Collagen Staining Research Articles

Magnetic Resonance Elastography (MRE) of Bleomycin-Induced Pulmonary Fibrosis in an Animal Model.

Idiopathic pulmonary fibrosis is responsible for 40,000 deaths annually in the United States. A hallmark of idiopathic pulmonary fibrosis is elevated collagen deposition, which alters lung stiffness. Clinically relevant ways to measure changes in lung stiffness during pulmonary fibrosis are not available, and new noninvasive imaging methods are needed to measure changes in lung mechanical properties. Magnetic resonance elastography (MRE) is an in vivo magnetic resonance imaging technique proven to detect changes in shear stiffness in different organs. This study used MRE, histology, and bronchoalveolar lavage (BAL) to study changes in the mechanical and structural properties of the lungs after bleomycin-induced pulmonary fibrosis in pigs. Pulmonary fibrosis was induced in 9 Yorkshire pigs by intratracheal instillation of 2 doses of bleomycin into the right lung only. Magnetic resonance elastography scans were performed at baseline and week 4 and week 8 postsurgery in a 1.5 T magnetic resonance imaging scanner using a spin-echo echo planar imaging sequence to measure changes in lung shear stiffness. At the time of each scan, a BAL was performed. After the final scan, whole lung tissue was removed and analyzed for histological changes. Mean MRE-derived stiffness measurements at baseline, week 4, and week 8 for the control (left) lungs were 1.02 ± 0.27 kPa, 0.86 ± 0.29 kPa, and 0.68 ± 0.20 kPa, respectively. The ratio of the shear stiffness in the injured (right) lung to the uninjured control (left) lung at baseline, week 4, and week 8 was 0.98 ± 0.23, 1.52 ± 0.41, and 1.64 ± 0.40, respectively. High-dose animals showed increased protein in BAL fluid, elevated inflammation observed by the presence of patchy filtrates, and enhanced collagen and α-smooth muscle actin staining on histological sections. Low-dose animals and the control (left) lungs of high-dose animals did not show significant histopathological changes. This study demonstrated that MRE can be used to detect changes in lung stiffness in pigs after bleomycin challenge.

5-Aminolevulinic Acid-Mediated Photodynamic Therapy Improves Vocal Fold Wound Healing in Rats.

Studies showed that photodynamic therapy (PDT) might be able to prevent vocal fold scar formation when treating laryngeal lesions. We aim to investigate if PDT improves vocal wound healing and reduces scar formation in both prophylactic and remodeling procedures performed in vivo. In vivo. Vocal fold stripping was performed in Sprague-Dawley rats. PDT was performed with intraperitoneal injection of 100 mg/kg 5-Aminolevulinic Acid (5-ALA) and 635 nm laser irradiation of 20, 40, and 60 J/cm2 . PDT was performed immediately after surgery to study the prophylactic effect and 4 weeks after surgery to study the remodeling effect. Gene expression was evaluated with real-time PCR at 1 week after PDT. Histologic evaluations were performed 12 weeks after PDT, including hematoxylin-eosin, Masson, Alcian blue staining, and immunohistochemical staining of collagen I and III. PDT induced similar effects on the vocal fold wound healing outcomes in both prophylactic and remodeling procedures. Expression of MMP8, MMP13, HAS2, and TGFβ1 was significantly elevated. Histologic evaluation revealed significantly increased thickness, decreased density of collagen, and increased deposition of hyaluronic acid in the lamina propria. Immunohistochemistry also revealed better distribution and reduced density of collagen I and III. The most obvious changes were seen in the 60 J/cm2 PDT group. PDT could significantly improve vocal wound healing by providing both prophylactic effects and remodeling effects. It may be a minimally invasive treatment for vocal fold lesions with slight vocal scarring, and may be used to treat acute or chronic vocal injury to reduce vocal scarring. N/A Laryngoscope, 133:1943-1951, 2023.

TRPM4 Participates in Irradiation-Induced Aortic Valve Remodeling in Mice.

Simple SummaryDespite its benefit in cancer treatment, thoracic irradiation can induce aortic valve stenosis with fibrosis and calcification. The TRPM4 cation channel is known to participate in cellular remodeling including the transition of cardiac fibroblasts to myofibroblasts, similar to that observed during aortic valve stenosis. This study evaluates if TRPM4 is involved in irradiation-induced aortic valve damage. The aortic valve of mice was targeted by irradiation. Cardiac echography 5 months after treatment revealed an increase in aortic jet velocity, indicating stenosis. This was not observed in non-treated animals. Histological analysis revealed an increase in valvular cusp surface associated with fibrosis which was not observed in non-treated animals. The experiments were reproduced on mice after Trpm4 gene disruption. In these animals, irradiation did not induce valvular remodeling. It indicates that TRPM4 influences irradiation-induced aortic valve damage and thus could be a target to prevent such side effects of irradiation.Thoracic radiotherapy can lead to cardiac remodeling including valvular stenosis due to fibrosis and calcification. The monovalent non-selective cation channel TRPM4 is known to be involved in calcium handling and to participate in fibroblast transition to myofibroblasts, a phenomenon observed during aortic valve stenosis. The goal of this study was to evaluate if TRPM4 is involved in irradiation-induced aortic valve damage. Four-month-old Trpm4+/+ and Trpm4−/− mice received 10 Gy irradiation at the aortic valve. Cardiac parameters were evaluated by echography until 5 months post-irradiation, then hearts were collected for morphological and histological assessments. At the onset of the protocol, Trpm4+/+ and Trpm4−/− mice exhibited similar maximal aortic valve jet velocity and mean pressure gradient. Five months after irradiation, Trpm4+/+ mice exhibited a significant increase in those parameters, compared to the untreated animals while no variation was detected in Trpm4−/− mice. Morphological analysis revealed that irradiated Trpm4+/+ mice exhibited a 53% significant increase in the aortic valve cusp surface while no significant variation was observed in Trpm4−/− animals. Collagen staining revealed aortic valve fibrosis in irradiated Trpm4+/+ mice but not in irradiated Trpm4−/− animals. It indicates that TRPM4 influences irradiation-induced valvular remodeling.

1732P Cancer-associated fibroblasts correlates with immune phenotypes in human tumors

Cancer associated fibroblast (CAFs) are key players in immune regulation that shapes the tumor microenvironment. Data related to their contribution to infiltrated, deserted, and excluded immune phenotypes in human tumor samples are lacking. Colorectal cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, triple-negative breast cancer, leiomyosarcoma, and undifferentiated pleomorphic sarcoma samples from the IMMUCAN institutional profiling program (Institut Bergonié, Bordeaux, France) were evaluated. Adjacent slides were stained with H&E and a multiplex immunofluorescence panel staining for CD8 and collagen 1 (COL1). Slides were evaluated by two pathologists who characterized the tumors as immune deserted, immune excluded, or immune infiltrated using the following criteria: deserted: characterized by a paucity/absence of CD8+ T cells; excluded: characterized by the presence of CD8+ T cells that do not penetrate the tumor parenchyma; and infiltrated: characterized by the presence within the tumor parenchyma of CD8+ T cells. Image analysis was used to quantify fibroblasts (nucleated, COL1+ cells in the TME) and immune infiltrate in the TME. In 143 samples evaluated, 67 (47.9%) were excluded, 27 (18.9%) deserted, and 49 (34.4%) infiltrated. Median CD8+ density increased in excluded (209 (40-1309) cells/mm2) and infiltrated (184 (25-1472) cells/mm2) compared to deserted (47 (4-307) cells/mm2) tumors (p<0.001). Fibroblast density was elevated in excluded (2937 (43-6616) cells/mm2) and deserted (2908 (467-7987) cells/mm2) tumors compared to infiltrated (1877 (581-6742) cells/mm2). CD8+ cell density directly correlated with fibroblast density in excluded (r=0.87), deserted (r=0.67), and infiltrated (r=0.79) tumors (all p<0.001). Immune excluded tumors have high levels of CD8+ lymphocytes in the TME, albeit without penetration into the tumor parenchyma. Higher CD8+ lymphocyte infiltration in the TME is associated with increased CAFs density in all immune phenotypes. Additional data related to the spatial relationships of immune cells and fibroblast subsets in the TME of different cancer immune phenotypes will be presented at the meeting.

Effects of homoarginine supplementation on heart and skeletal muscle of rats with heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is associated with left ventricular stiffness, impaired diastolic relaxation, and severe exercise intolerance. Decreased homoarginine (hArg) levels are an independent predictor of mortality in cardiovascular disease and correlate with impaired exercise performance. We recently reported alterations in arginine, hArg, and related amino acids in obese ZSF1 rats (O-ZSF1), with a HFpEF phenotype. Although low hArg is associated with diastolic dysfunction in humans, potential effects of hArg supplementation were not tested yet. At an age of 6weeks, 12 O-ZSF1 were randomized into two groups: (1) O-ZSF1 rats supplemented with hArg in their drinking water (sO-ZSF1) or (2) O-ZSF1 rats receiving no hArg supplementation (O-ZSF1). At an age of 32weeks, effects of primary prevention by hArg supplementation on echocardiographic, histological, and functional parameters of heart and skeletal muscle were determined. Lean ZSF1 rats (L-ZSF1) served as controls. hArg supplementation did not prevent impairment of diastolic relaxation (E/e': O-ZSF1 21±3 vs. sO-ZSF1 22±3, P=0.954, L-ZSF1 18±5) but resulted in more cardiac fibrosis (histological collagen staining: +57% in sO-ZSF1 vs. O-ZSF1, P=0.027) and increased collagen gene expression (Col1a1: +48% in sO-ZSF1 vs. O-ZSF1, P=0.026). In contrary, right ventricular function was preserved by hArg supplementation (TAPSE (mm): O-ZSF1 1.2±0.3 vs. sO-ZSF1 1.7±0.3, P=0.020, L-ZSF1 1.8±0.4). Musculus soleus maximal specific muscle force (N/cm2 ) in O-ZSF1 (30.4±0.8) and sO-ZSF1 (31.9±0.9) was comparable but significantly reduced compared with L-ZSF1 (36.4±0.7; both P<0.05). Maximal absolute muscle force (g) (O-ZSF1: 177.6±7.8, sO-ZSF1: 187.8±5.0, L-ZSF1: 181.5±7.9, all P>0.05) and cross-sectional fibre area (arbitrary units) (O-ZSF1: 1697±57, sO-ZSF1: 1965±121, L-ZSF1: 1691±104, all P>0.05) were not altered. Preservation of physiological hArg level in HFpEF may not be suited to prevent alterations in left ventricular and skeletal muscle function and structure. However, hArg supplementation may be beneficial for right ventricular function especially in pulmonary hypertension in HFpEF. We may speculate that clinically observed decreased hArg level are not the cause but the consequence of a yet unrecognized pathomechanism that underpins HFpEF.

Cardiometabolic Disease Results in Sex‐ and Age‐Dependent Cerebrovascular Degeneration and Arteriolar Morphological Changes

ABSTRACT In the past 20 years, no drug therapies have been successfully translated into human use for intracerebral hemorrhage (ICH), the most devastating stroke subset. One likely factor is lack of effective animal models of human disease including common comorbidities, such as hypertension and diabetes. We evaluated the cardiometabolic rodent model, (mRen2)27, where overexpression of Ren2 results in extreme hypertension, glucose intolerance and obesity, as an appropriate ICH model. Methods: Sprague Dawley (SD) and (mRen2)27 rats were sacrificed at 14‐, 32‐, and 52‐weeks. Brains were sectioned sagittally at 5 μm. Whole‐brain sections were stained using Hematoxylin &amp; Eosin (H&amp;E) and Van Geison (VG) collagen staining to visualize the pial blood vessels. Wall‐to‐lumen ratios for vessels were calculated and compared using a student t‐test to determine significance. Results: There was no significant difference in arteriolar morphology at 14‐weeks between male SD and mRen animals (SD, n=16 and mRen, n=16). At 32‐weeks, there was a significant difference in mRen male rats (SD, n=13 and mRen, n=17; p&lt;0.0001), with significantly increased wall thickness and decreased Lumen size in the mRen animals (increased W:L ratio). However, at this timeframe, there was no significant difference between the female SD and mRen cohorts (SD, n=6 and mRen, n=8). At 52‐weeks, there was a significant increase in W:L ratios in both male (SD, n=4 and mRen, n=7; p&lt;0.01) and female (SD, n=9 and mRen, n=8; p&lt;0.05) mRen animals compared to control. Conclusion: The (mRen2)27 cardiometabolic animal model demonstrates age‐related sex changes in the wall:lumen ratio of cerebral vasculature consistent with cerebrovascular disease seen in ischemic and hemorrhagic stroke patients. Males develop these vascular changes earlier than females, but both sexes in this model develop cerebral vascular changes by 1 year of age. The (mRen2)27 model appears to recapitulate human disease and sex differences, and may serve as an appropriate translational model of ICH.

Therapeutic Potential of the Combination of Pentoxifylline and Vitamin-E in Inflammatory Bowel Disease: Inhibition of Intestinal Fibrosis.

Background: Although intestinal fibrosis is a consequence of recurrent inflammation in Inflammatory bowel disease (IBD), alleviating inflammation alone does not prevent the progression of fibrosis, suggesting that the development of direct anti-fibrotic agents is necessary. This study aimed to evaluate the anti-fibrotic properties of combination treatment with pentoxifylline (PTX) and vitamin E (Vit-E) on human primary intestinal myofibroblasts (HIMFs) and the therapeutic potential of the combination therapy in murine models of IBD. Methods: HIMFs were pretreated with PTX, Vit-E, or both, and incubated with TGF-β1. We performed Western blot, qPCR, collagen staining, and immunofluorescence to estimate the anti-fibrotic effects of PTX and Vit-E. The cytotoxicity of these was investigated through MTT assay. To induce murine models of IBD for in vivo study, C57BL/6 mice were treated with repeated cycles of dextran sulfate sodium (DSS), developing chronic colitis. We examined whether the combined PTX and Vit-E treatment would effectively ameliorate colonic fibrosis in vivo. Results: We found that the co-treatment with PTX and Vit-E suppressed TGF-β1-induced expression of fibrogenic markers, with decreased expression of pERK, pSmad2, and pJNK, more than either treatment alone in HIMFs. Neither PTX nor Vit-E showed any significant cytotoxicity in given concentrations. Consistently with the in vitro results, the co-administration with PTX and Vit-E effectively attenuated colonic fibrosis with recovery from thickening and shortening of colon in murine models of IBD. Conclusions: These findings demonstrated that the combination of PTX and Vit-E exhibits significant anti-fibrotic effects in both HIMFs and in vivo IBD models, providing a promising therapy for IBD.

Blocking RAGE improves wound healing in diabetic pigs.

Receptor for Advanced Glycated End-products (RAGE) is highly expressed in diabetes and impairs wound healing. We proposed that administering an antibody that blocks RAGE will hasten the healing of dorsal wounds in diabetic pigs compared with a non-immune IgG. Two purpose-bred diabetic (D) Yucatan minipigs (Sinclair, Auxvasse MO) each underwent 12 2 × 2 cm full thickness dorsal wounds: four wounds received decellularized porcine skin patches (Xylyx Bio, Bklyn NY): four anti-RAGE Ab (CR-3) infused patches, four saline infused patches and four wounds were left open. One pig received anti-RAGE Ab (CR-3) 1mg/kg IM q 10 days and other received non-immune IgG. Wounds were measured at 2 and 4 weeks followed by euthanasia and wound harvesting. At 2 weeks few of the patches appeared to be incorporated into the wound. By 4 weeks all patches in pigs treated systemically with CR-3 were detached and the wounds almost healed. For all 24 wounds for both pigs regardless of presence of patch or type of patch, the average IgG treated pig wound size at 4 weeks was 69.2 ± 14.6% of initial size and the average CR-3 treated pig wound size was 40.9 ± 11.3% of initial size (P=0.0002). Quantitative immunohistology showed greater staining for collagen in the CR-3 treated wounds compared with IgG treated. Staining was positive for RAGE, Mac, and IL-6 in the IgG treated wounds and negative in the CR-3 treated wounds. From these pilot experiments, we conclude that a RAGE blocking antibody given parenterally improved wound healing in a diabetic pig while patches were not effective.

Abstract P1110: Identification Of Timp-1 As A Downstream Target Of The Coronary Artery Disease Risk Factor Adamts-7 In Atherosclerosis

Background: The gene encoding the extracellular matrix (ECM) protease ADAMTS-7 was associated with coronary artery disease (CAD) in genome-wide association studies. ADAMTS-7 is expressed at all stages in human plaques and mice lacking Adamts-7 displayed reduced atherosclerotic plaque formation. While these findings render ADAMTS-7 a promising therapeutic target, the underlying mechanisms remain unknown. Methods and Results: Here, we sought to identify downstream mechanisms of ADAMTS-7 in atherosclerotic plaque formation. Targets of Adamts-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques in Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. The endogenous inhibitor of matrix metalloproteinases (MMP) Timp-1 was identified as a novel target of Adamts-7. Adamts-7 and Timp-1 were found to be co-localized in atherosclerotic plaques and co-immunoprecipitation (Co-IP) studies revealed TIMP-1 as the first putative target to bind to the catalytic domain of ADAMTS-7. In vitro degradation assays demonstrated that ADAMTS-7 degrades TIMP-1. Co-IP furthermore revealed less binding of TIMP-1 to its canonical target MMP-9 when ADAMTS-7 was present. In line, scaffolding and degradation of TIMP-1 by ADAMTS-7 impaired TIMP-1-mediated inhibition of MMP-9 in vitro. As a downstream mechanism, we investigated collagen content in atherosclerotic plaques of Apoe-/- and Apoe-/- Adamts7-/- mice after Western diet. Collagen stainings of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. Since ADAMTS-7 might exert its pro-atherogenic effects through interaction with TIMP-1, we established a TIMP-1-ADAMTS-7-interaction assay based on Förster resonance energy transfer (FRET) to identify inhibitors of this protein-protein interaction. Conclusion and Outlook: TIMP-1 represents a novel downstream target of ADAMTS-7 that can explain the role of this novel risk factor in CAD. Our FRET-based protein-protein interaction assay may be used for high-throughput screening to identifiy inhibitors and prevent the initiation or slow the progression of atherosclerosis.

Clinicopathological Features of Alopecia With an Emphasis on Etiology and Histopathological Characteristics of Scarring Alopecia.

IntroductionAlopecia is a common dermatological condition with varied etiologies based on age, gender and geographic location. Non-cicatricial (non-scarring) alopecia is more common but often not biopsied. Alternatively, primary cicatricial (scarring) alopecia is diagnostically more challenging and more commonly biopsied to determine the etiology. In this study, we evaluated the clinicopathological characteristics of alopecia in our population.MethodsWe conducted a retrospective study at the Department of Histopathology, Liaquat National Hospital and Medical College, Pakistan. A total of 104 patients were enrolled in the study who underwent scalp biopsy for alopecia over a period of 11 years. Clinical data were obtained from clinical referral forms, which included age, sex of the patients and duration of the lesions. Three hematoxylin and eosin-stained tissue sections, along with periodic acid-Schiff (PAS), PAS with diastase and collagen stains were examined, and histopathological diagnosis was rendered.ResultsOur study demonstrated that alopecia was more prevalent among females, accounting for 73.1% of cases. Most of the patients belonged to the age group of <35 years (53.8%). The type of alopecia in 88.5% of the cases was scarring, while there were 11.5% cases of non-scarring alopecia. The most common diagnoses were discoid lupus erythematosus (DLE) (23.1%) and pseudopelade of Brocq (PB) (23.1%), followed by lichen planopilaris (LPP) (15.4%). A significant association was noted between the histological features and the diagnosis, as epidermal atrophy was the most common histological feature in most cases of DLE, followed by periadnexal infiltrates, lymphocytic infiltrates, follicular plugging and basement membrane thickening. In LPP, the most common histological features were perifollicular infiltrates and fibrosis. In PB, the frequently recurring histological features in most cases were the loss of sebaceous glands, dermal fibrosis and epidermal thinning.ConclusionIn this study, we demonstrated the key role of skin punch biopsy and histology in determining the accurate etiology of scarring alopecia. We found discoid lupus erythematosus and pseudopelade of Brocq to be the most common causes of scarring alopecia, followed by lichen planopilaris.

HHLA2 promotes tumor progression by long non‑coding RNA H19 in human gallbladder cancer.

Advanced gallbladder cancer (GBC) is one of the most malignant of all types of biliary tract cancers that is associated with poor prognosis and high mortality. Accumulating evidence suggest that the B7 family of proteins serve an essential role in various types of cancers, including GBC. However, the potential function and regulatory mechanism of human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2; also known as B7-H7 or B7H5) in GBC remain poorly understood. In the present study, immunohistochemistry was used to examine the expression pattern of HHLA2 in samples from 89 patients with GBC. The possible association between HHLA2 expression and the clinicopathological parameters, including prognosis, were then assessed. Using lentiviruses, overexpression of HHLA2 plasmid or short-hairpin RNA (shRNA) of HHLA2 were transfected into GBC-SD cells to overexpress or knock down HHLA2 expression, respectively. The effects of HHLA2 overexpression and knockdown on the epithelial-mesenchymal transition (EMT) process on GBC-SD cells were measured by the western blotting and immunofluorescence staining of collagen I, N-cadherin, E-cadherin, vimentin and α-SMA. By contrast, changes in cell proliferation were measured using EdU assay. Cell invasion and migration were assessed using Transwell and wound-healing assays, respectively. In addition, a xenograft mouse model was established to evaluate the tumorigenic ability of the GBC cell line in vivo after stable transfection with lentivirus for HHLA2 overexpression or shRNA for HHLA2 knockdown. The regulatory relationships among TGF-β1, long non-coding RNA (lncRNA) H19 (H19) and HHLA2 were then investigated. The mRNA expression of lncRNA H19 were assessed using reverse transcription-quantitative PCR, whereas the expression levels of HHLA2 were detected by western blotting and immunofluorescence staining. HHLA2 expression was found to gradually increase as the stages of the GBC samples become more advanced. In addition, the expression level of HHLA2 was calculated to be positively associated with the Nevin stage, American Joint Committee on Cancer stage, tumor invasion and regional lymph node metastasis but was negatively associated with the overall patient survival (OS). In vitro experiments demonstrated that overexpression of HHLA2 promoted GBC migration, invasion, proliferation and EMT, whereas in vivo experiments found a promoting role of HHLA2 overexpression on GBC tumor growth. After transfection with lentiviruses encoding the overexpression plasmid of lncRNA H19, GBC migration, invasion, proliferation and EMT were increased. By contrast, knocking down HHLA2 expression suppressed TGF-β1- or lncRNA H19 overexpression-induced GBC migration, invasion, proliferation and EMT. In addition, HHLA2 knockdown significantly reduced the sizes of the GBC tumors in vivo. These results suggest that HHLA2 overexpression can promote GBC progression. Conversely, ablation of HHLA2 expression inhibited both TGF-β1- and lncRNA H19-induced GBC progression, suggesting that HHLA2 is a potential therapeutic target for this disease.

The repositioned drugs disulfiram/diethyldithiocarbamate combined to benznidazole: Searching for Chagas disease selective therapy, preventing toxicity and drug resistance.

Chagas disease (CD) affects at least 6 million people in 21 South American countries besides several thousand in other nations all over the world. It is estimated that at least 14,000 people die every year of CD. Since vaccines are not available, chemotherapy remains of pivotal relevance. About 30% of the treated patients cannot complete the therapy because of severe adverse reactions. Thus, the search for novel drugs is required. Here we tested the benznidazole (BZ) combination with the repositioned drug disulfiram (DSF) and its derivative diethyldithiocarbamate (DETC) upon Trypanosoma cruzi in vitro and in vivo. DETC-BZ combination was synergistic diminishing epimastigote proliferation and enhancing selective indexes up to over 10-fold. DETC was effective upon amastigotes of the BZ- partially resistant Y and the BZ-resistant Colombiana strains. The combination reduced proliferation even using low concentrations (e.g., 2.5 µM). Scanning electron microscopy revealed membrane discontinuities and cell body volume reduction. Transmission electron microscopy revealed remarkable enlargement of endoplasmic reticulum cisternae besides, dilated mitochondria with decreased electron density and disorganized kinetoplast DNA. At advanced stages, the cytoplasm vacuolation apparently impaired compartmentation. The fluorescent probe H2-DCFDA indicates the increased production of reactive oxygen species associated with enhanced lipid peroxidation in parasites incubated with DETC. The biochemical measurement indicates the downmodulation of thiol expression. DETC inhibited superoxide dismutase activity on parasites was more pronounced than in infected mice. In order to approach the DETC effects on intracellular infection, peritoneal macrophages were infected with Colombiana trypomastigotes. DETC addition diminished parasite numbers and the DETC-BZ combination was effective, despite the low concentrations used. In the murine infection, the combination significantly enhanced animal survival, decreasing parasitemia over BZ. Histopathology revealed that low doses of BZ-treated animals presented myocardial amastigote, not observed in combination-treated animals. The picrosirius collagen staining showed reduced myocardial fibrosis. Aminotransferase de aspartate, Aminotransferase de alanine, Creatine kinase, and urea plasma levels demonstrated that the combination was non-toxic. As DSF and DETC can reduce the toxicity of other drugs and resistance phenotypes, such a combination may be safe and effective.

Breast Cancer-Stromal Interactions: Adipose-Derived Stromal/Stem Cell Age and Cancer Subtype Mediated Remodeling.

Adipose tissue is characterized as an endocrine organ that acts as a source of hormones and paracrine factors. In diseases such as cancer, endocrine and paracrine signals from adipose tissue contribute to cancer progression. Young individuals with estrogen receptor-alpha positive (ER-α+) breast cancer (BC) have an increased resistance to endocrine therapies, suggesting that alternative estrogen signaling is activated within these cells. Despite this, the effects of stromal age on the endocrine response in BC are not well defined. To identify differences between young and aged ER-α+ breast tumors, RNA sequencing data were obtained from The Cancer Genome Atlas. Analysis revealed enrichment of matrix and paracrine factors in young (≤40 years old) patients compared to aged (≥65 years old) tumor samples. Adipose-derived stromal/stem cells (ASCs) from noncancerous lipoaspirate of young and aged donors were evaluated for alterations in matrix production and paracrine secreted factors to determine if the tumor stroma could alter estrogen signaling. Young and aged ASCs demonstrated comparable proliferation, differentiation, and matrix production, but exhibited differences in the expression levels of inflammatory cytokines (Interferon gamma, interleukin [IL]-8, IL-10, Tumor necrosis factor alpha, IL-2, and IL-6). Conditioned media (CM)-based experiments showed that young ASC donor age elevated endocrine response in ER-α+ BC cell lines. MCF-7 ER-α+ BC cell line treated with secreted factors from young ASCs had enhanced ER-α regulated genes (PGR and SDF-1) compared to MCF-7 cells treated with aged ASC CM. Western blot analysis demonstrated increased activation levels of p-ER ser-167 in the MCF-7 cell line treated with young ASC secreted factors. To determine if ER-α+ BC cells heightened the cytokine release in ASCs, ASCs were stimulated with MCF-7-derived CM. Results demonstrated no change in growth factors or cytokines when treated with the ER-α+ secretome. In contrast to ER-α+ CM, the ER-α negative MDA-MB-231 derived CM demonstrated increased stimulation of pro-inflammatory cytokines in ASCs. While there was no observed change in the release of selected paracrine factors, MCF-7 cells did induce matrix production and a pro-adipogenic lineage commitment. The adipogenesis was evident by increased collagen content through Sirius Red/Fast Green Collagen stain, lipid accumulation evident by Oil Red O stain, and significantly increased expression in PPARγ mRNA expression. The data from this study provide evidence suggesting more of a subtype-dependent than an age-dependent difference in stromal response to BC, suggesting that this signaling is not heightened by reciprocal signals from ER-α+ BC cell lines. These results are important in understanding the mechanisms of estrogen signaling and the dynamic and reciprocal nature of cancer cell-stromal cell crosstalk that can lead to tumor heterogeneity and variance in response to therapy.

Inflammation and fibrosis in the coal dust-exposed lung described by confocal Raman spectroscopy.

BackgroundCoal workers’ pneumoconiosis (CWP) is an occupational disease that severely damages the life and health of miners. However, little is known about the molecular and cellular mechanisms changes associated with lung inflammation and fibrosis induced by coal dust. As a non-destructive technique for measuring biological tissue, confocal Raman spectroscopy provides accurate molecular fingerprints of label-free tissues and cells. Here, the progression of lung inflammation and fibrosis in a murine model of CWP was evaluated using confocal Raman spectroscopy.MethodsA mouse model of CWP was constructed and biochemical analysis in lungs exposed to coal dust after 1 month (CWP-1M) and 3 months (CWP-3M) vs control tissues (NS) were used by confocal Raman spectroscopy. H&E, immunohistochemical and collagen staining were used to evaluate the histopathology alterations in the lung tissues.ResultsThe CWP murine model was successfully constructed, and the mouse lung tissues showed progression of inflammation and fibrosis, accompanied by changes in NF-κB, p53, Bax, and Ki67. Meanwhile, significant differences in Raman bands were observed among the different groups, particularly changes at 1,248, 1,448, 1,572, and 746 cm−1. These changes were consistent with collagen, Ki67, and Bax levels in the CWP and NS groups.ConclusionConfocal Raman spectroscopy represented a novel approach to the identification of the biochemical changes in CWP lungs and provides potential biomarkers of inflammation and fibrosis.

36. Spironolactone Mitigates Fibrosis and Improves Healing of Burn Wounds

Purpose: Adhesion, contracture, and the formation of disfiguring, hypertrophic scars remain some of the most recalcitrant problems in care of the burned patient. The central pathology driving fibrosis is an imbalance of extracellular matrix (ECM). The mineralocorticoid receptor (MR) is a nuclear receptor with shared ligand-activity from both its primary activator, aldosterone, and glucocorticoids and has cell specific effects on proliferation, migration, and ECM production in fibroblasts and keratinocytes. Here we demonstrate that MR-inhibition with FDA approved spironolactone enhances epithelialization healing and mitigates hypertrophic collagen deposition after burns in mice. Methods: Female athymic mice sustained bilateral 1 cm full-thickness thermal injury and were stratified into either a) vehicle, b) spironolactone, c) aldosterone, or d) aldosterone + spironolactone. Aldosterone applied via subcutaneous pump (Alzet) spironolactone delivered intraperitoneally. Mice followed photographically for 4-to-6-weeks. At sacrifice wound biopsies were collected for H&E, Trichrome, and protein. An additional subset of samples were assessed via Pentachrome staining to assess relative ratio of collagen, fibrin, and elastic fibers. Pentachrome samples were analyzed using ImageJ (RGB Hue threshold) to isolate elastin, GAG and collagen for quantification. Results: Histologic evaluation of healing wounds from burned mice demonstrated persistence of inflammation, wound edema, and immature ECM. In mice treated with spironolactone this effect if alleviated by 4-weeks post-injury concurrent with gross findings of rapid wound epithelialization. Spironolactone treatment additionally enhanced gross and histologic evidence of scar resolution and resulted in a decreased in collagen staining vs. controls. Conclusion: We found a significant early improvement in wound epithelialization with spironolactone therapy. This suggests a potentially distinct effect from the proposed ECM-modifying strategy hypothesized. More analysis is needed but this data supports that MR-inhibition may be a valuable new therapeutic in treatment of burn scars.

Total collagen content and distribution is increased in human colon during advancing age.

BackgroundThe effect of ageing on total collagen content of human colon has been poorly investigated. The aim of this study was to determine if ageing altered total collagen content and distribution in the human colon.MethodsMacroscopically normal ascending colon was obtained at surgery from cancer patients (n = 31) without diagnosis of diverticular disease or inflammatory bowel disease. Masson’s trichrome and Picrosirius red stains were employed to identify the total collagen content and distribution within the sublayers of the colonic wall for adult (22–60 years; 6 males, 6 females) and elderly (70 – 91years; 6 males, 4 female) patients. A hydroxyproline assay evaluated the total collagen concentration for adult (30–64 years; 9 male, 6 female) and elderly (66–91 years; 8 male, 8 female) patients.Key resultsHistological studies showed that the percentage mean intensity of total collagen staining in the mucosa, submucosa and muscularis externa was, respectively, 14(1.9) %, 74(3.2) % and 12(1.5) % in the adult ascending colon. Compared with the adults, the total collagen fibres content was increased in the submucosa (mean intensity; 163.1 ± 11.1 vs. 124.5 ± 7.8; P < 0.05) and muscularis externa (42.5 ± 8.0 vs. 20.6 ± 2.8; P < 0.01) of the elderly patients. There was no change in collagen content of the mucosa. The total collagen concentration was increased in the elderly by 16%. Sex-related differences were not found, and data were combined for analysis.ConclusionsGreater total collagen content was found in the submucosa and muscularis externa of the elderly human male and female colon. These changes may contribute to a possible loss of function with ageing.

Abstract 5935: Malignant risk assessment of clinical and histological subtypes of lichenoid mucositis with dysplasia

Abstract Introduction: Oral lichen planus (OLP), a common, chronic inflammatory condition, is categorized as potentially malignant by the World Health Organization. However, some hypothesize that only lichenoid lesions with dysplasia (LLD) are at risk of progression to cancer. Others have proposed subtypes of LLD, with differing malignant risks. Objectives: To determine if subtypes of LLD exist, based on clinical and histological features, and to determine the proportion of malignant progression in these subtypes. Methods: A nested case control study was conducted using tissue samples and longitudinal data of patients enrolled in the Oral Cancer Prediction Longitudinal study. Inclusion criteria were a biopsy confirmed diagnosis of lichenoid mucositis with dysplasia, no history of head and neck cancer, at least 5 years of follow-up, and accessible clinical photographs and bio-banked tissue. A chart review was conducted, and photographs reviewed to assess for lesion features characteristic of OLP. Histological sections were examined for lichenoid inflammation, and immunohistochemical staining for collagen IV was used to evaluate the integrity of the basement membrane (BM). Two subtypes of LLD were proposed: 1) a primary lichenoid and secondary dysplastic (L1D2) type defined as a lesion with a reticular and bilateral clinical presentation, subepithelial inflammation, and evidence of BM degeneration, and 2) a primary dysplastic and secondary lichenoid type (D1L2), which may present unilaterally or bilaterally, clinically resemble OLP or leukoplakia, and exhibit subepithelial inflammation with or without evidence of BM degeneration. Association with outcome, which was defined as progression to severe dysplasia, carcinoma in situ, or squamous cell carcinoma, was tested using Fisher’s Exact test. Results: All progressing cases had a non-homogenous clinical presentation (P=0.005) and were more apt to change (in size, texture, and/or appearance) over time (P=0.002). A reticular, smooth appearance was more common in non-progressing cases (P=0.007, P=0.016, respectively). 30% of cases in the D1L2 subtype progressed to malignancy, compared to none in the L1D2 group, though this finding did not reach statistical significance (P=0.081). Conclusion: LLD with a non-homogenous, non-reticular clinical presentation are at greater risk of progression, and a high proportion of cases exhibiting primary dysplastic features progressed to malignancy, indicating that LLD should be under close clinical follow-up. Citation Format: Iris Lin, Oh Run Kim, Leigha Rock, Lewei Zhang, Miriam Rosin, Martial Guillaud, Denise Laronde. Malignant risk assessment of clinical and histological subtypes of lichenoid mucositis with dysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5935.

Artificial Intelligence Improves Pathologist Agreement for Fibrosis Scores in Nonalcoholic Steatohepatitis Patients

Artificial Intelligence Improves Pathologist Agreement for Fibrosis Scores in Nonalcoholic Steatohepatitis Patients

Histopathology of SUMO in TGF-β1 signaling in neural lesions of leprosy reversal reactions

Background Neural lesions are one of the main pathologies occurring in leprosy, which can result in devastating consequences due to disability and deformity. Transforming Growth Factor β (TGF-β) is important in the development of tissue fibrosis. The signaling process for TGF-β starts when it binds to TβRI and TβRII, activating and inducing the phosphorylation of Smad2 and Smad3. SUMOylation is a post-translational modification, mediating the phosphorylation of Smad3 and regulating the TGF-β response. S100b is a marker of nerve damage. Objectives This study evaluates the in-vivo expression levels of SUMO-1 and SUMO-2/3/4, the distribution of Schwann cells and mononuclear cells, and the distribution of S100b and collagen-1 in nerve biopsies of patients with leprosy reversal reactions. Methods Biopsies from twenty-six leprosy patients with reversal reactions (RR) and twenty-four leprosy patients without reversal reactions were stained for SUMO-1, SUMO-2/3/4, S100b, and collagen. We analyzed the cells using a Nikon microscope under 400× magnification. Results We found an increased distribution of SUMO-1, SUMO-2/3/4, macrophages, S100b, and collagen staining in neural lesions of reversal reaction patients, compared to leprosy patients without reversal reactions. Conclusion TGF-β plays an important role in the development of fibrosis in the neural lesions of patients with leprosy. Regulation of the transcription process is mediated by TGF-β1 signaling by SUMO-1 and/or SUMO-2/3/4. The increase in macrophages and collagen, with the decrease in S100b, in patients with neural lesions reflects the damage to nerves in leprosy reversal reactions.

Prognostic Value of Stromal Type IV Collagen Expression in Small Invasive Breast Cancers.

Breast cancer is the most common cause of cancer death among women worldwide. Localized breast cancer can be cured by surgery and adjuvant therapy, but mortality remains high for tumors that metastasize early. Type IV collagen is a basement membrane protein, and breach of this extracellular matrix structure is the first step of cancer invasion. Type IV collagen is found in the stroma of many cancers, but its role in tumor biology is unclear. Here, expression of type IV collagen in the stroma of small breast cancers was analyzed, correlated to clinically used prognostic biomarkers and patient survival. The findings were further validated in an independent gene expression data cohort. Tissue samples from 1,379 women with in situ and small invasive breast cancers (≤15 mm) diagnosed in 1986-2004 were included. Primary tumor tissue was collected into tissue microarrays. Type IV collagen expression in tissues was visualized using immunohistochemistry. Gene expression data was extracted from the Cancer Genome Atlas database. Out of 1,379 women, 856 had an invasive breast cancer and type IV collagen staining was available for 714 patients. In Kaplan-Meier analysis high type IV collagen expression was significantly associated (p = 0.026) with poorer breast cancer specific survival. There was no correlation of type IV collagen expression to clinically used prognostic biomarkers. High type IV collagen expression was clearly associated to distant metastasis (p = 0.002). In an external validation cohort (n = 1,104), high type IV collagen mRNA expression was significantly (p = 0.041) associated with poorer overall survival, with overexpression of type IV collagen mRNA in metastatic tissue. Stromal type IV collagen expression in the primary tumor correlates to poor breast cancer specific survival most likely due to a higher risk of developing distant metastasis. This ECM protein may function as biomarker to predict the risk of future metastatic disease in patients with breast cancers.