Immunohistochemical Analysis Research Articles

Macrophage polarization in human periapical lesions in relation to histopathological diagnosis, clinical features and lesion volume: An exvivo study.

To evaluate M1 and M2 macrophage polarization in radicular cysts and periapical granulomas through an immunohistochemical analysis and the correlation between macrophage polarization and histopathological diagnosis, clinical characteristics and lesion volume using cone-beam computed tomography. Periapical biopsies diagnosed as radicular cysts (n = 52) and periapical granulomas (n = 51) were analysed by immunohistochemical method. Teeth with periapical lesion with no history of root canal treatment (primary lesion) and lesions persistent to root canal treatment (persistent lesions) were included. Pathological diagnosis, patients' age, gender and clinical characteristics were obtained from treatment records. A cone-beam computed tomographic periapical volume index (CBCTPAVI) score was assigned to each periapical lesion based on the volume of the lesion. Immuno-expressions of CD68 and CD163 were quantified. The CD68/CD163 ratio was adopted to represent M1 or M2 macrophage polarization. Mann-Whitney U test was used to determine the different CD68/CD163 ratio between groups of radicular cyst and periapical granuloma. Spearman's correlation test was performed to assess the correlation between the CD68/CD163 ratio and lesion volume and CBCTPAVI score. Radicular cysts and periapical granulomas had CD68/CD163 median of 2.05 (IQR = 1.33) and 1.26 (IQR = 0.81), respectively. A significantly higher CD68/CD163 ratio was observed in radicular cysts (p < .001). In contrast, periapical granulomas had significantly lower median of CD68/CD163 ratio. Larger lesions had a higher median of CD68/CD163 ratio, while smaller lesions had lower median of CD68/CD163 ratio (p = .007, rs = .262). CD68/CD163 ratio was significantly correlated with the CBCTPAVI score in the overall periapical lesions (p = .002, rs = .306). The higher CD68/CD163 ratio in larger lesions indicated a higher degree of M1 polarization compared to smaller lesions. Regarding the pathological diagnosis, there was a significant positive correlation between CBCTPAVI score and CD68/CD163 ratio in periapical granulomas (p < .001, rs = .453), whereas the negative correlation was observed for radicular cysts (p < .001, rs = -.471). Periapical granulomas are characterized by a M2-dominant macrophage polarization, while radicular cysts have significantly higher M1 macrophages. The higher degree of M1 macrophage polarization was significantly correlated with larger volume and higher CBCTPAVI scores of overall periapical lesion and periapical granuloma.

Role of Fyn expression in predicting the sensitivity to platinum‑based chemotherapy in patients with ovarian serous carcinoma.

Ovarian serous carcinoma is a gynecological malignancy associated with a high mortality rate, which is commonly diagnosed in the first instance at a late stage and has a propensity to develop resistance to platinum-based chemotherapy. Identifying reliable biomarkers for platinum sensitivity is critical for improving patient outcomes. The present retrospective study included 64 patients with high-grade serous ovarian carcinoma (Federation of Gynecology and Obstetrics stages III or IV). Patients were classified as platinum-sensitive (no relapse within 6 months of the last platinum administration) or platinum-resistant (relapse within 6 months). Immunohistochemical analysis was performed to evaluate Fyn expression in tumor tissues, and Fyn knockdown experiments were performed using the OVSAHO ovarian cancer cell line to assess carboplatin sensitivity. Fyn expression was significantly higher in platinum-resistant patients compared with in platinum-sensitive patients (P<0.01). A weighted Fyn expression score was developed and a cutoff score of 6 was determined to predict platinum sensitivity with a specificity of 65.5% and a sensitivity of 62.9%. Patients with low Fyn expression (score ≤6) exhibited higher platinum sensitivity and longer overall survival (P<0.05). Multivariate analysis identified Fyn expression and postoperative residual tumor size as independent predictors of platinum sensitivity (P=0.033 and P=0.023, respectively). In vitro, Fyn knockdown significantly increased carboplatin sensitivity in ovarian cancer cells (P<0.05). Fyn, a member of the Src family of kinases, serves a crucial role in various cellular functions and has been implicated in chemotherapy resistance. The results demonstrated a notable association between Fyn expression and platinum sensitivity in ovarian serous carcinoma. The findings suggested that Fyn may serve as a predictive biomarker for response to platinum-based chemotherapy, offering the potential for more personalized treatment strategies. To the best of our knowledge, the present study is the first to establish an association between Fyn expression and platinum sensitivity in advanced ovarian serous carcinoma. Prospective studies with larger, multi-center cohorts and comprehensive biomarker analyses are recommended to validate and extend these results, ultimately improving therapeutic strategies and patient prognosis.

Pirfenidone targeted mechanisms for alleviating methotrexate-induced testiculopathy in Wistar rats.

Testicular injury and affected spermatogenesis are major complications of methotrexate (MTX) use. Oxidative stress is one contributing process leading to inflammation and apoptosis induction. Pirfenidone (PFD) is a well-known anti-fibrotic drug prescribed for interstitial lung fibrosis, in addition to anti-inflammatory, antioxidative, and antiapoptotic capabilities. The study aimed to explore the potential protection afforded by PFD in a rat model of MTX-induced testiculopathy. The experimental design included four groups, each containing seven adult Wistar rats: control, PFD (500mg/kg/day, orally)-, MTX (0.5mg/kg, intraperitoneal, twice weekly)-, and PFD/MTX-treated groups. Treatment continued for 4weeks. Blood and testicular samples were harvested for biochemical, histological, immunohistochemical, and polymerase chain reaction (PCR) analyses. Also, the testicular damage and spermatogenic activity were graded by the testicular injury and Johnsen scoring system, respectively. PFD positively affected the serum testosterone (TST) level, reduced the testicular inflammatory mediators [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β)], reduced the testicular oxidative burden, increased superoxide dismutase (SOD), and protected the testicular histological structure. In addition, antifibrotic effects, anti-caspase-3, and PCNA enhancement activity were recorded. PFD exhibited a protective potential and mitigated the MTX-induced testiculopathy via suppression of testicular oxidative stress, inflammation, fibrosis, and apoptosis and retaining the testicular proliferative efficacy as confirmed by histological, immunohistochemical, and biochemical methods.

A novel inhibitor of class IIa histone deacetylases attenuates collagen-induced arthritis.

Most inhibitors of histone deacetylases (HDACs) are not selective and are cytotoxic. Some have anti-inflammatory activity in disease models, but cytotoxicity prevents long-term uses in non-fatal diseases. Inhibitors selective for class IIa HDACs are much less cytotoxic and may have applications in management of chronic inflammatory diseases. LL87 is a novel HDAC inhibitor examined here for HDAC enzyme selectivity. It was also investigated in macrophages for cytotoxicity and for inhibition of lipopolysaccharide (LPS)-stimulated cytokine secretion. In a rat model of collagen-induced arthritis, LL87 was investigated for effects on joint inflammation in Dark Agouti rats. Histological, immunohistochemical, micro-computed tomography and molecular analyses characterise developing arthritis and anti-inflammatory efficacy. LL87 was significantly more inhibitory against class IIa than class I or IIb HDAC enzymes. In macrophages, LL87 was not cytotoxic and reduced both LPS-induced secretion of pro-inflammatory cytokines, and IL6-induced class IIa HDAC activity. In rats, LL87 attenuated paw swelling and clinical signs of arthritis, reducing collagen loss and histological damage in ankle joints. LL87 decreased immune cell infiltration, especially pro-inflammatory macrophages and osteoclasts, into synovial joints and significantly reduced expression of pro-inflammatory cytokines and tissue-degrading proteases. A novel inhibitor of class IIa HDACs has been shown to have an anti-inflammatory and anti-arthritic profile distinct from current therapies. It is efficacious in reducing macrophage infiltration and joint inflammation in a chronic model of rat arthritis.

CRGD-platelet@MnO/MSN@PPARα/LXRα Nanoparticles Improve Atherosclerosis in Rats by Inhibiting Inflammation and Reducing Blood Lipid.

Atherosclerosis (AS) is an inflammatory disease of arterial intima driven by lipids. Liver X receptor alpha (LXRα) and peroxisome proliferator-activated receptor alpha (PPARα) agonists are limited in the treatment of AS due to their off-target effects and serious side effects. Therefore, this study was designed to construct a novel nanoparticle (NP) and evaluate its mechanism of action on inflammation inhibition and lipid reduction in AS. We synthesized cRGD-platelet@MnO/MSN@PPARα/LXRα NPs (cRGD-platelet- NPs) and confirmed their size, safety, and targeting ability through various tests, including dynamic light scattering and immunofluorescence. In vivo and in vitro experiments assessed cell proliferation, apoptosis, inflammation, and plaque formation. Finally, the NF-κB signaling pathway expression in rat aorta was determined using a western blot. The synthesis of cRGD-platelet-NPs was successful; the particle size was approximately 150 nm, and the PDI was below 0.3. They could be successfully absorbed by cells, exhibiting high safety in vivo and in vitro. The cRGD-platelet-NPs successfully reduced plaque formation, improved lipid profiles by lowering LDL-cholesterol, total cholesterol, and triglycerides, and raised HDL-cholesterol levels. Additionally, they decreased inflammatory markers in the serum and aortic tissue, suggesting reduced inflammation. Immunohistochemistry and western blot analyses indicated that these NPs could not only promote M2 macrophage polarization but also suppress the NF-κB signaling pathway. The newly developed cRGD-platelet-NPs with high safety are a promising approach to AS treatment, which can regulate ABCA1, reduce the formation of AS plaques, and enhance cholesterol efflux. The mechanism may involve the suppression of the NF-κB signaling pathway.

Boosting impacts of Acacia nilotica against hepatic toxicity induced by gentamicin: biochemical, anti-inflammatory and immunohistochemical study.

It seems that gentamicin's toxicity to the liver is caused by reactive oxygen species production. The antioxidant and anti-inflammatory properties of Acacia nilotica extract (AN) have been demonstrated in recent studies. This research focused on how AN's extract affected gentamicin-induced liver damage in rats. Twenty-four Wister rats of male type were divided into four groups: first group received saline as a control, second group received AN (5%) for fifteen days, group three received daily intraperitoneal injections of gentamicin (100mg/kg) for fifteen days, and group four, as mentioned in groups 2 and 3, also received gentamicin injections and AN extraction (5%) for fifteen days. In order to conduct biochemical analysis, serum was extracted. Histopathology, immunohistochemistry analyses for hepatic toxicity were all performed on the collected tissue samples. Serum levels of ALT, AST, total bilirubin, and GGT were all elevated after using gentamicin. The inflammatory cytokines)IL-1, TNF-α and IL-6(, all were increased in gentamycin-injected group. There were showing deformity of bile duct, hepatocellular necrosis and infiltration of inflammatory cells congestion of portal vein, and hepatic sinusoids besides fibrosis of portal area (white arrows), hypertrophy in gentamycin-injected group compared to AN plus gentamycin administered rats. There were upregulation in the immunoreactivity of COX-2, IFNkB and TGF-beta1 (TGF-β1) in gentamycin intoxicated rats. When gentamicin and AN were administered together, hepatic biomarkers, inflammatory cytokines, histological, and immunohistochemical markers were all ameliorated by AN administration.

Resistance to SARS-CoV-2 infection in camelid nasal organoids is associated with lack of ACE2 expression

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects a variety of animal species. Susceptibility to SARS-CoV-2 is primarily determined by the utilization of the viral receptor, ACE2. SARS-CoV-2 can utilize a broad range of animal ACE2 isoforms in vitro, including the ACE2 from various camelid species. However, experimental infection of these animals does not lead to productive infection or seroconversion. In this study, we investigate the susceptibility of camelids to SARS-CoV-2 using novel well-differentiated camelid nasal organoids. We show that camelid nasal organoids are highly susceptible to Middle East respiratory syndrome coronavirus (MERS-CoV) infection, but not to infection with different SARS-CoV-2 variants (614G, BA.1 or EG.5.1.1). All viruses efficiently infected human airway organoids. Immunohistochemistry analysis revealed the absence of ACE2 on camelid nasal organoids and dromedary camel upper respiratory tract. In contrast, DPP4 was expressed in both camelid nasal organoids and the camel upper respiratory tract, which correlates with MERS-CoV infection. This study indicates that the camelid upper respiratory tract lacks expression of ACE2, which is associated with resistance to SARS-CoV-2 infection.

Unraveling the molecular landscape of UTROSCT: Insights from a clinicopathological, morphological, immunohistochemical, and molecular analysis of 35 cases.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare tumor of uncertain lineage and low malignant potential. Most tumors behave in a benign fashion, but a subset of UTROSCT exhibit an aggressive clinical course with recurrences and metastases. The recurrent molecular alterations in UTROSCT mostly represent gene fusions involving NCOA1-3. We performed a comprehensive clinicopathological, morphological, immunohistochemical, and molecular analysis on a cohort of 35 UTROSCT. The tumors exhibited various architectural patterns (diffuse, corded/trabecular, tubular, sertoliform, fascicular, whorled, nested, microfollicular, and pseudoglandular), often in combination. The immunohistochemical analysis confirmed the polyphenotypic immunoprofile, often with co-expression of sex cord-stromal, smooth muscle, and epithelial markers, as well as hormone receptors. NGS RNA analysis revealed recurrent NCOA1-3 gene fusions in 22/32 analyzed cases (69%), including ESR1::NCOA3 (11/22), GREB1::NCOA2 (7/22), ESR1::NCOA2 (3/22), and GREB1::NCOA1 (1/22). Tumor mutation burden was low in all cases. The fusion-positive cases exhibited statistically significant association with whorled architecture, conversely necrosis was associated with fusion-negative status. We did not find a significant relationship between any architectural pattern and GREB1-alterations, but the NCOA2-altered tumors were associated with pseudoglandular architecture. The GREB1-altered cases occurred in older patients and tended to be more often intramural masses compared to ESR1-altered cases. On the contrary, the ESR1-altered cases presented more often like submucosal or polypoid tumors. Two tumors exhibited aggressive behavior with recurrent disease. Both of these cases harbored a GREB1::NCOA2 fusion. Unsupervised hierarchical cluster analysis of our cohort revealed two main clusters. The tumors with GREB1 or NCOA2 fusion cluster together, suggesting that there are underlying molecular differences between these cases and cases with ESR1::NCOA3 fusion or without fusion. Our findings contribute to the growing knowledge about a rare neoplasm with currently uncertain biological behavior.

Activin levels correlate with lymphocytic infiltration in epithelial ovarian cancer.

The TGF-β superfamily member activin, a dimer of the gene products of INHBA and/or INHBB, has been implicated in immune cell maturation and recruitment, but its immune impact within epithelial ovarian cancer (EOC) is not well characterized. We sought to explore differences in activin (INHBA/ Inhibin-βA and INHBB/ Inhibin-βB) between malignant and ovarian tissues at the RNA and protein level and assess the relationship between activin and immune cells in EOC. Publicly available RNA sequencing data were accessed from GEO (#GSE143897) with normalization and quantification performed via DESeq2. Immune gene expression profile was further explored within the TCGA-OV cohort derived from The Cancer Genome Atlas (TCGA). Immunohistochemical analysis was performed to evaluate activin A and T-cell markers CD8 and FoxP3 at the protein level. ELISA to activin-A was used to assess levels in the ascites of advanced EOC patients. Kaplan-Meier curves were generated to visualize survival outcomes. Gene expression levels of components of the activin signaling pathway were elevated within EOC when compared to a benign cohort, with differences in activin type I/II receptor gene profiles identified. Additionally, INHBA gene expression was linked to lymphocytic immune markers in EOC samples. Immunohistochemistry analysis revealed a positive correlation of CD8 and FOXP3 staining with activin A at the protein level in both primary and metastatic epithelial ovarian cancer samples. Furthermore, Activin-A (inhibin-βA) is significantly elevated in EOC patient ascites. INHBA expression is elevated within EOC, correlating with worse survival, with activin protein levels correlating with specific immune infiltration. Our findings suggest that activin-A may play a role in suppressing anti-tumor immunity in EOC, highlighting its potential as a therapeutic target.

Prognostic value of tumor-infiltrating lymphocytes and PD-L1 expression in esophageal squamous cell carcinoma.

Tumor cells (TC) participate in tumor progression by altering the immune responses in the tumor microenvironment. However, the clinical relevance and prognostic effect of PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in esophageal squamous cell carcinoma (ESCC) are unknown. The purpose of this study was to investigate the interactions and clinical significance of PD-L1 expression and TILs in ESCC. Tissue specimens were collected from 126 patients with ESCC who underwent curative esophagectomy. Immunohistochemical analysis and multiplex immunofluorescence for CD4, CD8, CD25, FOXP3, and PD-L1 in the tumor were used to identify multiple tumor-infiltrating immune cells (TIIC), Tregs, and TC. PD-L1 was expressed in tumor cells (PD-L1 TC). PD-L1 TIIC and PD-L1 TC affected the biological behavior of TC. The positive expression rate of PD-L1 TC and CD8+ TILs was 27.8% (35/126) and 31.7% (40/126), respectively. Kaplan-Meier analysis showed that overall survival (OS) was significantly associated with decreased CD8+ TILs and PD-L1 TC-positive expression, which promote ESCC progression and metastasis. Tumor depth, CD8, and PD-L1 TC were independent prognostic factors in ESCC, and a predictive nomogram with these three risk factors improved the accuracy of predicting OS in patients with ESCC after surgical resection. The conjoint analysis of multiple immune-related factors is beneficial for stratifying patient survival risk.

Long-term effect observation after 10 years of tonsillotomy （Histological analysis of one case of secondary operation）

Objective:To investigate the long-term effect of partial tonsillectomy in children with tonsil hypertrophy. Methods:A total of 146 children with obstructive sleep apnea（OSA） who received surgical treatment for tonsil hyperplasia from January 2010 to January 2013 were selected and divided into the observation group（n=69） and the control group（n=77）. The observation group was received tonsillotomy（TT）, and the control group was received total tonsillectomy（TE）. Parental satisfaction and OSA quality of life questionnaire for children（OSA-18） were surveyed. Residual tonsil size was measured, and polysomnography（PSG） was monitored after 10 years. HE and immunohistochemical analysis were performed on tonsil tissues of one patient who performed a second operation after TT in 2017 year. Results:The results of questionnaire survey showed that the symptoms of respiratory obstruction were significantly improved in both groups, and the satisfaction of TT group was higher than that in the TE group. No increase in the number of respiratory tract infections was observed in all patients. In the TT group, nine cases（13.04%） had tonsil hyperplasia toⅡ°, and the remaining patients had tonsil hyperplasia to Ⅰ°. In addition, one case hadtonsil suppurative infection at the 14th month after surgery, and no recurrence or reoperation was found after treatment. There were seven cases in the TT group and eight cases in the TE group with occasional snoring and mouth breathing after surgery, but the PSG examination of the patients did not meet the diagnosis of OSA. The main causes were obesity and allergic rhinitis. Compared with the first operation, the cicatricial obstruction and infection of tonsil tissue in the second operation were not significantly changed, and the immunohistochemical results also demonstrated that the expression of CD20 was not changed, and the expression of CD3 was decreased. Conclusion:Both TT and TE can effectively improve the symptoms of OSA obstruction in children. TT has less trauma, less postoperative pain, faster recovery and lower rate of hyperplasia, which can be used as one of the main methods for the treatment of tonsil hypertrophy in children.

Trimetazidine Alleviates Bleomycin-Induced Pulmonary Fibrosis by Targeting the Long Noncoding RNA CBR3-AS1-Mediated miRNA-29 and Resistin-Like Molecule alpha 1: Deciphering a Novel Trifecta Role of LncRNA CBR3-AS1/miRNA-29/FIZZ1 Axis in Lung Fibrosis.

Pulmonary fibrosis (PF) and tissue remodeling can greatly impair pulmonary function and often lead to fatal outcomes. In the present study, we explored a novel molecular interplay of long noncoding (Lnc) RNA CBR3-AS1/ miRNA-29/ FIZZ1 axis in moderating the inflammatory processes, immunological responses, and oxidative stress pathways in bleomycin (BLM)-induced lung fibrosis. Furthermore, we investigated the pharmacological potential of Trimetazidine (TMZ) in ameliorating lung fibrosis. Our results revealed that the BLM-treated group exhibited a significant upregulation in the expression of epigenetic regulators, lncRNA CBR3-AS1 and FIZZ1, compared to the control group (P<0.0001), along with the downregulation of miRNA-29 expression. Furthermore, Correlation analysis showed a significant positive association between lnc CBR3-AS1 and FIZZ1 (R=0.7723, p<0.05) and a significant negative association between miRNA-29 and FIZZ1 (R=-0.7535, p<0.05), suggesting lnc CBR3-AS1 as an epigenetic regulator of FIZZ1 in lung fibrosis. BLM treatment significantly increased the expression of Notch, Jagged1, Smad3, TGFB1, and hydroxyproline. Interestingly, the administration of TMZ demonstrated the ability to attenuate the deterioration effects caused by BLM treatment, as indicated by biochemical and histological analyses. Our investigations revealed that the therapeutic potential of TMZ as an antifibrotic drug could be ascribed to its ability to directly target the epigenetic regulators lncRNA CBR3-AS1/ miRNA-29/ FIZZ1, which in turn resulted in the mitigation of lung fibrosis. Histological and immunohistochemical analyses further validated the potential antifibrotic effects of TMZ by mitigating the structural damage associated with fibrosis. Taken together, our study showed for the first time the interplay between epigenetic lncRNAs CBR3-AS1 and miRNA-29 in lung fibrosis and demonstrated that FIZZ1 could be a downregulatory gene for lncRNA CBR3-AS1 and miRNA-29. Our key findings demonstrate that TMZ significantly reduces the expression of fibrotic, oxidative stress, immunomodulatory, and inflammatory markers, along with epigenetic regulators associated with lung fibrosis. This validates its potential as an effective antifibrotic agent by targeting the CBR3-AS1/miRNA-29/FIZZ1 axis.

Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer

BackgroundMajor histocompatibility complex class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and...

A novel m6A reader RBFOX2 expression is increased in oral squamous cell carcinoma and promotes tumorigenesis

Background and ObjectiveOral squamous cell carcinoma (OSCC) is a significant global health concern due to its aggressive nature and poor prognosis. Recent research has highlighted the important role of RNA modifications in cancer biology, especially N6-methyladenosine (m6A) modifications, which are controlled by a complex interplay of m6A regulators. This study specifically investigates RBFOX2, a new m6A reader, and its involvement in OSCC. MethodsOur study primarily utilized OSCC tissue, adjacent normal tissue samples, OSCC cell lines, and normal healthy oral keratinocytes to validate RBFOX2 mRNA expression. Additionally, we used the TCGA-HNSCC dataset for large cohort analysis and clinicopathological characterization. Furthermore, we visualized the RBFOX2 network to identify the primary functions of the protein. ResultsOur research shows a noticeable increase in RBFOX2 expression in OSCC tissues compared to adjacent non-tumorous tissues, as determined by quantitative PCR and immunohistochemistry analyses. Functional pathway enrichment analysis revealed that RBFOX2 is involved in the receptor tyrosine kinase signaling pathway and the Hippo signaling pathway, which plays a critical role in oral cancer development and progression. Clinically, elevated RBFOX2 expression correlated with advanced tumor stages and poorer patient outcomes, demonstrating its prognostic value. These findings indicate that RBFOX2 acts as an oncogenic driver in OSCC as an m6A reader, facilitating the expression of m6A-modified oncogenes. ConclusionOur study identifies RBFOX2 as a critical player in OSCC pathogenesis and opens avenues for novel therapeutic strategies targeting the m6A regulatory machinery in this malignancy.

Huangqi-Danshen Decoction Against Renal Fibrosis in UUO Mice via TGF-β1 Induced Downstream Signaling Pathway.

Huangqi-Danshen decoction (HDD) is a Chinese medicinal herb pair with good efficacy in treating chronic kidney disease, but its mechanism needs to be clarified. To uncover the underlying mechanism of HDD antagonizing renal fibrosis through network pharmacology (NP) analysis and experimental validation. The chemical components of water extract of HDD were analyzed by combining the ultra-high performance liquid chromatography coupled with Q-Exactive mass spectrum analysis (UHPLC-QE-MS) and HERB database. NP was used to identify core common targets of HDD components and renal fibrosis. Subsequently, male C57BL/6 mice were divided into Sham, unilateral ureteral obstruction (UUO) and UUO+HDD groups. Renal function, histopathology, Western blotting, and immunohistochemistry analyses were used to evaluate the protective effect of HDD on UUO mice. The effects of HDD on signaling pathways were validated in both UUO mice and transforming growth factor-β1 (TGF-β1)-induced HK-2 cells. By combining UHPLC-QE-MS analysis and HERB database, 25 components were screened in HDD extract. There were 270 intersection targets of the 25 components and renal fibrosis. Based on their scores in protein-protein interaction analysis and degree values in component-pathway-target triadic network, 6 core common targets of the 25 components and renal fibrosis were identified, namely phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription 3(Stat3), non-receptor tyrosine kinase Src (Src), epidermal growth factor receptor (EGFR), matrix metalloproteinase 9 (MMP9), and MMP2. HDD ameliorated renal tubular damage and collagen deposition and downregulated fibrosis-related proteins expression in UUO mice. Furthermore, HDD was demonstrated to reduce PI3K, Stat3, Src, EGFR, and MMP2 expressions, and enhance MMP9 expression in the kidney of UUO mice and in TGF-β1-induced HK-2 cells. HDD can alleviate renal fibrosis which may be related to regulating the expression of essential proteins in the epithelial-mesenchymal transition and extracellular matrix production/degradation signaling pathways.

Mycosis fungoides refractory to treatment - importance of a multidisciplinary approach.

We report a case of difficult-to-control mycosis fungoides (MF), where the role of the dental surgeon was crucial for the control and prognosis of the disease. A 62-year-old female patient diagnosed with MF had a previous record of red patches and small raised bumps on the face, along with a cancerous growth in the cervical and vulvar region. The patient was initially treated with methotrexate and local radiotherapy without resolution. Chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone was then started (CHOP protocol). The dental team of a reference hospital was consulted to evaluate swelling in the anterior region of the palate, which had been developing for two months, reporting discomfort when eating. The role of the dentistry team was fundamental in the differential diagnosis of oral lesions with dental infections, second neoplasia, or even a new site of disease manifestation, in addition to controlling mucosal changes resulting from chemotherapy. After ruling out dental infection, the dentistry team performed a lesion biopsy to confirm the diagnosis. The histopathological and immunohistochemical analysis showed atypical lymphoid infiltration of T cells (CD3+/CD4+/CD7-/CD8-), coexpression of CD25, and presence of CD30 cells, corresponding to the finding for MF. Identifying CD30 + allowed for a new chemotherapy protocol with brentuximab vedotin (BV) combined with gemcitabine. This protocol effectively controlled MF, which previous protocols had failed to do. The diagnosis by the dental team was essential for therapeutic change and improvement of the patient's clinical condition without the need for invasive medical procedures.

202. MULTI-ARRAY MIRNA APPROACH AND ASSOCIATED PROTEIN EXPRESSION PROFILING FOR THE DEVELOPMENT OF PREDICTIVE AND PROGNOSTIC BIOMARKERS IN ESOPHAGEAL ADENOCARCINOMA

Abstract Background Esophageal adenocarcinoma (EA) may develop metastases even in early stage (pT1) disease, but molecular bi-omarkers are lacking to guide therapeutic decisions regarding the type of treatment. Endoscopic submucosal dis-section (ESD) is an effective treatment of early esophageal adenocarcinomas. However, ESD only an option for patients without lymph node metastases. The aim of this study is to detect possible biomarkers with which one could estimate lymphatic metastasis. Methods A multi-array miRNA study with consecutive in silico analysis of dependent genes and in situ analysis of selected proteins was chosen to detect possible biomarkers. A retrospective analysis including twenty cases of EA of the distal esophagus/esophagogastric junction after ESD without or after esophagectomy with lymph node metastasis (each n=10) was performed. The DNA of the Formalin-Fixed Paraffin-Embedded (FFPE) material of these patients was analysed using an advanced miRNA Human Card (spotted with 754 unique microRNAs) with consecutive in silico gene regulation and immunohistochemical analysis of the associated proteins. Result The multi-array miRNA of the tumour sample with EA showed an upregulation of 25 and a downregulation of 20 microRNAs, with the highest differences for the miRNAs miR-100-5p, miR-501-3p and miR-576-5p (upregulation) and miR-150-3p, miR-190a-5p and miR-545-3p (downregulation). The in silico analysis of these mirRNAs revealed the downstream paired partners RDX-Rock2, DICER1-AGO2, YAF2-EZF6 and TET2-5HMC, which were significantly differentially expressed by in-situ immunohistochemical staining. Biologically relevant downstream gene and pro-tein targets, which were also significantly differentially expressed in-situ in relation to the tumour stage of EA could be found. Conclusion This molecular study provides evidence for a specific miRNA marker profile when comparing cases with localized and advanced EA. These preclinical experimental biomarker analyses may help to guide therapeutic decisions in cases with EA, although validation in a larger cohort of EA is necessary to draw firm conclusions.

Glial activation in pain and emotional processing regions in the nitroglycerin mouse model of chronic migraine.

Our aim was to survey astrocyte and microglial activation across four brain regions in a mouse model of chronic migraine. Chronic migraine is a leading cause of disability, with higher rates in females. The role of central nervous system neurons and glia in migraine pathophysiology is not fully elucidated. Preclinical studies have shown abnormal glial activation in the trigeminal nucleus caudalis of male rodents. No current reports have investigated glial activation in both sexes in other important brain regions involved with the nociceptive and emotional processing of pain. The mouse nitroglycerin model of migraine was used, and nitroglycerin (10 mg/kg) or vehicle was administered every other day for 9 days. Prior to injections on days 1, 5, and 9, cephalic allodynia was determined by periorbital von Frey hair testing. Immunofluorescent staining of astrocyte marker, glial fibrillary protein (GFAP), and microglial marker, ionized calcium binding adaptor molecule 1 (Iba1), in male and female trigeminal nucleus caudalis, periaqueductal gray, somatosensory cortex, and nucleus accumbens was completed. Behavioral testing demonstrated increased cephalic allodynia in nitroglycerin- versus vehicle-treated mice. An increase in the percent area covered by GFAP+ cells in the trigeminal nucleus caudalis and nucleus accumbens, but not the periaqueductal gray or somatosensory cortex, was observed in response to nitroglycerin. No significant differences were observed for Iba1 staining across brain regions. We did not detect significant sex differences in GFAP or Iba1 quantification. Immunohistochemical analysis suggests that, at the time point tested, immunoreactivity of GFAP+ astrocytes, but not Iba1+ microglia, changes in response to chronic migraine-associated pain. Additionally, there do not appear to be significant differences between males and females in GFAP+ or Iba1+ cells across the four brain regions analyzed.

Re-evaluation of the concept of basaloid follicular hamartoma associated with naevoid basal cell carcinoma syndrome: a morphological, immunohistochemical and molecular study

Nevoid basal cell carcinoma syndrome (NBCCS) is a rare genodermatosis caused by germline mutations in genes of the Sonic Hedgehog pathway (SHH) and characterized by early onset of multiple basal cell carcinoma (BCC). Although skin tumors with follicular differentiation notably basaloid follicular hamartoma (BFH) have been reported in NBCCS, their relations with BCC are poorly defined. In this context, the aim of this study was to clarify morphological, immunohistochemical and molecular features of BFH arising in a context of NBCCS.140 skin tumors from NBCCS and 140 control BCC tumors were reviewed blinded to clinical data and classified as BCC or BFH. The morphological characteristics of these two groups were then compared. Twenty cases were submitted to immunohistochemical and molecular analysis.Thirty-three tumors among the exploratory cohort were classified as BFH and were exclusively detected in NBCCS patients. Histopathological criteria that were significantly different from BCC were: a small size (<1.5mm), connection to a hair follicle, arborescent organoid architecture, lack of cytologic atypia, infundibulocystic differentiation. Immunohistochemical analysis confirmed activation of the SHH pathway in these lesions. Targeted next-generation sequencing suggested that MYCN and GLI2/3 amplifications and TP53 mutations might be involved in progression of these follicular tumors to BCC.Our study confirms the high prevalence of BFH representing up to 24% of skin tumors in NBCCS and potentially being BCC precursors.

Immunohistochemical Expression of Lymphoid Enhancer-binding Factor 1 in Low-grade Endometrial Stromal Tumors.

Endometrial stromal tumors (ESTs) are uncommon uterine mesenchymal lesions. Nuclear expression of β-catenin, an indication of activated Wnt/β-catenin signaling pathway, was described in 50% to 92% of low-grade ESTs, including endometrial stromal nodule and low-grade endometrial stromal sarcoma. Activation of the Wnt/β-catenin signaling pathway leads to the translocation of β-catenin into the nucleus and interaction with the T-cell factor/lymphoid enhancer-binding factor-1 (LEF1) family of transcription factors to regulate cell proliferation, differentiation, migration, and survival. Immunohistochemical analysis of β-catenin and LEF1 was performed in 2 endometrial stromal nodules and 20 low-grade endometrial stromal sarcomas and demonstrated 90.9% and 81.8% positive rates for β-catenin and LEF1, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of β-catenin and LEF1 were 90.9% versus 81.8%, 81.0% versus 85.7%, 83.3% versus 85.7%, 89.5% versus 81.8%, respectively, in the diagnosis of low-grade ESTs. There is no statistical significance of the performance of β-catenin and LEF1 in all ESTs ( P = 0.664) or in primary or metastatic/recurrent settings ( P = 0.515 and 0.999, respectively). Only 3 smooth muscle tumors showed focal and weak positivity for LEF1. Our results indicate LEF1 can be a useful marker in aiding a diagnosis of low-grade EST and differentiating from smooth muscle tumors alone or in combination with β-catenin.