Immunohistochemical Analysis Research Articles

Oral supplementation with lactic acid bacteria improve the intestinal epithelial barrier and gut microbiota of broiler chicks to alleviate Salmonella Enteritidis infection

Lactic acid bacteria (LAB) play a key role in regulating the balance of gut microbiota and serve as a suitable alternative to antibiotics. This study aims to evaluate the characteristics of 2 LAB isolates Lactiplantibacillus plantarum Lp71 (L. plantarum Lp71) and Enterococcus faecium Ef72 (E. faecium Ef72), and their roles in alleviating Salmonella Enteritidis infection. Sixty 1-day-old chicks were randomly divided into 4 groups which treated with or without L. plantarum Lp71 and E. faecium Ef72 mixture for 21 d, and then intestinal samples were collected for gut microbiota analysis, pathological and immunohistochemical analysis at 24 h post infection with or without Salmonella Enteritidis on the 22nd d. The results showed that L. plantarum Lp71 and E. faecium Ef72 had the ability to anti-acid and anti-bile salt. Salmonella Enteritidis infection damaged the intestinal epithelial barrier and reduced the expression level of tight junction proteins (ZO-1, Claudin-1, Occludin). Oral supplementation with L. plantarum Lp71 and E. faecium Ef72 mixture could alleviated the damages to intestinal epithelial barrier by Salmonella Enteritidis infection. Salmonella Enteritidis could cause abnormal Akkermansia muciniphila proliferation and decrease the diversity of cecal microbiota in chicks. These conditions could have further led to reduce gut microbiota health index (GMHI), and improve microbial dysbiosis index (MDI). Moreover, oral supplementation with L. plantarum Lp71 and E. faecium Ef72 mixture could effectively prevent the aforementioned infection outcomes and increase the abundance proportions of the several key functions in metabolic pathways metabolic pathways such as transcription and signal transduction mechanisms. In summary, L. plantarum Lp71 and E. faecium Ef72 could be the probiotics candidates that used to prevent the damage from enteric pathogens such as Salmonella Enteritidis in broiler chicks.

Exploring the role of ADAMTSL2 across multiple cancer types: A pan-cancer analysis and validated in colorectal cancer

BackgroundRecent studies have established a correlation between ADAMTSL2 (ADAMTS-like 2) and the development of various cancers. This study aims to conduct a comprehensive pan-cancer analysis in 37 cancer types and investigate its potential role in colon and rectal adenocarcinoma (COADREAD).MethodPan-cancer and mutation data were sourced from The Cancer Genome Atlas (TCGA) database and analyzed using Sangerbox analysis platform. We explored the expression patterns and prognostic implications of ADAMTSL2, and investigated its relationships with tumor heterogeneity, stemness, immune checkpoint genes, immune cell infiltration, RNA modifications, and mutational profiles across different cancers. Additionally, with Ethics Committee approval, we conducted immunohistochemical (IHC) analysis on 120 COADEAD samples to evaluate ADAMTSL2 expression and its association with clinicopathological parameters.ResultsADAMTSL2 expression was positively correlated with the hazard ratio of OS, DSS, DFI and PFI for ESCA and COADREAD. A negative correlation was observed between ADAMTSL2 expression and NEO levels in COAD. Gene alterations in ADAMTSL2 were observed, with a mutation frequency of 5.0% in COAD. There is a significant correlation between ADAMTSL2 expression and immune cell infiltration in a variety of cancers. The expression level of ADAMTSL2 protein was associated with T stage, N stage, M stage (p < 0.05). Kaplan‒Meier survival curves demonstrated that the high ADAMTSL2 group had a shorter OS time (p = 0.047) and progression free survival time (p = 0.026) than the low ADAMTSL2 group.ConclusionIn summary, we conducted a comprehensive pan-cancer analysis of ADAMTSL2 and we demonstrated that ADAMTSL2 may serve as a novel prognostic biomarker and immunotherapy target in COADREAD.

Histodynamics of calcium phosphate coating on the osseointegration of medical-grade polycaprolactone β-tricalcium phosphate scaffolds

Bone formation on implant surfaces occurs via distance and contact osteogenesis, with osseointegration influenced by the implant’s surface topography and coating. However, the traditional mechanisms of osseointegration around metal implant surfaces may not fully encompass the ultimate outcomes of using medical-grade polycaprolactone β-tricalcium phosphate calcium phosphate coated (mPCL-TCP-CaP) scaffolds for the reconstruction of large bone defects. Using histology, immunohistochemical (IHC) and scanning electron microscopy (SEM) analyses our studies on large bone defects using mPCL-TCP-CaP scaffolds show osteogenic cells forming a fibrous connective matrix around these scaffolds. Despite extensive research, the in vivo mechanisms of osseointegration of CaP-coated mPCL-TCP-CaP scaffolds remain unclear. This study investigates the structural details and spatial organization of the mPCL-TCP-CaP scaffold’s interface, providing insights into the histodynamic processes involved in their osseointegration with CaP coatings.

Hepatocyte-derived Igκ promotes HCC progression by stabilizing electron transfer flavoprotein subunit α to facilitate fatty acid β-oxidation

BackgroundLipid metabolism dysregulation is a key characteristic of hepatocellular carcinoma (HCC) onset and progression. Elevated expression of immunoglobulin (Ig), especially the Igκ free light chain with a unique Vκ4-1/Jκ3 rearrangement in cancer cells, is linked to increased malignancy and has been implicated in colon cancer tumorigenesis. However, the role of Igκ in HCC carcinogenesis remains unclear. The aim of this study was to elucidate the pivotal roles of hepatocyte-derived Igκ in HCC development.MethodsThe rearrangement sequence and expression level of hepatocyte-derived Igκ in HCC cells were determined via RT-PCR, Sanger sequencing, immunohistochemistry, and western blot analysis. The function of Igκ in HCC tumorigenesis was assessed by silencing Igκ using siRNA or gRNA in various HCC cell lines. To assess the role of Igκ in HCC pathogenesis in vivo, a mouse model with hepatocyte-specific Igκ knockout and diethylnitrosamine (DEN) and carbon tetrachloride (CCL4)-induced HCC was utilized. The molecular mechanism by which Igκ affects HCC tumorigenesis was investigated through multiomics analyses, quantitative real-time PCR, immunoprecipitation, mass spectrometry, immunofluorescence, and metabolite detection.ResultsWe confirmed that Igκ, especially Vκ4-1/Jκ3-Igκ, is highly expressed in human HCC cells. Igκ depletion inhibited HCC cell proliferation and migration in vitro, and hepatocyte-specific Igκ deficiency ameliorated HCC progression in mice with DEN and CCL4-induced HCC in vivo. Mechanistically, Vκ4-1/Jκ3-Igκ interacts with electron transfer flavoprotein subunit α (ETFA), delaying its protein degradation. Loss of Igκ led to a decrease in the expression of mitochondrial respiratory chain complexes III and IV, resulting in aberrant fatty acid β-oxidation (FAO) and lipid accumulation, which in turn inhibited HCC cell proliferation and migration.ConclusionOur findings indicate that the Igκ/ETFA axis deregulates fatty acid β-oxidation, contributing to HCC progression, which suggests that targeting fatty acid metabolism may be an effective HCC treatment strategy. The results of this study suggest that hepatocyte-derived Vκ4-1/Jκ3-Igκ may serve as a promising therapeutic target for HCC.

Pan-cancer analysis and experimental validation of FPR3 as a prognostic and immune infiltration-related biomarker for glioma.

Formyl peptide receptor 3 (FPR3) is known to have implications in the progression of various cancer types. Despite this, its biological significance within pan-cancer datasets has yet to be investigated. In this investigation, we scrutinized FPR3's expression profiles, genetic alterations, prognostic significance, immune-related characteristics, methylation status, tumor mutation burden (TMB), and microsatellite instability (MSI) across different types of cancer. We utilized TISCH's single-cell data to identify immune cells closely associated with FPR3. The predictive significance of FPR3 was evaluated independently in gliomas using data from TCGA and CGGA datasets, leading to the development of a prognostic nomogram. Immunohistochemistry and Western blot analysis confirmed FPR3 expression in gliomas. Lastly, the CCK-8 and wound-healing assays were employed to assess the impact of FPR3 on the proliferation and metastasis of GBM cell lines. In numerous cancer types, heightened FPR3 expression correlated with adverse outcomes, immune cell infiltration, immune checkpoints, TMB, and MSI. In glioma, FPR3 emerged as a notable risk factor, with the prognostic model effectively forecasting patient results. The potential biological relevance of FPR3 was confirmed in glioma, and it was shown to have significant involvement in the processes of glioma growth, immune infiltration, and metastasis. Our results imply a potential association of FPR3 with tumor immunity, indicating its viability as a prognostic indicator in glioma.

Screening core genes for minimal change disease based on bioinformatics and machine learning approaches.

Based on bioinformatics and machine learning methods, we conducted a study to screen the core genes of minimal change disease (MCD) and further explore its pathogenesis. First, we obtained the chip data sets GSE108113 and GSE200828 from the Gene Expression Comprehensive Database (GEO), which contained MCD information. We then used R software to analyze the gene chip data and performed functional enrichment analysis. Subsequently, we employed Cytoscape to screen the core genes and utilized machine learning algorithms (random forest and LASSO regression) to accurately identify them. To validate and analyze the core genes, we conducted immunohistochemistry (IHC) and gene set enrichment analysis (GSEA). Our results revealed a total of 394 highly expressed differential genes. Enrichment analysis indicated that these genes are primarily involved in T cell differentiation and p13k-akt signaling pathway of immune response. We identified NOTCH1, TP53, GATA3, and TGF-β1 as the core genes. IHC staining demonstrated significant differences in the expression of these four core genes between the normal group and the MCD group. Furthermore, GSEA suggested that their up-regulation may be closely associated with the pathological changes in MCD kidneys, particularly in the glycosaminoglycans signaling pathway. In conclusion, our study highlights NOTCH1, TP53, GATA3, and TGF-β1 as the core genes in MCD and emphasizes the close relationship between glycosaminoglycans and pathogenesis of MCD.

Hesperidin facilitating gastrointestinal motility by “Gut-brain axis” and “SCF/C-Kit signaling pathways”

Hesperidin shows promising results as a potential feed additive for enhancing gastrointestinal motility in animals. Gastrointestinal function plays a pivotal role in animal growth and the digestibility of dietary nutrients, with gastrointestinal motor function serving as a crucial component. However, limited research has been conducted on the application of hesperidin as a feed additive to promote gastrointestinal motility. The present study aims to assess the efficacy of Hesperidin as a feed additive in promoting gastrointestinal motility and elucidating its underlying mechanism. A total of 200 newly hatched (1-day-old) broilers with similar body weight were randomly allocated into 4 groups as follows: the control group receiving only the basal diet, and the other 3 groups supplemented with 50, 100, and 150 mg of hesperidin per kg of the basal diet, respectively. Each group consisted of 5 replicates with ten broilers per replicate, and the feeding trial lasted for a duration of 21 d. At 21 d of age, a 5% w/v Evans Blue solution in distilled water was utilized to measure intestinal transit rates (ITR). Gastric emptying (GE) was evaluated by administering a phenol red solution at a concentration of 0.05% w/v (1 mL/broiler). Fifteen broilers from each group were euthanized and immediately dissected to obtain gizzard, hypothalamus, duodenum, and jugular blood samples. Jugular blood samples were collected for brain-gut peptide content analysis, while gizzard, hypothalamus, and duodenum samples were used for immunohistochemical analysis. Real-time qPCR was performed on gizzard samples. The results demonstrated a significant improvement in the GE and ITR of broilers in all treatment groups compared to the control group (P < 0.05), particularly in the 100mg/Kg and 150mg/Kg hesperidin group. Incorporation of hesperidin into the broilers' diet significantly enhances serum levels of ghrelin, encompassing serotonin (5-HT), motilin (MTL), cholecystokinin (CCK), and Stem Cell Factor (SCF) as well as substance P (SP) in the gizzard and duodenal tissues while reducing vasoactive intestinal peptide (VIP) levels (P < 0.05). The group administered a dosage of 150mg/Kg exhibited the most pronounced effect.Immunohistochemistry analysis revealed that hesperidin supplementation up-regulated SP protein content and down-regulated VIP protein content in the hypothalamus, gizzard, and duodenum of broilers (P < 0.05), with the most pronounced effect illustrated in the 150mg/Kg hesperidin group. Furthermore, addition of hesperidin to broiler feed resulted in a significant up-regulation of protein expression and gene expression related to SCF and The protein expression of Receptor tyrosine kinase (C-Kit) was significantly upregulated in the 150mg/Kg group, while the gene expression of C-Kit was significantly upregulated in the 50 mg/Kg group (P < 0.05). In conclusion, hesperidin exhibits promising potential as a feed additive for broilers, as its dietary supplementation of hesperidin improves gastrointestinal motility through modulation of both "gut-brain axis" signaling pathways and "SCF/C-Kit signaling pathways" within broiler chicken's digestive system. Notably, basal diet supplemented with 150mg/Kg hesperidin demonstrates superior efficacy.

β-carotene protects against α-amanitin nephrotoxicity via modulation of oxidative, autophagic, nitric oxide signaling, and polyol pathways in rat kidneys

Alpha-amanitin (α-AMA), a toxic component of Amanita phalloides, causes severe hepato- and nephrotoxicity. This study investigated the protective effects of βeta-carotene (βC) against α-AMA-induced kidney damage in rats. Thirty-two male Sprague-Dawley rats were divided into four groups: Control, βC (50 mg/kg/day), α-AMA (3 mg/kg), and βC+α-AMA. βC was administered orally for 7 days before α-AMA injection. Renal function, oxidative stress markers, histopathological changes, and enzyme activities were evaluated 48 h post-α-AMA administration. α-AMA significantly increased serum creatinine and urea levels, decreased glutathione and catalase activity, and increased malondialdehyde levels (P < 0.001). βC pretreatment attenuated these changes (P < 0.05). Histopathological examination revealed reduced tubular degeneration in the βC+α-AMA group (P < 0.001). Immunohistochemical analysis showed increased LC3B and Beclin-1 expression in α-AMA-treated rats, indicating enhanced autophagy, partially reversed by βC. Additionally, α-AMA reduced nitric oxide synthase (NOS) activity and increased aldose reductase (AR) activity, both normalized by βC pretreatment (P < 0.01). βC demonstrates protective effects against α-AMA-induced nephrotoxicity through antioxidant action, modulation of autophagy, and regulation of NOS and AR pathways, suggesting its potential as a therapeutic agent in α-AMA poisoning.

Primary adenocarcinoma of the spermatic cord: a case report and review of the literature

BackgroundPrimary malignant neoplasms of the spermatic cord are extremely rare, with most reported cases being sarcomas or metastatic carcinomas. However, primary adenocarcinoma of the spermatic cord has not been previously reported.Case presentationA 34-year-old male with a solid mass in the right spermatic cord, was eventually diagnosed with primary adenocarcinoma. Histological examination revealed a moderately-to-poorly differentiated adenocarcinoma exhibiting glandular, cribriform, or nested growth patterns, characterized by medium to large-sized cells and focal extracellular mucus. Immunohistochemical analysis demonstrated positive staining for CK (AE1/AE3), CK8/18, CK19, MOC31 (EP-CAM), and Ber-EP4, while negative staining was observed for CK7, D2-40, WT-1, MC, PAX-8, NKX3.1, PSA, CEA, TTF-1, and NapsinA. Furthermore, a complete loss of INI-1 expression and consistent BRG1 expression were noted in all tumor cells. Next-generation sequencing revealed SMARCB1 deletion, low tumor mutation burden (TMB-L), and microsatellite stability (MSS).ConclusionWe reported the first case of primary adenocarcinoma of the spermatic cord with SMARCB1 (INI-1) deficiency. This case contributes to the expanding understanding of rare neoplasms and underscores the importance of further research into therapeutic strategies targeting SMARCB1-deficient tumors.

An immunomodulatory and osteogenic bacterial cellulose scaffold for bone regeneration via regulating the immune microenvironment

Creating a bone homeostasis microenvironment that balances osteogenesis and immunity is a substantial challenge for bone regeneration. Here, we prepared an immunomodulatory and osteogenic bacterial cellulose scaffold (FOBS) via a facile one-pot approach. The aldehyde groups were generated via selective oxidation of the hydroxyl groups of bacterial cellulose, offering the bonding sites for dopamine through a Schiff base reaction. At the same time, the deposition of Ca2+ and PO43− was promoted on the aldehyde cellulose scaffold because of the high affinity of the catechol moiety for Ca2+. Compared with that of the unmodified scaffold, the hydroxyapatite content of FOBS increased by 47.1 % according to the ICP results. Interestingly, FOBS regulated the immune microenvironment to accelerate the conversion of M1 to M2 macrophages. The expressions of ARG-1 and Dectin-1 (M2) in the FOBS group increased by >100 %. The expression of osteogenic differentiation of BMSCs was also upregulated. In a rat cranial defect model, the BV/TV of FOBS was significantly increased. Further immunohistochemical analysis revealed that an improved immune microenvironment promoted the osteogenic differentiation of stem cells in vivo. This work provides an effective and easy-to-operate strategy for the development of the bone tissue engineering scaffolds.

Evaluation of the therapeutic potential of novel nanoparticle formulations of glutathione and virgin coconut oil in an experimental model of carbon tetrachloride-induced liver failure.

Acute liver failure (ALF) is a critical condition characterized by rapid liver dysfunction, leading to high mortality rates. Current treatments are limited, primarily supportive, and often require liver transplantation. This study investigates the potential of a novel nanoparticle formulation of glutathione (GSH) and virgin coconut oil (VCO) alone and in combination to enhance therapeutic outcomes in a rat model of ALF induced by orogastric carbon tetrachloride (CCl4). The study employed adult male Albino rats divided into ten groups, with ALF induced via a single oral dose of CCl4. Various treatment regimens were administered over seven days, including conventional and nanoparticle forms of GSH and VCO and their combinations. The efficacy of treatments was evaluated through biochemical analysis of liver function markers, oxidative stress indicators, inflammatory biomarkers, and histopathological examinations. Nanoparticles were synthesized using established methods, and characterization techniques were employed to ensure their quality and properties. The nanoparticle formulations significantly improved liver function, as indicated by reduced serum levels of alanine aminotransferase and aspartate aminotransferase, alongside decreased oxidative stress markers such as malondialdehyde. Furthermore, they reduced tumor necrosis factor alpha and interleukin-1 beta inflammatory markers. Histological analysis revealed reduced hepatocellular necrosis and inflammation in treated groups compared to controls. Also, decreased nuclear factor-kappa B was detected by immunohistochemical analysis. The findings show that the nanoparticle mixture of GSH and VCO effectively reduces liver damage in ALF. This suggests a promising drug-based approach for improving liver regeneration and protection. This innovative strategy may pave the way for new therapeutic interventions in the management of ALF.

A case of peeling skin syndrome type 1 with novel CDSN gene variation successfully treated with upadacitinib.

Peeling skin syndrome type 1 (PSS1) is an autosomal recessive genodermatosis caused by the CDSN gene loss-of-function mutation and characterized by widespread superficial skin peeling and erythroderma with unbearable pruritus. Because of its ultra-rarity and unclear mechanism, this rare disease has no established treatment regimen. Herein, we reported a Chinese woman who presented with congenital generalized pruritic erythroderma and exfoliation, notable for significantly elevated IgE levels. The whole exome sequencing identified an unpublished homozygous variant (c.295C>T, p.Gln99*) in the CDSN gene, confirming the diagnosis of PSS1. Immunohistochemistry analysis of the affected skin confirmed the lack of corneodesmosin expression, revealed the overexpression of T helper 2 (Th2)-related cytokines harboring interleukin (IL) 4 and IL-13. After Janus kinase 1 (JAK1) inhibitor upadacitinib administration, both the patient's skin rashes and itching symptoms were significantly alleviated. Our work expanded the PSS1-related CDSN gene mutation spectrums, substantiated the hypothesis regarding the overexpression of Th2-related cytokines, and uncovered the important role of JAK1 underlying PSS1. JAK1 signaling may dominate the pathogenesis in PSS1 and represent a potential therapeutic target.

A COMPARATIVE STUDY OF CLINICOHISTOPATHOLOGICAL CORRELATION IN PATIENTS OF PSORIASIS AND PSORIASIFORM LESIONS WITH KI67 AND CD34 IMMUNOHISTOCHEMICAL EXPRESSION

Objectives: Psoriasis, characterized by chronic inflammation of the skin, stands as a notable example of a psoriasiform tissue pattern. In dermatology, the term “psoriasiform dermatitis” frequently appears across various inflammatory skin conditions, presenting challenges for dermatologists and pathologists alike in accurate differentiation. Relying solely on clinical features may prove insufficient in distinguishing between psoriasis and other psoriasiform dermatoses. Therefore, comprehensive differentiation requires consideration of clinical, histopathological, and immunohistochemical factors. Our study is to compare the clinicohistopathological features of psoriasis and psoriasiform dermatitis and to evaluate the immunohistochemical expression of CD34 and Ki67 in psoriasis and psoriasiform dermatitis. Methods: This retrospective study was conducted at the Department of Pathology, Mediciti Institute of Medical Sciences, Ghanpur, focusing on cases between January 1, 2020, and December 31, 2022. Clinical histories were extracted from requisition forms submitted by the Department of Dermatology. The study analyzed paraffin blocks from 50 patients each diagnosed with psoriasis and psoriasiform dermatitis. Formalin-fixed paraffin-embedded tissue samples were processed into 4-micron sections and initially stained with hematoxylin and eosin. The immunohistochemical analysis included primary antibodies against Ki-67 and CD34, with positive controls sourced from strongly positive samples of squamous cell carcinoma and capillary hemangioma, respectively. Appropriate negative controls were employed to ensure the accuracy of our findings. Results: In our study, we analyzed a total of 100 cases, comprising 50 cases each of psoriasis and psoriasiform dermatitis. Among the psoriasis cases, there was a male predominance with 29 males and 21 females, while psoriasiform dermatitis also showed male predominance with 32 males. The mean age was 33.8 years for psoriasis and 34.8 years for psoriasiform dermatitis. The majority of psoriasis cases fell in the 21–30 years age range, whereas for psoriasiform dermatitis, it was between 31 and 40 years. Significantly, our immunohistochemical analysis revealed that the mean staining intensity of the two markers was notably higher in psoriasis compared to psoriasiform dermatitis. Conclusions: In our study, we observed that the expression levels of Ki-67 and CD34 biomarkers were significantly elevated in psoriasis compared to psoriasiform dermatitis. Notably, four cases of psoriasiform dermatitis demonstrated positivity for CD34 in our analysis. This finding suggests a potential avenue for further research to explore the implications of CD34 positivity in psoriasis.

Temperature-Induced Seasonal Dynamics of Brain Gangliosides in Rainbow Trout (Oncorhynchus mykiss Walbaum) and Common Carp (Cyprinus carpio L.).

This study aimed to determine the expression and distribution of gangliosides in specific regions of the brains of rainbow trout (Oncorhynchus mykiss Walbaum) and common carp (Cyprinus carpio L.) with regard to seasonal temperature changes. Seasonal changes in ganglioside expression and distribution within the species were expected. The natural ecosystems of these fishes differ significantly due to their distinct habitat preferences, geographic distributions, and environmental requirements. Based on the fact that the common carp is eurythermic and adapts to a wide range of temperatures, while the rainbow trout is stenothermic and thrives in a narrower temperature range, it was expected that these species would exhibit distinct patterns of ganglioside modification as part of their adaptive response to temperature fluctuations. Immunohistochemistry using specific antibodies for the major brain gangliosides (GM1, GD1a, GD1b, GT1b), along with the Svennerholm method for quantifying sialic acid bound to gangliosides, revealed that cold acclimatization led to an increase in polysialylated gangliosides in the common carp brain and an increase in trisialogangliosides in the rainbow trout brain. Immunohistochemical analysis also identified region-specific changes in ganglioside expression, suggesting specific functional roles in neuronal adaptation. These results supported the hypothesis that the composition and distribution of brain gangliosides change in response to seasonal thermal shifts as part of the adaptive response. The results underscore the importance of gangliosides in neuronal function and adaptation to environmental stimuli, with implications for understanding fish resilience to temperature changes. This study offers valuable insights into species' temperature adaptation, with implications for physiological and ecological management and improved aquaculture practices. Future research could expand the species scale, study molecular mechanisms and regulatory pathways in ganglioside metabolism, and examine ganglioside interactions with membrane proteins and lipids for a deeper understanding of thermal adaptation.

LHPP deficiency aggravates liver fibrosis through TGF-β/Smad3 signaling.

Liver fibrosis is characterized by a wound-healing response and may progress to liver cirrhosis and even hepatocellular carcinoma. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a tumor suppressor that participates in malignant diseases. However, the role of LHPP in liver fibrosis has not been determined. Herein, the function and regulatory network of LHPP were explored in liver fibrosis. The expression of LHPP in human and murine fibrotic liver tissues was assessed via immunohistochemistry and Western blot analysis. In addition, liver fibrosis was induced in wild-type (WT) and LHPP-/- (KO) mice after carbon tetrachloride (CCl4) or thioacetamide (TAA) treatment. The effect of LHPP was systematically assessed by using specimens acquired from the above murine models. The functional role of LHPP was further explored by detecting the pathway activity of TGF-β/Smad3 and apoptosis after interfering with LHPP invitro. To explore whether the function of LHPP depended on the TGF-β/Smad3 pathway invivo, an inhibitor of the TGF-β/Smad3 pathway was used in CCl4-induced WT and KO mice. LHPP expression was downregulated in liver tissue samples from fibrosis patients and fibrotic mice. LHPP deficiency aggravated CCl4- and TAA-induced liver fibrosis. Moreover, through immunoblot analysis, we identified the TGF-β/Smad3 pathway as a key downstream pathway of LHPP invivo and invitro. The effect of LHPP deficiency was reversed by inhibiting the TGF-β/Smad3 pathway in liver fibrosis. These results revealed that LHPP deficiency exacerbates liver fibrosis through the TGF-β/Smad3 pathway. LHPP may be a potential therapeutic target in hepatic fibrosis.

Nodule-specific NRF2-targeted upregulation in patients with KEAP1 mutations and familial nontoxic multinodular goiter.

Kelch-like ECH-associated protein 1 (KEAP1) is associated with nuclear factor erythroid-2 related factor 2 (NRF2) and promotes NRF2 degradation in normal conditions. Genetic abnormality in KEAP1 is a rare disease and presents with familial multinodular goiter. This study assessed the clinical and molecular findings concerning nodular formation in the thyroid gland of patients harboring KEAP1 germline mutations. Next-generation sequencing analysis targeting goiter-associated genes was performed on 39 patients with familial multinodular goiter. The expression of NRF2-targeted genes from surgical thyroid specimens of patients with KEAP1 mutations were analyzed using a whole transcript expression array and immunohistochemistry. We found five probands with pathogenic heterozygous mutations in KEAP1 (p.Q86*, p.L136P, p.V411fs, p.R415C, and p.R483H), which had no meaningful concomitance with mutations of other goiter-associated genes in germline and somatic levels. Their common histopathological features showed multinodular goiters in the entire thyroid gland with few degenerative lesions or complications of malignancy and slow proliferation indicating < 1% at the Ki-67 labeling index. Among 42 NRF2-targeted genes, antioxidant genes were most frequently upregulated (11/12) in the nodule, followed by detoxification genes (6/11). Immunohistochemical analysis showed relatively high expression of glutathione peroxidase 2 and NAD(P)H quinone oxidoreductase 1 (representative NRF2-targeted genes) in the nodules of various patients harboring KEAP1 mutations. KEAP1 germline heterozygous mutations exert excessive NRF2 activity in the thyroid gland and may confer cytoprotective effects even under abundant reactive oxygen species associated with thyroid hormone production, resulting in thyroid hyperplasia with scarce degradation.

Translation Initiation Factor-2S2 (eIF2S2) Contributes to Cervical Carcinogenesis by Inhibiting the TGF-β/SMAD4 Signaling Pathway.

The deregulation of protein translational machinery and the oncogenic role of several translation initiation factors have been extensively investigated. This study aimed to investigate the role of eukaryotic translation initiation factor 2S2 (eIF2S2, also known as eIF2β) in cervical carcinogenesis. Immunohistochemical analysis of human cervical carcinoma tissues revealed a stage-specific increase in eIF2S2 expression. The knockdown of eIF2S2 in human cervical cancer (SiHa) cells significantly reduced growth and migration properties, whereas its overexpression demonstrated the opposite effect. Immunoprecipitation and Bimolecular fluorescence complementation (BiFC) assay confirmed the previous photo array finding of the interaction between eIF2S2 and SMAD4 to understand the tumorigenic mechanism of eIF2S2. The results indicated that the N-terminus of eIF2S2 interacts with the MH-1 domain of SMAD4. The interaction effect between eIF2S2 and SMAD4 was further evaluated. The knockdown of eIF2S2 increased SMAD4 expression in cervical cancer cells without changing SMAD4 mRNA expression, whereas transient eIF2S2 overexpression reduced SMAD4 expression. This indicates the possibility of post-translational regulation of SMAD4 expression by eIF2S2. Additionally, eIF2S2 overexpression was confirmed to weaken the expression and/or promoter activity of p15 and p27, which are SMAD4-regulated antiproliferative proteins, by reducing SMAD4 levels. Therefore, our study indicated the pro-tumorigenic role of eIF2S2, which diminishes both SMAD4 expression and function as a transcriptional factor in cervical carcinogenesis.

Radiation-Induced Intraosseous Malignant Peripheral Nerve Sheath Tumor: A Case Report.

The significance of radiation therapy in cancer treatment comes with associated complications, including fibrosis, osteonecrosis, and the development of secondary malignancies, such as malignant peripheral nerve sheath tumors (MPNSTs). We emphasize the importance of understanding these complications for an effective patient management. We report a 47-year-old man with a history of squamous cell carcinoma of the tongue, treated with surgery, chemotherapy, and radiation therapy. The patient later presented with symptoms that led to the discovery of an intraosseous MPNST. Histopathological examination revealed characteristic features of MPNST, including spindle cells arranged is sweeping fascicles with contrasting hypercellular and hypocellular areas, producing a marble-like pattern, with atypical wavy, buckled, hyperchromatic nuclei, and brisk mitotic activity. Immunohistochemical analysis showed patchy positive staining for S100 and SOX10, and a complete loss of H3K27me3 expression. This report underscores the challenge of diagnosing secondary malignancies post-radiation therapy and the importance of careful histological examination to differentiate them from other conditions. In conclusion, radiation-induced secondary malignancies are a significant late side effect of radiation therapy that can profoundly impact treatment decision-making and requires a high index of suspicion during post radiation surveillance. Malignant peripheral nerve sheath tumor serves as a pertinent example, highlighting the importance of considering long-term risks when developing optimal management plans for cancer patients.

SENP7 inhibits glioblastoma metastasis and invasion by dissociating SUMO2/3 binding to specific target proteins.

The poor surgical efficacy and recurrence of glioblastoma (GBM) are due to its lack of visible infiltrative features. Our bioinformatics study suggests that low expression of small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) indicates poor prognosis in GBM. This study investigated the effect of SENP7 expression on the invasion, migration, and proliferation of GBM cells and aims to identify the SUMO target proteins affected by SENP7. SENP7 expression was analyzed in eight GBM tumor samples and four GBM cell lines, comparing them to normal brain tissue. The effect of SENP7 overexpression on GBM LN229 cell migration, invasion, and proliferation was examined through in vitro assays. Furthermore, four SUMO target proteins involved in tumor invasion and proliferation (CDK6, matrix metalloproteinase-9 [MMP9], AKT, and HIF-1α) were studied to explore SENP7's molecular mechanism. SENP7 expression was significantly lower in GBM tumors compared to normal tissue. SENP7 overexpression in LN229 cells inhibited migration and invasion without affecting proliferation. Overexpression reduced the levels of MMP9, AKT, and HIF-1α, but not CDK6. Immunohistochemical analysis showed decreased MMP9 and CD31 levels, suggesting reduced tumor invasion and angiogenesis. However, SENP7 overexpression did not affect tumor growth in vivo. SENP7 inhibits GBM invasion by dissociating proteins associated with tumor invasion from SUMO2/3, providing a potential target for future GBM therapies.

7092 Blocking of Aggressive Tumor Progression of Anaplastic Thyroid Cancer by p53

Abstract Disclosure: E. Hwang: None. R. Chari: None. T. Kimura: None. S. Cheng: None. Background: Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer, with very limited treatment options. Patients succumb to death within 6-12 months after diagnosis. It has been observed that mutations of p53, occurring as tumorigenesis progresses, is associated with the aggressiveness of ATC. The present studies tested the hypothesis that wild type p53 (WTp53) could block tumor progression and mitigate its aggressiveness. Methods: We used human 8505C cells as a model, derived from human ATC tumors, harboring BRAFV600E mutation and one allele of mutated p53C742G. We re-expressed WTp53 or mutant p53C742G into 8505C cells (8505C-WTp53 or 8505C-MTp53 cells, respectively) via lentiviral transduction. We analyzed the effects of exogenous expressed WTp53 and mutant p53 on cell proliferation, apoptosis, and migration in vitro and the progression of tumors in vivo mouse xenograft models. Results: We showed that p53 proteins were more abundantly expressed in 8505C-WTp53 or 8505C-MTp53 cells than the parental 8505C cells. Using WTp53 luciferase reporter (PG-13), we demonstrated that the expressed WTp53 was functional as the transcription activity of p53 was higher than the parental 8505C cells. Consistently, in 8505C-MTp53, the reporter activity of PG-13 was not detected. The expressed WTp53 inhibited cell proliferation and decreased cell migration. Immunohistochemical analysis (IHC) showed decreased proliferation marker Ki67 and increased cleaved caspase-3, indicating the induction of apoptosis in 8505C-WTp53 cells. Importantly, we found that the NIS expression was increased in 8505C-WTp53 cells, but not in 8505C-MTp53 cells, as compared with 8505C cells. Xenograft studies showed that tumor progression was inhibited in nude mice injected with 8505C-WTp53 cells as compared with tumors induced by 8505C parental cells and 8505C-MTp53 cells. Consistent with in vitro findings, IHC demonstrated decreased proliferation marker Ki67 and increased cleaved caspase-3 proteins in tumors induced by 8505C-WTp53 cells. Furthermore, the expression of apoptotic regulators such as BAX and PUMA, known as p53 target genes, were elevated in 8505C-WTp53 cultured cells and in 8505C-WTp53-induced tumors, indicating induction of apoptosis. Conclusions: Our findings showed that the aggressiveness of ATC, caused by mutant p53 in late stage of tumor progression, can be mitigated by exogenous expression of WTp53. These results provided new insights into the deleterious actions of mutation of p53 in ATC tumor development. Importantly, our studies revealed that targeting p53 in ATC could be considered as a potential therapeutic opportunity. Presentation: 6/3/2024