Human Neuralized is a novel tumour suppressor targeting Wnt/ß-catenin signalling in colon cancer.

While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/ß-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic ß-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic ß-catenin, and reducing its cytoplasmic levels in a GSK3ß- and ß-TrCP-independent manner, indicating that NEURL-ß-catenin interactions can lead to a disruption of the canonical Wnt/ß-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/ß-catenin signalling.

Aryl hydrocarbon receptor-targeted therapy for CD4+ T cell-mediated idiopathic pneumonia syndrome in mice.

We previously demonstrated that interferon γ (IFN-γ) derived from donor T cells co-opts the indoleamine 2,3-dioxygenase 1 (IDO1) → aryl hydrocarbon receptor (AHR) axis to suppress idiopathic pneumonia syndrome (IPS). Here we report that the dysregulated expression of AP-1 family genes in Ahr-/- lung epithelial cells exacerbated IPS in allogeneic bone marrow transplantation settings. AHR repressed transcription of Jund by preventing STAT1 from binding to its promoter. As a consequence, decreased interleukin-6 impaired the differentiation of CD4+ T cells toward Th17 cells. IFN-γ- and IDO1-independent induction of Ahr expression indicated that the AHR agonist might be a better therapeutic target for IPS than the IDO1 activator. We developed a novel synthetic AHR agonist (referred to here as PB502) that potently inhibits Jund expression. PB502 was highly effective at inducing AHR activation and ameliorating IPS. Notably, PB502 was by far superior to the endogenous AHR ligand, L-kynurenine, in promoting the differentiation of both mouse and human FoxP3+ regulatory CD4+ T cells. Our results suggest that the IDO1-AHR axis in lung epithelial cells is associated with IPS repression. A specific AHR agonist may exhibit therapeutic activity against inflammatory and autoimmune diseases by promoting regulatory T-cell differentiation.

Priming Mesenchymal Stem/Stromal Cells with a Combination of a Low Dose of IFN-γ and Bortezomib Results in Potent Suppression of Pathogenic Th17 Immunity Through the IDO1-AHR Axis.

Preconditioning of mesenchymal stem/stromal cells (MSCs) with the inflammatory cytokine IFN-γ enhances not only their immunosuppressive activity but also their expression of HLA and proinflammatory genes. We hypothesized that prevention of the upregulation of inflammatory cytokines and HLA molecules in IFN-γ-primed MSCs would render these cells more immunosuppressive and less immunogenic. In this study, we discovered the following findings supporting this hypothesis: (1) activated human T cells induced the expression of IDO1 in MSCs via IFN-γ secretion and those MSCs in turn inhibited T-cell proliferation in an AHR-dependent fashion; (2) there was no difference in the expression of IDO1 and HLA-DR in MSCs after priming with a low dose (25 IU/mL) versus a high dose (100 IU/mL) of IFN-γ; (3) the transient addition of bortezomib, a proteasome inhibitor, to culture MSCs after IFN-γ priming decreased the expression of HLA-DR, inflammatory cytokine genes and Vcam1 while increasing the expression of IDO1 and the production of L-kynurenine; finally, MSCs primed with a combination of a low dose of IFN-γ and bortezomib were more effective in inhibiting Th17-mediated idiopathic pneumonia syndrome (IPS) and chronic colitis than unprimed MSCs. Our results suggest that bortezomib significantly eliminates the unfavorable effects of IFN-γ priming of MSCs (increased expression of MHC molecules and inflammatory cytokines and cell aggregation genes) and simultaneously increases their immunosuppressive activity by upregulating IDO1. Taken together, our newly established MSC priming method may contribute to MSC-based cell therapy for inflammatory diseases.

Potential Molecular Markers Related to Lymph Node Metastasis and Stalk Resection Margins in Pedunculated T1 Colorectal Cancers Using Digital Spatial Profiling: A Pilot Study with a Small Case Series.

There is a debate regarding the prediction of lymph node metastasis (LNM) in pedunculated T1 colorectal cancer (CRC). In this study with four cases of pedunculated T1 CRCs, we aimed to investigate gene expression variations based on the distance from the Haggitt line (HL) and identify potential molecular risk factors for LNM. By leveraging the Cancer Transcriptome Atlas and digital spatial profiling technology, we meticulously analyzed discrete regions, including the head, HL, proximal stalk region (300-1000 µm from HL), and distal stalk region (1500-2000 µm from HL) to identify spatially sequential molecular changes. Our findings showed significant overall gene expression variations among the head, proximal stalk, and distal stalk regions of pedunculated T1 CRCs compared to the control adenoma. Compared to LNM-negative T1 CRCs, LNM-positive T1 CRC showed that the expression of genes involved in immune-related pathways such as B2M, HLA-B, and HLA-E were significantly downregulated in the distal stalk region compared to the proximal stalk region. In summary, our results may tentatively suggest considering endoscopic resection of the stalk with a minimum 2000 µm margin from the HL, taking into account the gene expression alterations related to immune-related pathways. However, we acknowledge the limitations of this pilot study, notably the small case series, which may restrict the depth of interpretation. Further validation is imperative to substantiate these findings.

X-ray radiation-induced intestinal barrier dysfunction in human epithelial Caco-2 cell monolayers.

Radiation therapy and unwanted radiological or nuclear exposure, such as nuclear plant accidents, terrorist attacks, and military conflicts, pose serious health issues to humans. Dysfunction of the intestinal epithelial barrier and the leakage of luminal antigens and bacteria across the barrier have been linked to various human diseases. Intestinal permeability is regulated by intercellular structures, termed tight junctions (TJs), which are disrupted after radiation exposure. In this study, we investigated radiation-induced alterations in TJ-related proteins in an intestinal epithelial cell model. Caco-2 cells were irradiated with 2, 5, and 10 Gy and harvested 1 and 24 h after X-ray exposure. The trypan blue assay revealed that cell viability was reduced in a dose-dependent manner 24 h after X-ray exposure compared to that of non-irradiated cells. However, the WST-8 assay revealed that cell proliferation was significantly reduced only 24 h after radiation exposure to 10 Gy compared to that of non-irradiated cells. In addition, a decreased growth rate and increased doubling time were observed in cells irradiated with X-rays. Intestinal permeability was significantly increased, and transepithelial electrical resistance values were remarkably reduced in Caco-2 cell monolayers irradiated with X-rays compared to non-irradiated cells. X-ray irradiation significantly decreased the mRNA and protein levels of ZO-1, occludin, claudin-3, and claudin-4, with ZO-1 and claudin-3 protein levels decreasing in a dose-dependent manner. Overall, the present study reveals that exposure to X-ray induces dysfunction of the human epithelial intestinal barrier and integrity via the downregulation of TJ-related genes, which may be a key factor contributing to intestinal barrier damage and increased intestinal permeability.

Blood Concentrations of Lead, Cadmium, and Mercury Are Associated With Alcohol-Related Liver Disease.

BACKGROUND: An association between environmental pollutants and alcohol-related liver disease (ALD) has not been determined until now. The objectives of this study were to examine the association of the pollutants with ALD, and whether the pollutants together increased the risk of ALD. METHODS: Data were extracted from the Korea National Health and Nutrition Examination Survey (2010-2013 and 2016-2017; n = 11,993). Blood levels of lead, cadmium, and mercury were measured. ALD was defined by a combination of excessive alcohol consumption and ALD/non-alcoholic fatty liver disease index > 0. The aspartate aminotransferase-to-platelet ratio index and fibrosis (FIB)-4 score were used to evaluate ALD FIB. RESULTS: The odds ratios (ORs) of ALD for the highest versus the lowest quartiles of exposure were for lead, 7.39 (95% confidence interval [CI], 5.51-9.91); cadmium, 1.68 (95% CI, 1.32-2.14); and mercury, 5.03 (95% CI, 3.88-6.53). Adjusting for age, gender, smoking, occupation, education, and personal income attenuated the associations but indicated significant positive trends (all Ptrend < 0.001). A positive additive interaction between cadmium and lead was observed. The relative excess OR due to the interaction was 0.96 (95% CI, 0.41-1.51); synergy index = 2.92 (95% CI, 0.97-8.80). Among 951 subjects with ALD, advanced FIB was associated with lead and cadmium (OR, 3.46, 95% CI, 1.84-6.53; OR, 8.50, 95% CI, 2.54-28.42, respectively), but not with mercury. The effect estimates for lead and cadmium remained significant even after adjustment for daily alcohol intake. CONCLUSION: Blood levels of lead, cadmium, and mercury were significantly associated not only with the risk of ALD but also with ALD FIB. Cadmium and lead have synergistic effects that increase the risk of ALD.

A Case of Cavernous Hemangioma on the Lateral Wall of the Inferior Nasal Meatus.

Hemangiomas, which originate in the sinonasal area, are not common among the various types of tumors from the head and neck region. Mechanisms for the formation of the tumor are yet to be discovered, and a few factors such as trauma, infection, oncogene, and some hormones are considered to take a role in the occurrence and growth of the tumor. Hemangiomas are classified for their histologic features as cavernous, capillary, and mixed types. There are a few reported cases of cavernous hemangiomas of the maxillary sinus, ethmoid sinus, middle and inferior nasal turbinate, and nasal septum. However, a case of cavernous hemangioma from the inferior nasal meatus, on the lateral wall to be precise, has never been reported. The authors are the first to report a case of a 69-year-old female patient who had cavernous hemangioma which was originated from the lateral wall of the inferior nasal meatus and successfully managed.

Secondary diffuse large B-cell breast lymphoma developed in subcutaneous fat layer of the breast with cardiac involvement.

We present a rare case of diffuse large B-cell lymphoma developed in subcutaneous fat layer of the breast with cardiac involvement. Radiologists should perform an image-guided biopsy for pathologic confirmation of breast lymphomas and avoidance of unnecessary invasive treatment.

Efficacy of Polydeoxyribonucleotide in Promoting the Healing of Diabetic Wounds in a Murine Model of Streptozotocin-Induced Diabetes: A Pilot Experiment.

We assessed the efficacy of polydeoxyribonucleotide (PDRN) in accelerating the healing of diabetic wounds in a murine model of streptozotocin (STZ)-induced diabetes. After the creation of diabetic wounds, the mice of the PDRN SC, PDRN IP and PBS groups received a subcutaneous, an intra-peritoneal injection of PDRN and a subcutaneous injection of PBS, respectively. After euthanasia, time-dependent changes in the wound diameter and histologic scores were measured and vascular endothelial growth factor (VEGF), transforming growth factor-ß1 (TGF-ß1) and collagen types I and III were assessed for their expression levels. The PDRN SC and the PDRN IP groups showed a significantly smaller diameter of diabetic wounds, significantly higher histologic scores, a significantly greater expression of VEGF, a significantly lower expression of TGF-ß1 and a significantly greater expression of collagen types I and III as compared with the PBS group (p < 0.05 or 0.0001). In conclusion, PDRN might be effective in promoting the healing of diabetic wounds in a murine model of STZ-induced diabetes.

Inhibition Effect of Adipogenesis and Lipogenesis via Activation of AMPK in Preadipocytes Treated with Canavalia gladiata Extract.

The aim of this study was to investigate the effect of Canavalia gladiata extract (CGE) on the regulation of AMP-activated protein kinase (AMPK) in 3T3-L1 preadipocytes and evaluate the adipogenesis and lipogenesis mechanisms. In 3T3-L1 preadipocytes, lipid accumulation and differentiation were suppressed by 1.1, 1.3, and 1.4 times under the CGE treatment at 0.25, 0.5, and 1.0 mg/mL, respectively. The expression of the main genes involved in the inhibition of adipogenesis was evaluated at the mRNA level via a transcription-polymerase chain reaction. The extract at 1.0 mg/mL increased the mRNA expressions of AMPK and carnitine palmitoyl transferase-1 (CPT-1) by 1.9 and 1.2 times, respectively, while it decreased the expression of sterol regulatory element binding proteins-1c (SREBP-1c), peroxisome proliferator activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), and fatty acid synthase (FAS) by 1.1, 1.2, 1.8, and 1.5 times, respectively, indicating inhibition of the adipogenesis and lipogenesis potential of CGE. Gallic acid (4.02 mg/g) was identified as the main component of the CGE via LC-MS/MS and HPLC analysis. The results of this study suggested that CGE can be utilized as an anti-obesity food additive or medication by activating the AMPK-induced regulation and suppressing adipogenesis transcription factors.

Different miRNAs Related to FBXW7 Mutations or High Mitotic Indices Contribute to Rectal Neuroendocrine Tumors: A Pilot Study.

Recent studies suggest that miRNA may be involved in the development of rectal neuroendocrine tumors (NETs). We explored the frequency of clinicopathologically relevant mutations and miRNA expression in rectal NETs to examine molecular profiles related to prognosis and behavior. Twenty-four eligible specimens with endoscopically excised rectal NETs were selected. Next-generation sequencing and an miRNA expression assay were used to evaluate the expression profile relevant to common genetic mutations in rectal NETs. Kyoto Encyclopedia of Genes and Genomes analysis predicted that the possible target signaling pathways were correlated with dysregulated miRNAs. Nineteen rectal NETs harbored more than one mutation in the 24 cancer-related genes. Seven miRNAs (hsa-miR-769-5p, hsa-miR-221-3p, hsa-miR-34a-5p, hsa-miR-181c-5p, hsa-miR-1246, hsa-miR-324-5p, and hsa-miR-361-3p) were significantly down-regulated in tumors harboring the FBWX7 mutation. Unsupervised hierarchical clustering analysis showed that up-regulation of these seven miRNAs may result in high mitotic indices, indicating the role of miRNAs in tumor progression. Among the down-regulated miRNAs, hsa-miR-769-5p was strongly correlated with extracellular matrix-receptor interaction and lysine degradation. Among the clinicopathological factors, up-regulated hsa-miR-3934-5p was linked to an increased mitotic count. No change in miRNA expression was associated with a tumor size >1 cm, lymphovascular invasion, or Ki-67 index. In summary, we identified different miRNA signatures involved in FBXW7 mutations or high mitotic indices in rectal NETs, which may play a critical role in tumor behavior.

A method for early diagnosis of lung cancer from tumor originated DNA fragments using plasma cfDNA methylome and fragmentome profiles.

Early detection is critical for minimizing mortality from cancer. Plasma cell-free DNA (cfDNA) contains the signatures of tumor DNA, allowing us to quantify the signature and diagnose early-stage tumors. Here, we report a novel tumor fragment quantification method, TOF (Tumor Originated Fragment) for the diagnosis of lung cancer by quantifying and analyzing both the plasma cfDNA methylation patterns and fragmentomic signatures. TOF utilizes the amount of ctDNA predicted from the methylation density information of each cfDNA read mapped on 6243 lung-tumor-specific CpG markers. The 6243 tumor-specific markers were derived from lung tumor tissues by comparing them with corresponding normal tissues and healthy blood from public methylation data. TOF also utilizes two cfDNA fragmentomic signatures: 1) the short fragment ratio, and 2) the 5' end-motif profile. We used 298 plasma samples to analyze cfDNA signatures using enzymatic methyl-sequencing data from 201 lung cancer patients and 97 healthy controls. The TOF score showed 0.98 of the area under the curve in correctly classifying lung cancer from normal samples. The TOF score resolution was high enough to clearly differentiate even the early-stage non-small cell lung cancer patients from the healthy controls. The same was true for small cell lung cancer patients.

Male breast cancer presenting in accessory axillary breast.

We present a rare case of male axillary accessory breast cancer, which is extremely rare and is indistinguishable from lymphadenopathy and other malignancies, such as lymphoma and skin-derived tumors. Clinicians should consider accessory breast cancer in the differential diagnosis even in men, particularly in those who present with superficially located tumors with adjacent accessory breast tissue.

Promoter Methylation of Cancer Stem Cell Surface Markers as an Epigenetic Biomarker for Prognosis of Oral Squamous Cell Carcinoma.

Growing evidence suggests that genetic and epigenetic factors, including environmental factors, contribute to the development of oral squamous cell carcinoma (OSCC). Here, we investigated the transcriptional silencing of the CD24, CD44, CD133, and CD147 genes, which are well-known cancer stem cell surface markers in various cancer types, including OSCC. We first examined the correlation between the transcriptional expression level and reactivation by 5-aza-2'-deoxycytidine (5-aza-dC) and the promoter methylation levels of the four genes in several OSCC cell lines. We observed promoter hypermethylation for the CD24, CD133, and CD147 genes at 70%, 75%, and 70%, respectively, in OSCC cell lines compared to normal oral mucosa tissues (<53%), indicating that this methylation pattern is cancer-specific, which was confirmed by bisulfite sequencing analysis. More specifically, the expression and methylation profiles of CD133 and CD147 extracted from The Cancer Genome Atlas (TCGA) database were negatively correlated, supporting their epigenetic regulation in primary OSCC tumors. The methylation status of CD133 and CD147 was associated with poor survival in patients with OSCC using the TCGA database. Our findings provide additional insight into the abnormal DNA methylation of CD133 and that CD147 could be used for the diagnosis and therapeutic treatment of patients with OSCC.

Expression of VISTA on tumor-infiltrating immune cells correlated with short intravesical recurrence in non-muscle-invasive bladder cancer.

V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint molecule expressed in hematopoietic cells, granulocytes, macrophages, and monocytes. However, few studies to date have investigated VISTA expression, especially its clinical utility, in bladder cancer. The present retrospective study aimed to examine VISTA, programmed death ligand-1 (PD-L1), and CD45 expression by immunohistochemical and immunofluorescence staining of archived pathological tissue samples from 159 patients with primary bladder cancer. The correlation between VISTA expression in immune cells (ICs) and clinicopathologic variables including PD-L1 expression in ICs was examined. Briefly, the rates of VISTA-positive ICs and VISTA-positive tumor cells were 67.9% (108/159) and 30.8% (49/159), respectively. The VISTA expression in ICs of patients with bladder cancer, including those with non-muscle-invasive bladder cancer (NMIBC), was positively correlated with tumor stage, grade, size, and multiplicity. The VISTA expression in ICs was stronger in bladder cancer cases with PD-L1-positive ICs than in those with PD-L1-negative ICs (p < 0.001). The mean intravesical recurrence-free survival was shorter in NMIBC cases with VISTA-positive ICs than in those with VISTA-negative ICs (34.0 vs 39.9 months, p = 0.03, log-rank test). In this first study to investigate VISTA expression in bladder cancer, these results implicate VISTA as a potential immunotherapeutic target and immunologic biomarker in bladder cancer.

High Concordance of Genomic Profiles between Primary and Metastatic Colorectal Cancer.

The comparison of the genetic profiles between primary and metastatic colorectal cancer (CRC) is needed to enable the discovery of useful therapeutic targets against metastatic CRCs. We performed the targeted next generation sequencing assay of 170 cancer-associated genes for 142 metastatic CRCs, including 95 pairs of primary and metastatic CRCs, to reveal their genomic characteristics and to assess the genetic heterogeneity. The most frequently mutated gene in primary and metastatic CRCs was APC (71% vs. 65%), TP53 (54% vs. 57%), KRAS (45% vs. 44%), PIK3CA (16% vs. 19%), SMAD4 (15% vs. 14%) and FBXW7 (11% vs. 11%). The concordance in the top six frequently mutated genes was 85%, on average. The overall mutation frequencies were consistent with two sets of public data (TCGA and MSKCC). To the author's knowledge, this is the first study to compare the genetic profiles of our cohort with that of the metastatic CRCs from MSKCC. Comparative sequencing analysis between primary and metastatic CRCs revealed a high degree of genetic concordance in the current clinically actionable genes. Therefore, the genetic investigation of archived primary tumor samples with the challenges of obtaining an adequate sample from metastatic sites appears to be sufficient for the application of cancer precision medicine in the metastatic setting.

Delayed diagnosis of lung cancer due to misdiagnosis as worsening of sarcoidosis: a case report.

BACKGROUND: The concurrence of sarcoidosis and primary lung cancer is very rare. We report a very rare case with a delayed diagnosis of primary lung cancer due to its misdiagnosis as worsening of pulmonary sarcoidosis. CASE PRESENTATION: A 68-year-old man presented to the outpatient department for evaluation of a mass in the right hilar area with lymphadenopathies in subcarinal and both interlobar areas on chest computed tomography (CT). Sufficient core samples were obtained from subcarinal and bilateral interlobar lymph nodes using endobronchial ultrasonography (EBUS) guided transbronchial needle aspiration (TBNA). EBUS could not reach the right hilar lymph node due to its high angle. The pathologic findings were consistent with sarcoidosis. After 5 months, chest CT revealed aggravation of the right upper paratracheal lymphadenopathy. Assuming worsening of sarcoidosis, he was prescribed an oral corticosteroid for 5 months. However, follow-up chest CT showed a newly developed right lower paratracheal lymphadenopathy and worsening right hilar lymphadenopathy. Bronchoscopy and EBUS were performed once again. Transbronchial lung biopsy from the right upper lobe and EBUS-TBNA from the right lower paratracheal lymph node revealed adenocarcinoma from the lung. CONCLUSIONS: Although coexistence of sarcoidosis and lung cancer is very rare, the clinician should consider the possibility of accompanying lung cancer in sarcoidosis patients who are not responding to initial corticosteroid therapy.

Inhibition of acute lethal pulmonary inflammation by the IDO-AhR pathway.

The lung is a prototypic organ that was evolved to reduce immunopathology during the immune response to potentially hazardous endogenous and exogenous antigens. In this study, we show that donor CD4+ T cells transiently induced expression of indoleamine 2,3-dioxygenase (IDO) in lung parenchyma in an IFN-γ-dependent manner early after allogeneic hematopoietic stem cell transplantation (HSCT). Abrogation of host IDO expression by deletion of the IDO gene or the IFN-γ gene in donor T cells or by FK506 treatment resulted in acute lethal pulmonary inflammation known as idiopathic pneumonia syndrome (IPS). Interestingly, IL-6 strongly induced IDO expression in an IFN-γ-independent manner when deacetylation of STAT3 was inhibited. Accordingly, a histone deacetylase inhibitor (HDACi) could reduce IPS in the state where IFN-γ expression was suppressed by FK506. Finally, l-kynurenine produced by lung epithelial cells and alveolar macrophages during IPS progression suppresses the inflammatory activities of lung epithelial cells and CD4+ T cells through the aryl hydrocarbon receptor pathway. Taken together, our results reveal that IDO is a critical regulator of acute pulmonary inflammation and that regulation of IDO expression by HDACi may be a therapeutic approach for IPS after HSCT.

Clinical significance of the pattern-based classification in endocervical adenocarcinoma, usual and variants.

BACKGROUND: The Silva system is a pattern-based classification system that stratifies endocervical adenocarcinomas (AC) into 3 categories to assess the risk of lymph node (LN) metastasis. This study aimed to evaluate whether this novel risk stratification system is applicable to all endocervical AC, including usual and variant, and to suggest a suitable management plan for cervical AC. METHODS: We retrospectively retrieved consecutive pathology cases with a final diagnosis of endocervical AC treated via radical hysterectomy and pelvic lymphadenectomy. Specimens were classified by consensus according to the Silva system based on "pattern of invasion" as A, B, or C, further clinical/pathologic features were assessed according to pattern-based classification. RESULTS: A total of 76 cases of invasive cervical AC were evaluated. Of these, 63 (82.9%) were categorized as usual-type endocervical AC and 13 (17.1%) as special types. Among those with usual and variants, all patients with pattern A tumor had no LN metastasis and did not develop recurrence. Likewise, multivariate analysis revealed that LN metastasis and pattern C or B tumors are significant independent predictors of disease-free survival (DFS). Although pattern A tumors had no LN metastasis, they also developed complications after surgery, similar to pattern B or C tumors. CONCLUSION: Regardless of histologic subtypes, pattern A tumors had no LN metastasis and no recurrence. Thus, the Silva classification system can influence the clinical management of all types of endocervical AC. Conservative management is reasonable in all patients with endocervical AC with pattern A tumors.

In vitro evaluation of Rhus succedanea extracts for ruminants.

OBJECTIVE: This study was conducted to evaluate the effects of Rhus succedanea extract addition on in vitro ruminal fermentation and microbial growth. METHODS: Two ruminally-fistulated steers consuming 600 g/kg timothy- and 400 g/kg cracked corn-based concentrate with free access to water and mineral block were used as rumen fluid donors. In vitro batch fermentation, with timothy as a substrate, was conducted for up to 72 h, with Rhus succedanea extracts added to achieve final concentrations of 0, 10, 30, 50, 70, and 90 mg/L. RESULTS: Effective dry matter (DM) degradability rate linearly decreased (p = 0.046) depending on extract dosing levels. Total gas production after 24 to 72 h incubation tended to decrease following extract addition, beginning with 50 mg/L starting dose (significance of quadratic effects: p = 0.006, p<0.001, and p = 0.008 for 24, 48, and 72 h, respectively). Methane production decreased depending on dosing levels following 24 h (p<0.05) and 48 h (p<0.005) incubations and was the lowest with the 50 mg/L dose. The Rhus succedanea extracts increased the abundance of Fibrobacter succinogenes (p<0.05) and Ruminococcus flavefaciens (p = 0.0597) and decreased the abundance of methanogenic archaea (p<0.05) following 24 h incubation. CONCLUSION: Rhus succedanea was shown to reduce methane production and increase cellulolytic bacteria without any signs of toxic effects and with a minor effect on DM degradability.