Background: Canine mammary gland tumor (MGT) is the most common cancer in aged female dogs. Although it's important to identify reliable metastasis or prognostic factors by evaluating related to cell division, adhesion, and cancer stem cell-related transcription factor (TF) in metastasis-induced canine MGT, but there are limited studies. Objectives: We aimed to identify metastasis prognostic factors and cancer stem cell-TFs in canine MGTs. Methods: Age-matched female dogs diagnosed with MGT only were classified into metastatic and non-metastatic groups by histopathological staining of MGT tissues. The mRNA levels of cancer prognostic metastasis molecular factors (E-cadherin, ICAM-1, PRR14, VEGF, HPRT1, RPL4 and hnRNP H ) and cancer stem cell-related TFs (Oct4, Sox2, and Nanog ) were compared between metastatic and non-metastatic canine MGT tissues using qRT-PCR analysis. Results: The mRNA levels of ICAM-1, PRR14, VEGF, hnRNP H, Oct4, Sox2, and Nanog in metastatic MGT group were significantly higher than those in non-metastatic MGT group. However, mRNA level of RPL4 was significantly lower in metastatic MGT group. Loss of E-cadherin and HPRT1 was observed in the metastatic MGT group but it was not significant. Conclusions Consistent expression patterns of all metastasis-related factors showing elevation in ICAM-1, PRR14, VEGF, hnRNP H, Oct4, Sox2, and Nanog, but decreases in RPL4 levels occurred in canine MGT tissues, which was associated with metastasis. Thus, these cancer prognostic metastasis factors and TFs of cancer stem cells, except for E-cadherin and HPRT1 , can be used as reliable metastasis factors for canine MGT and therapeutic strategy.

Background: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully.We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis. Methods: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants. Results: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions. Conclusion We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Background: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully.We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis. Methods: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants. Results: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions. Conclusion We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Background: Canine mammary gland tumor (MGT) is the most common cancer in aged female dogs. Although it's important to identify reliable metastasis or prognostic factors by evaluating related to cell division, adhesion, and cancer stem cell-related transcription factor (TF) in metastasis-induced canine MGT, but there are limited studies. Objectives: We aimed to identify metastasis prognostic factors and cancer stem cell-TFs in canine MGTs. Methods: Age-matched female dogs diagnosed with MGT only were classified into metastatic and non-metastatic groups by histopathological staining of MGT tissues. The mRNA levels of cancer prognostic metastasis molecular factors (E-cadherin, ICAM-1, PRR14, VEGF, HPRT1, RPL4 and hnRNP H ) and cancer stem cell-related TFs (Oct4, Sox2, and Nanog ) were compared between metastatic and non-metastatic canine MGT tissues using qRT-PCR analysis. Results: The mRNA levels of ICAM-1, PRR14, VEGF, hnRNP H, Oct4, Sox2, and Nanog in metastatic MGT group were significantly higher than those in non-metastatic MGT group. However, mRNA level of RPL4 was significantly lower in metastatic MGT group. Loss of E-cadherin and HPRT1 was observed in the metastatic MGT group but it was not significant. Conclusions Consistent expression patterns of all metastasis-related factors showing elevation in ICAM-1, PRR14, VEGF, hnRNP H, Oct4, Sox2, and Nanog, but decreases in RPL4 levels occurred in canine MGT tissues, which was associated with metastasis. Thus, these cancer prognostic metastasis factors and TFs of cancer stem cells, except for E-cadherin and HPRT1 , can be used as reliable metastasis factors for canine MGT and therapeutic strategy.

Purpose: Adenosine A2A receptor agonist polydeoxyribonucleotide (PDRN) possesses an anti-inflammatory effect and suppress apoptotic cell death in several disorders. In this current study, the effect of PDRN on inflammation and apoptosis in rats with Achilles tendon injury was investigated. Methods: von Frey filament test and plantar test were conducted for the determination of pain threshold. Analysis of histological alterations was conducted by hematoxylin and eosin staining. Immunohistochemistry for cleaved caspase-3-positive cells and cleaved caspase-9-positive cells was done. Enzyme-linked immunoassay was used to detect the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and cyclic adenosine-3’,5’-monophosphate (cAMP). Western blot was conducted to detect the protein levels of cAMP response element-binding protein (CREB), protein kinase A (PKA), Bcl-2-associated X (Bax), and B-cell lymphoma 2 (Bcl-2). Results: PDRN treatment relieved mechanical allodynia and alleviated thermal hyperalgesia after Achilles tendon injury. TNF-α and IL-6 concentrations were decreased by PDRN application. PDRN injection significantly enhanced cAMP concentration and phosphorylated CREB versus CREB ratio, showing cAMP-PKA-CREB pathway was activated by PDRN application. PDRN treatment inhibited percentages of cleaved caspase-3-positive cells and caspase-9-posiive cells and the suppressed Bax versus Bcl-2 ratio in Achilles tendon injury rats. Conclusions PDRN is probably believed to have a good effect on pain and inflammation in the urogenital organs. PDRN may be used as a new treatment for Achilles tendon injury.

Animal models of osteoarthritis (OA) have played a key role in understanding the etiology of OA and in the development of new therapeutic strategies. Although pigs have an advantage as an animal disease model due to their similarity to humans, there are few studies on the induction of OA in minipigs. Therefore, this study aimed to characterize disease progression of OA in total medial meniscectomy (TMM)-operated skeletally mature minipigs, up to day 180 postoperatively. There were no significant alterations in vital signs or hematological indices throughout the observation period. However, clinical manifestations of OA in the medial femoral condyles of TMM-operated minipigs were progressive, depending on postoperative duration, with respect to osteophytes formation and roughened surfaces on radiological observation, cartilage erosion under macroscopic examination, and severe cartilage defects including fibrillation, vertical fissures, and cartilage denuding on histopathological observation, with the highest score indicating late-stage OA on day 180 and without indicating apparent variation between subjects. In particular, the lateral femoral condyles were also degenerated, possibly due to localization of weight-bearing from both menisci to the lateral meniscus. Therefore, TMM in minipigs is suitable for reproducible induction of degenerative changes in the femorotibial joints that closely resemble late-stage OA, and is suitable for use in further research.
Cartilage ; Disease Models, Animal ; Disease Progression ; Humans ; Joints ; Menisci, Tibial ; Models, Animal ; Osteoarthritis ; Osteophyte ; Swine ; Swine, Miniature ; Vital Signs ; Weight-Bearing

BACKGROUND: Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, three-dimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional monolayer cultured MSCs. METHODS: In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties. RESULTS: In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis. CONCLUSION: To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.
Adipogenesis ; Aging ; Apoptosis ; Bone and Bones ; Cell Cycle ; Cell Cycle Checkpoints ; Dental Sac ; Extracellular Matrix ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Methods ; Molar, Third ; Osteogenesis ; Regeneration ; Stem Cells

Animal models of osteoarthritis (OA) have played a key role in understanding the etiology of OA and in the development of new therapeutic strategies. Although pigs have an advantage as an animal disease model due to their similarity to humans, there are few studies on the induction of OA in minipigs. Therefore, this study aimed to characterize disease progression of OA in total medial meniscectomy (TMM)-operated skeletally mature minipigs, up to day 180 postoperatively. There were no significant alterations in vital signs or hematological indices throughout the observation period. However, clinical manifestations of OA in the medial femoral condyles of TMM-operated minipigs were progressive, depending on postoperative duration, with respect to osteophytes formation and roughened surfaces on radiological observation, cartilage erosion under macroscopic examination, and severe cartilage defects including fibrillation, vertical fissures, and cartilage denuding on histopathological observation, with the highest score indicating late-stage OA on day 180 and without indicating apparent variation between subjects. In particular, the lateral femoral condyles were also degenerated, possibly due to localization of weight-bearing from both menisci to the lateral meniscus. Therefore, TMM in minipigs is suitable for reproducible induction of degenerative changes in the femorotibial joints that closely resemble late-stage OA, and is suitable for use in further research.

Evidence indicates that Helicobacter pylori is the causative agent of chronic gastritis and perhaps gastric malignancy. Extracellular vesicles (EVs) play an important role in the evolutional process of malignancy due to their genetic material cargo. We aimed to evaluate the clinical significance and biological mechanism of H. pylori EVs on the pathogenesis of gastric malignancy. We performed 16S rDNA-based metagenomic analysis of gastric juices either from endoscopic or surgical patients. From each sample of gastric juices, the bacteria and EVs were isolated. We evaluated the role of H. pylori EVs on the development of gastric inflammation in vitro and in vivo. IVIS spectrum and confocal microscopy were used to examine the distribution of EVs. The metagenomic analyses of the bacteria and EVs showed that Helicobacter and Streptococcus are the two major bacterial genera, and they were significantly increased in abundance in gastric cancer (GC) patients. H. pylori EVs are spherical and contain CagA and VacA. They can induce the production of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β by macrophages, and IL-8 by gastric epithelial cells. Also, EVs induce the expression of interferon gamma, IL-17 and EV-specific immunoglobulin Gs in vivo in mice. EVs were shown to infiltrate and remain in the mouse stomach for an extended time. H. pylori EVs, which are abundant in the gastric juices of GC patients, can induce inflammation and possibly cancer in the stomach, mainly via the production of inflammatory mediators from gastric epithelial cells after selective uptake by the cells.
Adenocarcinoma* ; Animals ; Bacteria ; Epithelial Cells* ; Extracellular Vesicles* ; Gastric Juice* ; Gastritis ; Helicobacter pylori ; Helicobacter* ; Humans ; Immunoglobulin G ; In Vitro Techniques ; Inflammation* ; Interferons ; Interleukin-17 ; Interleukin-8 ; Interleukins ; Macrophages ; Metagenomics ; Mice ; Microscopy, Confocal ; Necrosis ; Stomach ; Stomach Neoplasms ; Streptococcus

The characterization and potential of mesenchymal stem cells (MSCs) are gender dependent and estrogen influences these properties. This study demonstrated that supplementation with 17β-estradiol (E2) increases the proliferation of bone marrow-MSCs derived from male and female mini-pigs (Mp- and Fp-BMSCs) in a concentration-dependent manner, with 10(-12) M E2 suggested as the optimal dose of E2 that led to the greatest improvement in BMSCs proliferation. Supplementation of 10(-12) M E2 resulted in down-regulation of β-galactosidase activity and pro-apoptotic activity in both BMSCs, while anti-apoptotic activity was up-regulated in only Fp-BMSCs. Further, E2 increased the osteogenic ability of Fp-BMSCs. Based on these findings, optimal utilization of E2 can improve cellular senescence and apoptosis, as well as in vitro osteogenesis of BMSCs, and could therefore be useful in stem cell therapy, particularly in bone regeneration for adult females.
Adult ; Aging* ; Apoptosis ; Bone Regeneration ; Cell Aging ; Down-Regulation ; Estradiol ; Estrogens ; Female* ; Humans ; In Vitro Techniques* ; Male* ; Mesenchymal Stromal Cells* ; Osteogenesis ; Stem Cells