and may be a life-threatening condition. It is possible to distinguish between an acute, disposable process, as well as recurrent colic symptoms (abdominal pain) caused by an ongoing chronic inflammatory process. This paper presents a retrospective analysis of the histopathological findings of duodenal and rectal samples taken from horses with recurrent colic, with the aim to determine the frequency and extent of inflammation. The samples, i.e., duodenal biopsy (60 samples) and rectal biopsy (17 samples), were taken from 77 horses showing recurrent colic symptoms. Histopathological examination included staining with hematoxylin and eosin. The examination included evaluation of the superficial epithelium, mucosal lamina propria, and submucosa. All samples from the duodenum and rectum showed the presence of leukocyte infiltration in the mucosal lamina propria. The most frequently observed cellular infiltration was a moderate infiltration consisting of lymphocytes and plasma cells in duodenum and mixed populations of plasma cells, lymphocytes, and eosinophilia in the rectum. Mott cells were also noted among the inflammatory infiltrates. More than one-fourth of the horses were found to have shortened intestinal villi. The results presented here showed the involvement of inflammation in the course of recurrent colic, which can be both its cause (by impairing motility and absorption) and its effect (as a result of obstruction or ischemia).

Controlling Pore Size of Electrospun Vascular Grafts by Electrospraying of Poly(Ethylene Oxide) Microparticles.

Electrospinning has become a popular polymer processing technique for application in vascular tissue engineering due to its unique capability to fabricate porous vascular grafts with fibrous morphology closely mimicking the natural extracellular matrix (ECMs). However, the inherently small pore sizes of electrospun vascular grafts often inhibit cell infiltration and impede vascular regeneration. Here we describe an effective and controllable method to increase the pore size of electrospun poly(ε-caprolactone) (PCL) vascular graft. With this method, composite grafts are prepared by turning on or off electrospraying of poly(ethylene oxide) (PEO) microparticles during the process of electrospinning PCL fibers. The PEO microparticles are used as a porogen agent and can be subsequently selectively removed to create a porogenic layer within the electrospun PCL grafts. Three types of porogenic PCL grafts were constructed using this method. The porogenic layer was either the inner layer, the middle one, or the outer one.

Chemical Fingerprinting, Antioxidant, and Anti-Inflammatory Potential of Hydroethanolic Extract of Trigonella foenum-graecum.

In the current study, the antioxidant and anti-inflammatory potential of hydroethanolic extract of T. foenum-graecum seeds was evaluated. Phenolic profiling of T. foenum-graecum was conducted through high-performance liquid chromatography-photodiode array (HPLC-PDA) as well as through the mass spectrometry technique to characterize compounds responsible for bioactivity, which confirmed almost 18 compounds, 13 of which were quantified through a chromatographic assay. In vitro antioxidant analysis of the extract exhibited substantial antioxidant activities with the lowest IC50 value of both DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) inhibition assays. The extract was found to be non-toxic against human RBCs and murine macrophage RAW 264.7 cells. Moreover, the extract significantly (p < 0.001) reduced the lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), intrlukin-6 (IL-6), prostaglandin E2 (PGE2), and nitric oxide (NO) in RAW 264.7 cells in a concentration-dependent manner. The hydroethanolic extract of T. foenum-graecum exhibited considerable anti-inflammatory potential by decreasing the cellular infiltration to the inflammatory site in both carrageenan-induced peritonitis and an air pouch model of inflammation. Pretreatment with T. foenum-graecum extract caused significant improvement in antioxidants such as superoxide dismutase (SOD), CAT (catalase), malondialdehyde (MDA), and myeloperoxidase (MPO) against oxidative stress induced by carrageenan. Based on our results of in vivo and in vitro experimentation, we concluded that hydroethanolic extract of T. foenum-graecum is a potential source of phenolic compounds with antioxidant and anti-inflammatory potential.

Tumor Microenvironment Characterization in Breast Cancer and an Immune Cell Infiltration Score Development, Validation, and Application.

The tumor microenvironment (TME) refers to the cellular environment in which tumors exist. An increasing number of reports have emphasized its role in tumor progression, prognosis, relapse, metastasis, and therapeutic response with breast cancer (BRCA). Few studies have revealed a systematic landscape of immune cell infiltration (ICI) in BRCA. In this study, we comprehensively analyzed the immune cells infiltrating TME in BRCA. Three ICI patterns were identified through an unsupervised clustering method and an ICI score was developed by a principal component analysis (PCA). A Kaplan-Meier survival with log-rank test revealed a significant overall survival (OS) difference of BRCA patients with these three ICI patterns. We also found that a high ICI score was characterized by an elevated tumor mutation burden (TMB), effector T-cell infiltration, INF-γ-related cytotoxicity, and cytolytic activity score. An independent cohort validated that this ICI score could be a prognostic indicator for BRCA. Two immunotherapeutic cohorts and two chemotherapeutic cohorts confirmed that patients with higher ICI scores showed significant chemotherapeutic and immunotherapeutic advantages. In summary, these results suggest that the ICI patterns could act as a prognostic indicator and that the ICI score could precisely predict the clinical outcome for BRCA patients.

Enhancing Cellular Infiltration on Fluffy Polyaniline-Based Electrospun Nanofibers.

Despite the unique properties of polyaniline (PANI), the processability of this smart polymer is associated with challenges. Particularly, it is very difficult to prepare PANI nanofibers due to poor solubility, high charge density, and rigid backbone. The most common approach for solving this problem is blending PANI with a carrier polymer. Furthermore, the major limitations of nanofibers for tissue engineering applications are their low porosity and two-dimensional (2D) structure. In this study, conductive nanofibers were fabricated through electrospinning of PANI/poly(ether sulfone) (PES) with different solvents including dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and hexafluoroisopropanol (HFIP). The effect of solvent, carrier polymer (PES), and PANI content on formation of 3D conductive nanofibers with appropriate porosity were investigated. It was shown that a solvent with suitable properties should be selected in such a way that the composite nanofibers can be electrospun at the lowest concentration of PES. In this way, the ratio of PANI increased in the scaffold, the electrical conductivity of nanofibers enhanced, and the flat 2D structure of scaffold changed to a fluffy 3D structure. Among the three studied solvents, HFIP with the lowest boiling point and the lowest surface tension was the best solvent for the fabrication of PANI/PES nanofibers. PES could be electrospun at a concentration of 9% w/w in HFIP, while the optimum percentage of PES in DMSO and NMP was above 23% w/w to produce uniform nanofibers. 3D nanofibrous scaffold obtained from 0.5% PANI/9% PES/HFIP solution with electrical conductivity of 3.7 × 10-5 S/Cm and porosity of 92.81 ± 1.23%. Cell infiltration into the 3D nanofibers with low packing density improved compared to densely packed 2D nanofibers.

Hydrogel matrix presence and composition influence drug responses of encapsulated glioblastoma spheroids.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with median patient survival of 12-15 months. To facilitate treatment development, bioengineered GBM models that adequately recapitulate the in vivo tumor microenvironment are needed. Matrix-encapsulated multicellular spheroids represent such model because they recapitulate solid tumor characteristics, such as dimensionality, cell-cell, and cell-matrix interactions. Yet, there is no consensus as to which matrix properties are key to improving the predictive capacity of spheroid-based drug screening platforms. We used a hydrogel-encapsulated GBM spheroid model, where matrix properties were independently altered to investigate their effect on GBM spheroid characteristics and drug responsiveness. We focused on hydrogel degradability, tuned via enzymatically degradable crosslinkers, and hydrogel adhesiveness, tuned via integrin ligands. We observed increased cellular infiltration of GBM spheroids and increased resistance to temozolomide in degradable, adhesive hydrogels compared to spheroids in non-degradable, non-adhesive hydrogels or to free-floating spheroids. Further, a higher infiltration index was noted for spheroids in adhesive compared to non-adhesive degradable hydrogels. For spheroids in degradable hydrogels, we determined that infiltrating cells were more susceptible to temozolomide compared to cells in the spheroid core. The temozolomide susceptibility of the infiltrating cells was independent of integrin adhesion. We could not attribute differential drug responses to differential cellular proliferation or to limited drug penetration into the hydrogel matrix. Our results suggest that cell-matrix interactions guide GBM spheroid drug responsiveness and that further elucidation of these interactions could enable the engineering of more predictive drug screening platforms. STATEMENT OF SIGNIFICANCE: Glioblastoma multiforme (GBM) multicellular spheroids hold promise for drug screening and development as they better mimic in vivo cellular responses to therapeutics compared to monolayer cultures. Traditional spheroid models lack an external extracellular matrix (ECM) and fail to mimic the mechanical, physical, and biochemical cues seen in the GBM microenvironment. While embedding spheroids in hydrogel matrices has been shown to better recapitulate the tumor microenvironment, there is still limited understanding as to the key matrix properties that govern spheroid responsiveness to drugs. Here we decoupled and independently altered matrix properties such as degradability, via an enzymatically degradable peptide crosslinker, and cell adhesion, via an adhesive ligand, giving further insight into what matrix properties contribute to GBM chemoresistance.

From Cellular Infiltration Assessment to a Functional Gene Set-Based Prognostic Model for Breast Cancer.

Background: Cancer heterogeneity is a major challenge in clinical practice, and to some extent, the varying combinations of different cell types and their cross-talk with tumor cells that modulate the tumor microenvironment (TME) are thought to be responsible. Despite recent methodological advances in cancer, a reliable and robust model that could effectively investigate heterogeneity with direct prognostic/diagnostic clinical application remained elusive. Results: To investigate cancer heterogeneity, we took advantage of single-cell transcriptome data and constructed the first indication- and cell type-specific reference gene expression profile (RGEP) for breast cancer (BC) that can accurately predict the cellular infiltration. By utilizing the BC-specific RGEP combined with a proven deconvolution model (LinDeconSeq), we were able to determine the intrinsic gene expression of 15 cell types in BC tissues. Besides identifying significant differences in cellular proportions between molecular subtypes, we also evaluated the varying degree of immune cell infiltration (basal-like subtype: highest; Her2 subtype: lowest) across all available TCGA-BRCA cohorts. By converting the cellular proportions into functional gene sets, we further developed a 24 functional gene set-based prognostic model that can effectively discriminate the overall survival (P = 5.9 × 10-33, n = 1091, TCGA-BRCA cohort) and therapeutic response (chemotherapy and immunotherapy) (P = 6.5 × 10-3, n = 348, IMvigor210 cohort) in the tumor patients. Conclusions: Herein, we have developed a highly reliable BC-RGEP that adequately annotates different cell types and estimates the cellular infiltration. Of importance, the functional gene set-based prognostic model that we have introduced here showed a great ability to screen patients based on their therapeutic response. On a broader perspective, we provide a perspective to generate similar models in other cancer types to identify shared factors that drives cancer heterogeneity.