Local Fibrosis Research Articles

Lacticaseibacillus paracsei HY7207 Alleviates Hepatic Steatosis, Inflammation, and Liver Fibrosis in Mice with Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty acid disease (NAFLD) is caused by a build-up of fat in the liver, inducing local inflammation and fibrosis. We evaluated the effects of probiotic lactic acid-generating bacteria (LAB) derived from a traditional fermented beverage in a mouse model of NAFLD. The LAB isolated from this traditional Korean beverage were screened using the human hepatic cell line HepG2, and Lactocaseibacillus paracasei HY7207 (HY7207), which was the most effective inhibitor of fat accumulation, was selected for further study. HY7207 showed stable productivity in industrial-scale culture. Whole-genome sequencing of HY7207 revealed that the genome was 2.88 Mbp long, with 46.43% GC contents and 2778 predicted protein-coding DNA sequences (CDSs). HY7207 reduced the expression of lipogenesis and hepatic apoptosis-related genes in HepG2 cells treated with palmitic acid. Furthermore, the administration of 109 CFU/kg/day of HY7207 for 8 weeks to mice fed an NAFLD-inducing diet improved their physiologic and serum biochemical parameters and ameliorated their hepatic steatosis. In addition, HY7207 reduced the hepatic expression of genes important for lipogenesis (Srebp1c, Fasn, C/ebpa, Pparg, and Acaca), inflammation (Tnf, Il1b, and Ccl2), and fibrosis (Col1a1, Tgfb1, and Timp1). Finally, HY7207 affected the expression of the apoptosis-related genes Bax (encoding Bcl2 associated X, an apoptosis regulator) and Bcl2 (encoding B-cell lymphoma protein 2) in the liver. These data suggest that HY7207 consumption ameliorates NAFLD in mice through effects on liver steatosis, inflammation, fibrosis, and hepatic apoptosis. Thus, L. paracasei HY7207 may be suitable for use as a functional food supplement for patients with NAFLD.

Semiconducting polymer nanoparticles laden with platelet-rich plasma for endometrium regeneration via regulating macrophage M1/M2 type polarization

Thin endometrium is linked to recurrent miscarriage and infertility. Reliable clinical strategies for endometrium regeneration are lacking. Human platelet-rich plasma (PRP), especially derived from umbilical cord blood, shows promising regenerative capabilities. Here, we designed a construct of the human umbilical cord blood-derived PRP and the as-synthesized semiconducting polymer nanoparticles (SPNs) for promoting endometrial regeneration and fertility restoration following in situ microinjection in a rat model of endometrial damage. The mechanistic basis for the regenerative benefits of these PRP-SPNs was also assessed, with a particular focus on the regulation of macrophage activity. In vitro, PRP-SPNs treatment was sufficient to enhance the proliferation of human endometrial stromal cells. In vivo, PRP-SPNs administration induced robust endometrial proliferation, re-epithelialization, and angiogenic activity while inhibiting localized fibrosis. Critically, the regenerated endometrium exhibited enhanced receptivity such that it was conducive to higher rates of implantation and fetal development. At the cellular level, PRP-SPNs were found to promote the polarization of CD163+ M2 macrophages while coordinating the induction of more effective anti-inflammatory responses in vivo and in vitro. Together, these results suggest that PRP-SPNs represent a safe, effective, noninvasive approach that can be conveniently implemented to remediate thin endometrium and restore fertility for patients in need.

Fuchs endothelial corneal dystrophy: aspects of pathogenesis

Fuchs endothelial corneal dystrophy (FECD) – multigenic bilateral disorder, characterized by dysfunction of corneal endothelium cells (CECs) that eventually results in loss of transparency. Purpose: To evaluate the pathogenesis of FECD with histological and immunohistochemical methods, immunofluorescence, scanning electron microscopy (SEM). Methods: Endothelium–Descemet membrane (EDM) complexes, corneal buttons were obtained from 76 patients (85 eyes) with FECD during keratoplasty and divided into 1A, 2A, 3A groups, 15 EDMs (donor tisuue) – 1B, 2B, 3B groups (control). Morphological (hematoxylin/eosin staining) and immunohistochemical (primary antibodies to cytokeratin AE1/AE3 and vimentin) studies (1A, 1B groups), phase-contrast microscopy and immunofluorescence analysis of tight junction protein ZO-1 (2A, 2B groups), SEM (3A, 3B groups) were performed. Results: FECD is characterized by decline of CECs, cell and nuclear enlargement. Nuclear-cytoplasmic ratio of CECs is relative to control. CECs expression ZO-1 is decreased in the area of guttae ingrowth. Coexpression of cytokeratin AE1/AE3 and vimentin is found. Morphological and immunohistochemical studies show DM’ thickening, stromal and epithelial edema and local fibrosis, vimentin (stromal cells) and cytokeratin AE1/AE3 (epithelium) expression in FECD. Conclusion: Pathogenesis of FECD include CECs’ epithelial-mesenchymal transition, followed by guttae formation – the excrescences, which destroy cytosketon and lead to progressive loss of CECs. Changes in permeability of endothelium cause edema and fibrosis of stroma and epithelial layer.

Vascularized Omentum Lymphatic Transplantation as a Treatment for Chronic Inguinal Ulceration and Lymphedema.

After tumor resection, lymphadenectomy, and radiotherapy, recurrent lymphatic fluid leakage and infection can occur in the inguinal region, contributing to severe localized tissue fibrosis. When wounds form in this region, they tend to heal slowly over extended periods, and no optimal approach for treating these complex wounds has yet been established. Groin wound debridement and dissection of the vessels in the wound recipient site were performed by the burn surgeon. A general surgeon performed the laparoscopic partial omentum excision. One portion of the omentum was used to fill the large inguinal space, whereas the other portion was laid flat on the wound sites in the groin and anterior perineum to facilitate the restoration of appropriate lymphatic fluid reflux. The vessels of the omentum were microsurgically anastomosed with the vessels in the recipient site. Thin split-thickness slices of skin were then taken from this donor site based on the size of the wound. After the successful establishment of revascularization between the flap and recipient sites, lymphatic fluid leakage was not observed in this patient. No inguinal wounding or lymphatic exudate were evident in the patient during follow-up, and significant improvements in lymphedema of the lower extremities were evident. In this article, we discuss the advantages and disadvantages of vascularized omentum lymphatic transplantation. Overall, this procedure represents a promising new approach for the treatment of refractory wounds caused by lymphatic fistulas.

Role of lingual mucosa as a graft material in the surgical treatment of Peyronie's disease.

Peyronie's disease (PD) is a localized fibrosis of tunica albuginea, which causes the anatomical and functional changes to the penis. Corporoplasty with grafting is indicated in severe (>60°) and complex curvature. Buccal mucosa is the most favored autologous graft material nowadays. The ventrolateral aspect of lingual mucosa has similar histological features to the rest of the oral cavity. This study aimed to test the efficacy, safety, durability, and reproducibility of corporoplasty with lingual mucosal graft (LMG) in the surgical treatment of PD in terms of surgical outcome, sexual function, and donor site complications. This prospective study included 19 patients of PD with severe and complex curvature, who underwent corporoplasty with LMG. Surgical and functional outcomes were assessed at follow-up planned at 2 weeks, 3 months, 6 months, 1 year, and 2 years. The mean operative time was 126.31 ± 21.45 min. Additional Nesbit's plication to correct the residual deformity was required in 26% (5 / 19) of patients. Straightening of the penis (curvature <10°) was achieved in 89% (17 / 19) of patients. Increase in the penile length (>1 cm) postoperatively was achieved in 63% (12 / 19) of patients and a shortening of penis occurred in 5% (1 / 19) of patients. Newer onset erectile dysfunction developed in 11% (2 / 19) of patients, and patient and partner satisfaction rates were 89% (17 / 19) and 84% (16 / 19), respectively, in a mean follow-up of 20.66 ± 5.37 months. Donor site complications were minimal and no patient had any salivary changes or speech disturbances. LMG provided excellent short-term results in terms of deformity correction, improved sexual function, and minimal donor site morbidity. The method is simple and reproducible, and multicenter studies with larger number of cases with longer follow-up are required to confirm these favorable results.

Takayasu Artiritis, Peyronie’s Disease and Dupuytren’s Disease: A Non-Fortuitous Association

Background: Peyronie’s disease (PD), Dupuytren’s disease (DD) and Ledderhose disease (LD) are three localized fibrosis. Its pathophysiology is still controversial. Many factors as genetic predisposition, trauma and inflammation could be related. These disorders are commonly associated to environmental factors, diabetes and microtrauma. Until now, there is not a study reporting an association between PD, DD and large vessel vasculitis particularly takayasu’s arteritis. Case Report: It is about a 52-year-old man weaned from smoking since 15 years, with a history of takayasu’s arteritis for 10 years. At the CT angiography, he had a stenosis of the left subclavian artery, left carotid artery and left renal artery with a functional right kidney. At the physical examination, the penis size was normal but curved and deviated to the right side with palpable nodules. A penile ultrasonography was performed showing hyeperchogenic thickening of the tunica albuginea. The upper limbs examination showed nodules in the palm, fourth and fifth fingers in two hands but more pronounced in the left hand, with contractures related to DD and absence pulses in the left subclavian, humeral and radial arteries. Conclusion: To the best of our knowledge, there are no studies or cases reporting the association between a vasculitis of large artery and DD and PD. The presence of a PD on an occluded renal artery and a DD on a stenosed left subclavian artery, could be explain this theory.

Magnetic Resonance Imaging of Cyclops Lesion in Patients With Non-reconstructed Anterior Cruciate Ligament.

Introduction The term cyclops lesion refers to localized anterior fibrosis, which is the abnormal proliferation of fibrous tissue in a joint that develops in the anterior aspect of the intercondylar notch. It is a known cause of extension loss of the knee after anterior cruciate ligament (ACL) reconstruction; however, it can be found in patients who have not undergone any surgical repair of the ACL. The term "cyclops lesion" was given based on the arthroscopic appearance of the fibrous nodule and vessels that resemble an eye. The purpose of this study is to highlight the existence of cyclops lesions in non-operated knees. Methods We conducted a study on 10 patients who were subjected to an MRI knee in a Siemens Magnetom Vida 3 Tesla (Erlangen, Germany) machine. We retrospectively analyzed all 10 cases in our institution from July 2021 to March 2022. These subjects had a previous history of trauma, and they presented with complaints of pain and difficulty in knee extension but no history of previous ligament repair. All patients underwent an MRI examination. When a cyclops lesion was revealed on MR imaging, the signal-intensity characteristics, location, and size were documented. Results There were a total of 10 patients included in the study, of whom eight were males and two were females. The most common clinical presentation in all our cases was difficulty in the extension of the knee, while there was associated instability and difficulty in walking in some patients. There was a demonstrable cyclops lesion near the tibial attachment of ACL in eight (80%) patients, whereas it was found to be located just lateral to the anterior intercondylar notch in the rest of the two (20%) patients. Conclusion MRI isaneffective tool to evaluate unexplained pain, functionallimitations,and limited range ofmotioninpatients with suspected arthrofibrosis.MRI also helpsdeterminethe extent offibrosisinvolvement andexcludes other complications that may have a similar clinicalpicture.

Improving outcomes in high-grade goblet cell adenocarcinomas: The interplay of complete cytoreduction, HIPEC and fibrosis.

e16379 Background: High-grade goblet cell adenocarcinomas (GCA), including signet-ring cell type (SRC) and poorly differentiated, behave more aggressively compared to typical GCA and are associated with poor prognosis. Due to GCA’s rarity, the treatment aligns with colon cancer guidelines, including surgery, chemotherapy and/or cytoreductive surgery (CRS) with Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for peritoneal carcinomatosis (PC). In our case series, we assessed the outcome of patients with high grade GCA tumors and PC, who underwent either CRS with HIPEC or tumor debulking. Methods: A prospectively maintained database of patients treated at the Division of Surgical Oncology for Peritoneal Carcinomatosis (PC) from 2007 to 2022 was reviewed retrospectively. Patient selection was based on the primary tumor’s original pathology report and scheduling to undergo CRS/HIPEC for PC. We calculated median overall survival from the time of diagnosed peritoneal carcinomatosis, with focus on the attainment of complete cytoreduction (CC) and HIPEC administration. Results: 385 records were screened and 21 cases of GCA were identified. Thirteen (13) patients had a diagnosis of high-grade GCA with PC. Complete cytoreduction (CC0/CC1) with HIPEC was achieved in 7/13 patients, and 6/13 patients underwent tumor debulking. The median overall survival after diagnosing peritoneal carcinomatosis was 53 months for the patients who underwent CC/HIPEC and 20 months for the patients who received debulking. Of note, in the debulking group, 5/6 patients had a poorly differentiated GCA and exhibited intraoperatively tumor characteristics of infiltration, thickening and fibrosis, confirmed by the pathology report, which rendered CC unsuccessful. In contrast, within the CC/HIPEC group, pathology reported localized fibrosis as a chemotherapy response in only 2/7 patients, which did not impede the achievement of CC. Preoperative 5-FU based chemotherapy was administered to 4/7 patients in the CC/HIPEC group and to 5/6 patients in the debulking group. Conclusions: Our results demonstrate that CC with HIPEC may improve overall survival in patients with high grade GCA with PC. Notably, poorly differentiated GCA and the presence of fibrotic tissue, stemming from the tumor and/or preoperative chemotherapy, influences CC success. Considering the potential impact of fibrosis on surgical outcomes, pathological assessment of fibrosis preoperatively and/or use of fibrosis-specific biomarkers could guide cytoreduction strategies and treatment choices in patients diagnosed with high-grade GCA.

Developing a model for aqueous deficient dry eye secondary to periglandular cicatrizing conjunctivitis

PurposeThe current study used various techniques to develop a rabbit animal model of lacrimal gland damage caused by scarring conjunctivitis in the periglandular area. MethodsLeft eyes of New Zealand white rabbits were injected with 0.1 ml of 1M NaOH subconjunctivally around superior and inferior lacrimal gland orifices (Group 1, n = 4), touched with 1M NaOH for 100 s to the superior and inferior fornices with conjunctival denuding (Group 2; n = 4), and electrocauterization to the ductal opening area (Group 3; n = 4). The ocular surface staining, Schirmer I, lacrimal gland, and conjunctival changes were observed at baseline,1, 4, 8, and 12 weeks. The degree of glandular inflammation, conjunctival fibrosis (Masson Trichrome), and goblet cell density (PAS) were also assessed. ResultsAt 12 weeks, the lacrimal glands of group 1 rabbits with periglandular injection showed severe inflammation with mean four foci/10HPF and a significant mean reduction in the Schirmer values by 7.6 mm (P = 0.007). Lacrimal glands had diffuse acinar atrophy, loss of myoepithelial cells, and ductular dilatation. The overlying conjunctiva showed fibrosis, goblet cell loss, and corneal vascularization in the inferotemporal quadrant. No lacrimal gland or ocular surface changes were observed in groups 2 and 3 at 12 weeks, except for localized subconjunctival fibrosis. ConclusionPeriglandular injection of 0.1 ml of 1M NaOH induced extensive lacrimal gland damage with reduced secretion and scarring in the subconjunctival plane compared to direct cauterization or direct NaOH contact to the ductal orifices of the rabbit lacrimal gland.

#957 The immunometabolic profile of human peritoneal macrophages during peritoneal dialysis and its relation to systemic cardiovascular effects

Abstract Background and Aims Patients with end-stage kidney disease (ESKD) treated with peritoneal dialysis (PD) exhibit a high risk of heart failure (HF). This largely concerns HF with a preserved ejection fraction (HFpEF). Uremic toxins, oxidative stress and systemic inflammation related to ESKD contribute to this HFpEF phenotype, which may be aggravated by the daily administration of glucose-rich PD-fluids. These induce insulin resistance and formation of advanced glycaemic end products, resulting in microvascular injury and arteriosclerosis. A hallmark of HFpEF is the presence of profibrotic cardiac macrophages that aggravate fibrosis. In the peritoneum, the non-physiological high glucose concentrations trigger local oxidative stress, fibrosis and inflammation, processes alike those seen in HFpEF. While the role of macrophages in HFpEF is established, the involvement of human peritoneal macrophages (HPMs) in PD-induced peritoneal injury is largely unknown. We hypothesize that HPMs, chronically exposed to the glucose-rich fluids, exhibit an inflammation-driven immunometabolic profile. This may contribute to cardiovascular (CV)-risk in PD-treated patients. We present preliminary results on the isolation of HPMs from PD effluent (PDE) and a detailed protocol to establish the immunometabolic profile of HPMs. These profiles will subsequently be related to cardiac function (left ventricular global longitudinal strain (LV-GLS), an echocardiographic measure of HFpEF) and parameters of systemic inflammation. Method In 15 adult PD-treated patients we will prospectively perform cardiac echography, draw a blood sample and collect PDE for HPM isolation. To obtain control HPMs a peritoneal saline flush will be collected from 5 adult patients with non-metastasized colon cancer during a routine diagnostic laparoscopy. HPMs will be isolated from the PDE and saline by centrifugation, followed by freezing for storage. We compared fresh and thawed cells of a PDE-sample to evaluate the impact of storage on HPM phenotype and functionality, respectively using flow cytometry and measurement of IL-6 production, with and without a lipopolysaccharide (LPS) challenge. In-depth HPM characterization will be performed by measuring glucose and lactate, conducting metabolic flux analysis, assessing glycolysis and mitochondrial characteristics using Seahorse®, and employing cytometry to evaluate reactive oxygen species and metabolic fuel preference. A phenotypical profile will be established using fluorescent probes and antibodies. To assess functionality, HPMs will be exposed in vitro to “M1” and “M2” inducers and we will use human monocyte-derived macrophages from healthy adult controls as a comparison. We will relate the immunometabolic, phenotypical and functional HPM characteristics to LV-GLS, blood cytokine (obtained with Bio-Plex multiplex) and blood immune profiles (obtained with cytometry) in PD-treated patients. Furthermore, we will compare the HPM characteristics of the PD-treated patients with the control group. Results Preliminary results of 6 patients show that we can isolate HPMs from the PDE with a yield of &amp;gt;5M cells. The phenotype and functionality of HPMs was evaluated both directly after isolation and after a freeze-thaw cycle. Flow cytometry showed high percentages of CD14+HLA-DR+CD64+ macrophages in both fresh and biobanked HPMs. IL-6 production was observed in both fresh and biobanked HPMs even without LPS-stimulation (Figure). Conclusion By successfully isolating and storing HPMs from the easily accessible PDE, we optimized the conditions to assess their immunometabolic characteristics. The proposed protocol not only aims to provide valuable insights into HPM phenotype and functionality, but also allows us to relate these to cardiac function and systemic inflammation. With this strategy, we aim to gain insight in the immunometabolic effects of PD on peritoneal immune cells and how these relate to CV-effects.

First in Human Phase 1 Clinical Trial of Stereotactic Irradiation to Achieve Lung Volume Reduction (SILVR) in Severe Emphysema

PurposeOnly a subset of patients with severe emphysema qualify for lung volume reduction surgery or endobronchial valves. We previously demonstrated that Stereotactic Ablative Radiotherapy (SABR) of lung tumors reduces lung volume in treated lobes by creating localized lung fibrosis. We aimed to determine the safety and, secondarily, explore the efficacy of Stereotactic Irradiation for Lung Volume Reduction (SILVR) over 18 months following intervention in patients with severe emphysema. Methods and materialsWe conducted a single-arm prospective clinical trial in eligible patients with severe emphysema treated with unilateral SABR (45 Gy in three fractions) to a target within the most emphysematous region. Primary outcome was safety i.e., incidence of grade≥3 adverse events. Secondary outcomes of efficacy were also explored. ResultsEight subjects received the intervention. Median (range) baseline characteristics were age 73 years (63-78), FEV1% 28.5% (19.0-42.0), DLCO% 40% (24.0-67.0), and BODE index 5.5 (5-9). The incidence of grade≥3 adverse events was 3/8 (37.5%). The relative Δtarget lobe volume was -23.1% (-1.6,-41.5) and -26.5% (-20.6,-40.8) at six and 18 months, respectively. Absolute ΔFEV1% was greater in subjects with BODE index ≤5 vs. ≥6 (+12.0% vs. -2.0%). The mean baseline lung density (in Hounsfield units, reflecting the amount of preserved parenchyma) within the intermediate dose volume (V60BED3) correlated with the absolute Δtarget lobe volume at 18 months. ConclusionsStereotactic Irradiation for Lung Volume Reduction appears to be safe, with a signal for efficacy as a novel therapeutic alternative for patients with severe emphysema. SILVR may be most safe/effective in patients with lower BODE index and/or less parenchymal destruction.

Abstract 3017: Lack of MCPIP1 enzymatic activity leads to neoplastic transformation of normal kidney cells

Abstract Introduction: The MCPIP1 protein (Monocyte Chemotactic Protein-1 Induced Protein) is involved in the negative regulation of inflammation due to its RNase activity. A growing number of publications suggest that the MCPIP1 protein may influence the development of cancer by regulation of factors involved in angiogenesis, proliferation and cell death. We believe, that MCPIP1 may be a potential tumor suppressor. Our recent results show that lack of enzymatic activity of MCPIP1 leads to increased tumor growth, angiogenesis and metastasis. The main aim of our research is to study the importance of MCPIP1 for neoplastic transformation of normal, epithelial kidney cells, as well as acquirement of stemness features. Material and Methods: To examine the effect of MCPIP1 in normal epithelial cell lines TCMK-1, RPTEC-Tert1 and HK-2 were transduced to overexpress mutated, inactive form of MCPIP1 (pLIX D141N) with proper control (pLIX PURO). We analyzed cells clonogenicity, as well as the levels of CSCs markers by western blot, NGS and qPCR. Next, cells were injected subcutaneously into NOD-SCID mice to check if mutation of MCPIP1 will induce tumor growth in vivo. Mice blood was analyzed for the presence of circulating tumor cells (CTCs), and secretion of cytokines by Proteome Profiler assay. Tumors were evaluated by multiple IHC stainings, as well as western blot and qPCR. Results and Discussion: We have shown that the mutation of MCPIP1 increases the clonogenicity of kidney cells. Moreover, pLIX D141N cells were characterized by higher levels of the c-Met receptor, vimentin, Twist, CD44 and c-Myc, which are the markers of epithelial to mesenchymal transition and cancer stemness. The obtained results suggest that the loss of the MCPIP1 protein causes the acquisition of the characteristics of neoplastic cells. Next, we checked whether MCPIP1 mutation in normal cells predisposes them to proliferate when administered to mice. We found that, D141N mutation caused the growth of large tumors with higher expression of Cd44, Klf4 and Myc and number of CTCs, while the control cells injected into mice developed only local fibrosis with high expression of aSMA. pLIX D141N tumors secreted CXCL16 into mice plasma, and were characterized by increased phosphorylation of c-Met, Src kinase and c-Myc. Conclusions: We believe that the MCPIP1 protein may be a marker of tumor initiation and play a key role in neoplastic transformation by regulating the changes in cell phenotype and levels of CSCs markers. This study was supported by National Science Center grants no. 2022/47/B/NZ5/02724, 2022/45/B/NZ5/01973 and MNS 15/2021. Citation Format: Paulina Marona, Iga Piasecka, Judyta Gorka, Oliwia Kwapisz, Rafal Myrczek, Ewelina Pospiech, Jolanta Jura, Katarzyna Miekus. Lack of MCPIP1 enzymatic activity leads to neoplastic transformation of normal kidney cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3017.

Abstract 782: Correlation between collagen peptides and physical features of fibrosis in breast tissue from women at increased risk of cancer

Abstract Breast cancer risk increases significantly in women who maintain higher radiologically dense breast tissue as they age. However, the mechanisms that underlie this association remain poorly understood, especially in postmenopausal women. A predominant contributor to dense breasts and differences between women is the relative amount of fibrillar collagens. Experimental models suggest that changes in collagen properties such as fiber alignment and stiffness promote local stromal fibrosis, which we hypothesize may contribute to the relationship between breast density, aging, and tumorigenesis. Normal breast biopsy samples from the contralateral, healthy breast of 32 postmenopausal breast cancer patients participating in a clinical trial for drug effect on breast density were examined and scored for areas of fibrosis and analyzed using second harmonic generation microscopy (SHG) and mass spectrometry proteomics. Our aim was to identify areas of fibrosis and relate the severity of focal fibrosis to the physical properties and peptide composition of collagen fibers. Using GraphPad Prism, we conducted one-way ANOVA and Kruskal-Wallis tests and post-hoc multiple comparison tests. H&amp;E-stained breast tissue samples were scored according to percent fibrosis. The minimum and maximum percent fibrosis were 5% and 100%, respectively, with a median of 60% (95% CI: 97.06%). Out of 31 people, 16 had a higher fibrosis score than the median. Consistent with pathologic scoring of the presence and extent of fibrosis, fiber counts were significantly lower in tissue with lower fibrosis scores than samples with more highly scored fibrosis (p-value &amp;lt;0.001). Distance to the nearest fiber was greater in samples with &amp;lt; 20% fibrosis, whereas greater alignment to the nearest fiber was observed in higher fibrosis percentages. At the molecular level, 22 peptides demonstrated a strong correlation with percent fibrosis (spearman r ≥ 60), of which 13 peptides were statistically significant (p-value &amp;lt;0.0001). The putatively identified peptides belong to type I, III, IV and VI collagens. Fiber length and straightness, but not fiber width, were higher in tissues with greater percent fibrosis. Significant correlations were identified between collagen physical features and peptides: two distinct peptides correlated with collagen fiber length and straightness while 20 peptides correlated with fiber width. These putative peptides belong to types I, III, IV and V collagens with ~70% of the peptides containing at least one hydroxyproline modification. This study is the first to explore the link between focal fibrosis and collagen-associated peptides in healthy breast tissue of high-risk postmenopausal women. These findings provide novel insights on stromal collagens associated with fibrotic changes in normal at-risk breast tissue that may act in tissue susceptibility to tumor formation. Citation Format: Denys Rujchanarong, Alison T. Stopeck, Christina Preece, Ren Sun, Pavani Chalasani, Sean Brown, Ji D. Bai, Heather Jensen Smith, Sungyong You, Peggi M. Angel, Patricia Thompson. Correlation between collagen peptides and physical features of fibrosis in breast tissue from women at increased risk of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 782.

Cldn4 overexpression promotes penile cavernous smooth muscle cell fibrotic response via the JNK signaling pathway.

Erectile dysfunction (ED), defined as the inability to achieve or maintain a penile erection sufficient to satisfy sexual behavior, is prevalent worldwide. Using previous research, bioinformatics, and experimental confirmation, we aimed to discover genes that contribute to ED through regulating hypoxia in corpus cavernosum smooth muscle cells (CCSMCs). We used the Gene Expression Omnibus to acquire the sequencing data of the corpus cavernosum transcriptome for diabetic ED and nerve injury type ED rats. We intersected the common differentially expressed genes. Further verification was performed using single cell sequencing. Real-time quantitative polymerase chain reaction and immunofluorescence were used to investigate whether the differentially expressed genes are found in the corpus cavernosum. We used induced hypoxia to assess cell viability changes, and we developed a lentivirus overexpressing Cldn4 for in vitro and in vivo experiments to measure changes in JNK signaling, fibrosis, hypoxia, and erectile function. Our results indicate that targeting the JNK pathway and decreasing local hypoxia may be better options for therapeutic intervention to improve erectile function. We identified Cldn4 and found its expression increased in the corpora cavernosa of the 2 datasets. In addition, we found that hypoxia can increase the expression of Cldn4, activate the JNK signaling pathway, and exacerbate fibrosis in CCSMCs. Cldn4 overexpression in CCSMCs activated the JNK signaling pathway and increased fibrotic protein expression. Last, rat corpus cavernosum overexpressing Cldn4 activated the JNK signaling pathway, increased local fibrosis, and impaired erectile function. Through bioinformatics and in vitro and in vivo experiments, we found that Cldn4 has a negative effect on ED, and targeting Cldn4 may provide new ideas for ED treatment. Although we have identified Cldn4 as a potential target for ED treatment, we have only conducted preliminary validation on CCMSCs, and we still need to further validate in other cell lines. CCSMC hypoxia leads to increased Cldn4, in both nerve injury and diabetic ED rat models, and promotes fibrosis by activating the JNK signaling pathway.

Bronchoscopic deployment and implantation of Diffusing alpha-emitters Radiation Therapy into the lung and mediastinum for treatment of lung cancer: a pre-clinical safety and feasibility study.

Radiotherapy is a standard treatment modality in cancer therapy, particularly for lung cancer. Diffusing alpha-emitters Radiation Therapy sources (hereafter, "Alpha DaRTs") are fixed with Ra-244 (half-life =3.6 days) that releases alpha-emitting atoms into the tumor tissue to an effective range of a few millimeters. The feasibility, usability, and safety of Alpha DaRTs deployment and implantation via bronchoscopy into the lung parenchyma and mediastinum in a big animal model of healthy swine was studied in two phases: (I) inert and (II) active Alpha DaRTs deployment. The Alpha DaRTs were inserted in both individual and cluster patterns based on a predefined plan. Swine health was monitored throughout the study. The usability of bronchoscopic deployment and implantation was evaluated using a user questionnaire. The movement and migration of the Alpha DaRTs were assessed. Necropsy was performed, and lungs were evaluated via gross pathology and histopathology. A total of 158 Alpha DaRTs were inserted successfully in the lung parenchyma and mediastinum of five swine in two phases. It was possible to deliver and place the Alpha DaRTs in clusters of no more than 4 mm distance between the Alpha DaRTs. No adverse event or change in the health and general condition of animals was observed. Hematologic evaluation did not show any clinically significant abnormality related to the Alpha DaRTs. Histopathology demonstrated local mild inflammatory changes, minimal fibrosis, and dystrophic mineralization with giant cells. Minimal movement and no migration of Alpha DaRTs were observed. Bronchoscopic deployment of Alpha DaRTs in the lung parenchyma and mediastinum of the porcine animal is feasible, precise, and safe.

Juvenile localized scleroderma

Juvenile scleroderma, often referred to as juvenile localized scleroderma or "morphea", is arare inflammatory disease of the skin and skin-related structures, accompanied by local sclerosis and tissue fibrosis. Depending on the clinical manifestation, four different subtypes can be defined: limited, generalized, linear, and mixed. To prevent possible sequelae of the disease, the diagnosis should be made as early as possible and therapy should be initiated at specialized centers in multiprofessional pediatric and dermatologic collaboration. In this review, we present the main clinical, laboratory, and therapeutic characteristics of juvenile localized scleroderma and summarize recommendations.

Assessment of tear derived extracellular vesicles as a minimally source of novel biomarkers for subconjunctival glaucoma surgery

Aims/purpose: Glaucoma is a highly prevalent pathology causing avoidable blindness. Topical treatments used to stabilize intraocular pressure (IOP) may produce several side effects including inflammatory reactions. Patients who fail to control IOP or suffer significant side effects undergo subconjunctival glaucoma surgery (SGS). Estimations point to 30–40% of these patients will develop local scarring and fibrosis, which are the main complications of SGS. Thus, predicting the surgical outcome and starting preemptively fibrosis treatment are crucial. The aim of this study was to determine whether tears, and specifically tear‐derived extracellular vesicles (L‐EVs), can be used as liquid biopsy source, as a previous step for identifying new biomarkers for SGS outcome.Methods: Tear samples were obtained from patients (no surgical treatment, n = 11) by Schirmer's test. L‐EVs were enriched by size‐exclusion chromatography. Protein quantification was measured by microBCA assay and miRNA extraction (both from whole tear and L‐EVs) was performed using commercial kits.Results: Whole tear samples displayed variable miRNA and protein concentrations among patients (range 222.0–1388.1 ng miRNAs and 5.6–27.2 μg protein). Levels of miRNA and protein were also highly variable but considerably reduced in L‐EVS (0.14–46.78 ng miRNAs and 0.5–4.2 μg protein). Yet, in most patients, both types of samples (whole tears and L‐EVs) rendered sufficient quantity of protein and miRNA to perform proteomic and transcriptomic analysis.Conclusions: Concentration of miRNAs and Protein in tears and L‐EVs is highly variable among patients. As expected by their higher enrichment, miRNA and protein concentrations in L‐EVs are reduced compared to whole tears, yet quantities may be still enough to perform mic analyses. This higher purification of L‐EVS may help identifying specific biomarkers related to SGS outcome in a minimally invasive manner.

68Ga-labeled fibroblast activation protein inhibitor (FAPI) PET/CT for locally advanced or recurrent pancreatic cancer staging and restaging after chemoradiotherapy.

Purpose: 68Ga-labeled fibroblast activation protein inhibitor (FAPI) is a novel PET tracer with great potential for staging pancreatic cancer. Data on locally advanced or recurrent disease is sparse, especially on tracer uptake before and after high dose chemoradiotherapy (CRT). The aim of this study was to evaluate [68Ga]Ga-FAPI-46 PET/CT staging in this setting. Methods: Twenty-seven patients with locally recurrent or locally advanced pancreatic adenocarcinoma (LRPAC n = 15, LAPAC n = 12) in stable disease or partial remission after chemotherapy underwent FAPI PET/CT and received consolidation CRT in stage M0 with follow-up FAPI PET/CT every three months until systemic progression. Quantitative PET parameters SUVmax, SUVmean, FAPI-derived tumor volume and total lesion FAPI-uptake were measured in baseline and follow-up PET/CT scans. Contrast-enhanced CT (ceCT) and PET/CT data were evaluated blinded and staged according to TNM classification. Results: FAPI PET/CT modified staging compared to ceCT alone in 23 of 27 patients in baseline, resulting in major treatment alterations in 52% of all patients (30%: target volume adjustment due to N downstaging, 15%: switch to palliative systemic chemotherapy only due to diffuse metastases, 7%: abortion of radiotherapy due to other reasons). Regarding follow-up scans, major treatment alterations after performing FAPI PET/CT were noted in eleven of 24 follow-up scans (46%) with switch to systemic chemotherapy or best supportive care due to M upstaging and ablative radiotherapy of distant lymph node and oligometastasis. Unexpectedly, in more than 90 % of the follow-up scans, radiotherapy did not induce local fibrosis related FAPI uptake. During the first follow-up, all quantitative PET metrics decreased, and irradiated lesions showed significantly lower FAPI uptake in locally controlled disease (SUVmax p = 0.047, SUVmean p = 0.0092) compared to local failure. Conclusion: Compared to ceCT, FAPI PET/CT led to major therapeutic alterations in patients with LRPAC and LAPAC prior to and after radiotherapy, which might help identify patients benefiting from adjustments in every treatment stage. FAPI PET/CT should be considered a useful diagnostic tool in LRPAC or LAPAC before and after CRT.

Advanced navigation technology enables endobronchial brachytherapy for peripheral lung cancer: An old technique plays a new role

Background: To investigate the feasibility of super-selectively endobronchial brachytherapy in the treatment of peripheral lung cancer guided by advanced navigation technology. Patients and Methods: Six patients with peripheral lung tumors successfully underwent treatment with super-selectively endobronchial brachytherapy guided by advanced navigation technology following pathway planning, and were subsequently followed up to assess survival and treatment-related toxicities. Results: The endobronchial applicators were successfully placed inside the tumors of all patients using advanced navigation techniques according to the pre-treatment plan, and brachytherapy was delivered at curative doses after evaluation using radiotherapy planning software. None of the patients showed local progression of the treated lesions during the follow-up for a duration ranging from 11 months to 35 months, with a median follow-up time of 23 months. The patient with the longest follow-up, nearly 3 years, exhibited a stable condition. After undergoing endobronchial brachytherapy, patients predominantly experienced localized fibrosis as indicated. No significant alterations in cardiopulmonary function were detected during the follow-up, and no other adverse effects were found. Conclusion: The use of endobronchial brachytherapy for the curative treatment of peripheral lung cancers is feasible. Furthermore, the development of novel bronchial navigation techniques has the potential to broaden the application of endobronchial brachytherapy.

'Trapped re-entry' as source of acute focal atrial arrhythmias.

Diseased atria are characterized by functional and structural heterogeneities, adding to abnormal impulse generation and propagation. These heterogeneities are thought to lie at the origin of fractionated electrograms recorded during sinus rhythm (SR) in atrial fibrillation (AF) patients and are assumed to be involved in the onset and perpetuation (e.g. by re-entry) of this disorder. The underlying mechanisms, however, remain incompletely understood. Here, we tested whether regions of dense fibrosis could create an electrically isolated conduction pathway (EICP) in which re-entry could be established via ectopy and local block to become 'trapped'. We also investigated whether this could generate local fractionated electrograms and whether the re-entrant wave could 'escape' and cause a global tachyarrhythmia due to dynamic changes at a connecting isthmus. To precisely control and explore the geometrical properties of EICPs, we used light-gated depolarizing ion channels and patterned illumination for creating specific non-conducting regions in silico and in vitro. Insight from these studies was used for complementary investigations in virtual human atria with localized fibrosis. We demonstrated that a re-entrant tachyarrhythmia can exist locally within an EICP with SR prevailing in the surrounding tissue and identified conditions under which re-entry could escape from the EICP, thereby converting a local latent arrhythmic source into an active driver with global impact on the heart. In a realistic three-dimensional model of human atria, unipolar epicardial pseudo-electrograms showed fractionation at the site of 'trapped re-entry' in coexistence with regular SR electrograms elsewhere in the atria. Upon escape of the re-entrant wave, acute arrhythmia onset was observed. Trapped re-entry as a latent source of arrhythmogenesis can explain the sudden onset of focal arrhythmias, which are able to transgress into AF. Our study might help to improve the effectiveness of ablation of aberrant cardiac electrical signals in clinical practice.