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This study investigated submucosal alterations in biliary carcinogenesis of pancreaticobiliary maljunction (PBM).Thirty-three patients with PBM (including seven with gallbladder [GB] cancer), four with neither biliary tract cancer nor PBM who underwent pancreaticoduodenectomy (controls), and seven with chronic cholecystitis without PBM were enrolled. Protein expression of α-smooth muscle actin (αSMA), CD68, and CD204 in the GB lamina propria and that of NLRP3 and caspase 1 in the GB epithelium and lamina propria were examined.Compared with the control and cholecystitis groups, αSMA expression was higher in the cancerous part (stroma) of the GB in patients with GB cancer + PBM and in the lamina propria of patients with PBM. The CD204/CD68 ratio in the lamina propria was higher in the PBM group than in the control and cholecystitis groups. NLRP3 and caspase 1 expression in both the lamina propria and epithelium was higher in the PBM than control group. In the PBM group, NLRP3- and caspase 1-positive cells in the lamina propria were located near the epithelium.Activated fibroblasts and M2 macrophages in the GB lamina propria may be associated with biliary carcinogenesis of PBM, possibly through inflammasome activation.

Purpose : This study aimed to investigate the short-term outcomes of laparoscopic gastrectomy / robotic gastrectomy after chemotherapy in patients with advanced gastric cancer and compare these outcomes with those of open gastrectomy. Methods : Fifty patients who underwent radical gastrectomy for advanced gastric cancer after chemotherapy between 2007 and 2021 were retrospectively analyzed. The patients were divided into two groups : the laparoscopic gastrectomy / robotic gastrectomy (n = 11) and open gastrectomy (n = 39) groups. The short-term outcomes of these procedures were subsequently examined. Results : The laparoscopic gastrectomy / robotic gastrectomy group had significantly shorter hospital stays and lower intraoperative blood loss than the open gastrectomy group. The overall complication rates were 12.8% (5 of 39 patients) and 0% (0 of 11 patients) in the open gastrectomy and laparoscopic gastrectomy / robotic gastrectomy groups, respectively (P = 0.1). Conclusions : Laparoscopic gastrectomy / robotic gastrectomy may be a surgical option after chemotherapy for patients with advanced gastric cancer. J. Med. Invest. 69 : 261-265, August, 2022.

Urethral injury is one of the most important complications in transanal total mesorectal excision (TaTME) in male patients with rectal cancer. The purpose of this study was to investigate holographic image-guided surgery in TaTME.Polygon (stereolithography) files were created and exported from SYNAPSE VINCENT, and then uploaded into the Holoeyes MD system (Holoeyes Inc., Tokyo, Japan). After uploading the data, the three-dimensional image was automatically converted into a case-specific hologram. The hologram was then installed into the head mount display, HoloLens (Microsoft Corporation, Redmond, WA). The surgeons and assistants wore the HoloLens when they performed TaTME.In a Wi-Fi-enabled operating room, each surgeon, wearing a HoloLens, shared the same hologram and succeeded in adjusting the hologram by making simple hand gestures from their respective angles. The hologram contributed to better comprehension of the positional relationships between the urethra and the surrounding pelvic organs during surgery. All surgeons were able to properly determine the dissection line.This first experience suggests that intraoperative holograms contributed to reducing the risk of urethral injury and understanding transanal anatomy. Intraoperative holograms have the potential to become a new next-generation surgical support tool for use in spatial awareness and the sharing of information between surgeons.

Gastric carcinomas with lymphoid stroma (GCLS) are characterized by prominent stromal infiltration of lymphocyte and account for 1–4% of gastric cancers. Although, osteoclast-like giant cells (OGC) have been reported in some GCLS, OGCs in gastric tumors is exceedingly rare. A 60-year-old woman presented to our hospital after the finding of a positive fecal blood test during a routine medical check. Esophagogastroduodenoscopy revealed a Type 0-III + IIc tumor in the middle part of the gastric body. Biopsy revealed a poorly differentiated tumor and she was referred to our department. Early phase computed tomography showed thickening of the wall in the middle of the gastric body and enlargement of nearby lymph nodes. Laparoscopic total gastrectomy was performed. Pathological examination revealed a hamartomatous inverted polyp (HIP) in the submucosal layer with tub2-por1 tumor in the HIP. Prominent lymphocytic infiltration and OGCs were found around the tumor. Immunohistochemical analysis showed that the tumor cells were negative for EBER, MLH-1, and MSH2, 6. These findings suggest that this tumor was a non-microsatellite instability (MSI)-high GCLS without Epstein–Barr virus (EBV) infection. The patient’s postoperative course was uneventful and she was discharged 11 days after surgery. She remains well 3 years after surgery.

Robotic gastrectomy (RG) has been rapidly adopted for gastric cancer (GC) treatment. However, the benefits of RG over laparoscopic gastrectomy (LG) for GC remain unclear.A total of 451 patients who underwent either RG (n = 83) or LG (n = 368) for GC were enrolled in this study. A 1:1 matched, propensity score-matched analysis was performed using the following factors: age, sex, body mass index, pT, pN, pStage, tumor location, vessel invasion, tumor markers, surgical procedure, reconstruction method, extent of lymphadenectomy, and Endoscopic Surgical Skill Qualification System qualified surgeon as an operator. The surgical outcomes of the two groups were compared.A well-balanced cohort of 158 patients was analyzed (n = 79 in the RG group, n = 79 in the LG group). Regarding the short-term outcomes, the respective blood loss volume, drain amylase content, and number of retrieved lymph nodes in the RG and LG groups were 38.62 ± 73.06 ml and 67.53 ± 108.20 ml (p0.05), 450 ± 371 IU/l and 1590 ± 6392 IU/l (p0.01), and 35.02 ± 15.51 and 25.28 ± 11.70 (p0.01). The morbidity rate was similar between the RG and LG groups (not significant [NS]). Regarding the long-term survival outcomes, there were no intergroup differences in 3-year overall survival (91.72% in the RG group vs. 83.39% in the LG group: NS) and 3-year, disease-free survival (93.31% in the vs. 90.44%: NS).RG was safe and contributed to better short-term outcomes and similar long-term survival outcomes compared with LG.

Tumor-infiltrating lymphocytes (TILs) are a prognostic factor or an indicator of chemotherapy response for various malignancies. The aim of this study was to investigate the prognostic impact of TILs in resected intrahepatic cholangiocarcinoma (IHCC). We also investigated the usefulness of the apparent diffusion coefficient (ADC) in diffusion-weighted magnetic resonance imaging (DW-MRI) to predict TILs. We enrolled 23 patients with IHCC who underwent initial hepatic resection in Tokushima University Hospital from 2006 to 2017. We evaluated stromal TILs in the tumor marginal area and central area in surgical specimens. Patients were divided into low vs high stromal TILs groups. We analyzed the patients’ clinicopathological factors, including prognosis, according to the degree of stromal TILs. We also analyzed the correlation between stromal TILs and the minimum ADC value. Stromal TILs in the marginal area reflected overall survival more accurately than that in the central area. Additionally, marginal low TILs was significantly associated with lymph node metastasis and portal vein invasion. Both overall- and disease-free survival rates in the marginal low TILs group were significantly worse than those in the marginal high TILs group (P

Background Hemodialysis (HD) patients have an increased risk of postoperative complications and poor prognosis after surgery. Likewise, frailty, defined as decreased muscle strength and activity, affects prognosis after cancer surgery. We evaluated frailty as a prognostic factor in HD patients undergoing surgery for gastric cancer. Methods This study included eight HD patients who underwent radical surgery for gastric cancer (seven stage I, one stage II) from August 2009 to the present and 355 non-HD patients who underwent surgery for stage I or II gastric cancer from 2009 to 2018. Results Five-year overall survival (OS) was significantly lower in the HD group than in the control group (43% vs. 86%, respectively; P = .007). However, there was no significant difference in disease-free survival between groups; none of the HD patients died of gastric cancer. Three of the eight HD patients had frailty; those patients had significantly lower OS than the non-frail patients (0% vs. 67%; P = .045). HD, cancer stage, and modified Glasgow prognostic score were independent predictors of OS; HD correlated with the modified Glasgow prognostic score. No postoperative complications occurred among HD patients. Postoperative hospitalization did not significantly differ between groups. Six HD patients underwent reduction surgery with limited lymph node dissection. Conclusion No HD patient died of gastric cancer; however, OS was significantly lower among HD patients than among controls. Frailty was a strong prognostic factor among HD patients. Gastrectomy for HD patients is feasible; reduction surgery may be considered for frail patients.

To clarify whether the preoperative lymphocyte/C-reactive protein (CRP) ratio (LCR) is a prognostic factor for patients with intrahepatic cholangiocarcinoma (IHCC), and investigate its mechanism via tumor-infiltrating lymphocytes. The subjects of this retrospective study were 42 patients who had undergone hepatectomy for IHCC. We divided the patients into low LCR and high LCR groups (cutoff value: 8780) and analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated the levels of stromal tumor-infiltrating lymphocytes (TILs) and CD8+ TILs in surgical specimens, and the relationship between LCR and TILs. A low LCR was identified in 21 patients and was significantly correlated with older age, a high CRP-albumin ratio, and advanced disease stage, and was a prognostic factor for OS and DFS. Multivariate analysis revealed that a low LCR was an independent prognostic factor for worse OS (HR 10.40, P = 0.0077). Although the LCR and levels of stromal TILs were not significantly related, LCR and levels of CD8+ TILs were significantly related (P = 0.0297). The preoperative LCR may predict the postsurgical prognosis of patients with IHCC and reflect the CD8+ TILs.

Background No universal classification method for intrahepatic cholangiocarcinoma (IHCC) has been reported based on the embryological origin of biliary epithelial cells. The aim of this study was to classify IHCC according to protein expression levels of somatostatin receptor 2 (SSTR2) and b-cell leukemia/lymphoma 2 (Bcl2) and to elucidate the clinicopathological features of each group. Methods Fifty-two IHCC patients who underwent hepatic resection were enrolled in this study. Protein expression levels of SSTR2 and Bcl2 were examined using immunohistochemistry. Clinicopathological factors were compared between the three groups and prognostic factors were investigated. Results The patients were divided into three groups: SSTR2 positive and Bcl2 negative (p-Group H, n = 21), SSTR2 negative and Bcl2 positive (p-Group P, n = 14), and the indeterminate group (p-Group U, n = 17) for cases where SSTR2 and Bcl2 were both positive or both negative. All p-Group P cases displayed curability A or B. The 5-year survival rates of p-Group H and U patients were worse than those in p-Group P. p-Group H had higher T-factor, clinical stage, and incidence of periductal infiltration than p-Group P. Conclusions This method could be used to classify IHCC into peripheral and perihilar type by embryological expression patterns of SSTR2 and Bcl2.

Introduction The tumor microenvironment is involved in acquiring tumor malignancies of colorectal liver metastasis (CRLM). We have reported that TU-100 (Daikenchuto) suppresses hepatic stellate cell (HSC) activation in obstructive jaundice. In this study, we report new findings as the direct and indirect inhibitory effects of TU-100 on cancer cell growth through the suppression of HSC activation. Materials and methods The HSCs (LX2) were cultured in colon cancer cells (HCT116 and HT29)-conditioned medium (CM) with or without TU-100 treatment (90, 270, 900 μg/ml). Activated HSCs (aHSCs) were detected by α-SMA and IL-6 mRNA expressions and cytokine arrays of HSC's culture supernatants. Cancer cell growth was analyzed for proliferation and migration ability, compared with TU-100 treatment. To investigate the direct anti-tumor effect of TU-100, cancer cells were cultured in the presence of aHSC-CM and TU-100 (90, 270, 900) or aHSC-CM alone, and assessed autophagosomes, conversion to LC3-II protein, and Beclin-1 mRNA expression. Results Colon cancer-CM significantly increased α-SMA and IL-6 mRNA expressions of aHSC. α-SMA and IL-6 mRNA expressions of aHSC, and IL-6 secretions from aHSCs were significantly decreased with TU-100 (270, 900) treatment, compared to colon cancer-CM alone. Compared with normal culture medium, aHSC-CM led to a significantly increased cell number and modified HSC-CM (TU-100; 270, 900) significantly suppressed cancer cell growth and migration. TU-100 (900) treatment induced autophagy and significantly promoted the autophagic cell death. Conclusions TU-100 inhibited colon cancer cell malignant potential by both suppressing HSC activation and inducing directly autophagy of cancer cells.

Background Frailty plays a crucial role in cancer patients who have received surgery in this recent aging society. We aimed to investigate frailty as a prognostic factor in elderly gastric cancer (GC) patients who underwent curative gastrectomy. Methods In this study, we analyzed 86 elderly (over 75 years old) GC patients who underwent curative gastrectomy. Frailty was defined as clinical frailty scale score of ≥ 4. Patients were assigned to two groups; frailty group (n = 29) and no-frailty group (n = 57). Clinicopathological values, including comorbidity, immune-nutritional score, and long-term outcomes, were compared between the frailty and no-frailty groups.Results The frailty group displayed a positive correlation with a previous history of symptomatic stroke (p < 0.05). Moreover, the overall survival (OS) rate was significantly poorer in the frailty group (5-yr OS; frailty group 52.09% vs. no-frailty group 72.02%, p < 0.05). By contrast, there were no significant differences in both the cancer-specific survival (5-yr CSS; frailty group 82.33% vs. no-frailty group 89.32%, p = 0.14) and the disease-free survival (5-yr DFS; frailty group 74.68% vs. no-frailty group 86.91%, p = 0.33). Finally, frailty, differentiation, surgical procedure and the lymphocyte c-reactive protein ratio were identified among clinicopathological factors as independent prognostic factors for OS in multivariate analysis. Conclusion We identified the clinical impact of frailty as an independent prognostic factor for elderly GC patients who underwent gastrectomy.

Background/aim Carbohydrate antigen 19-9 (CA19-9) is a poor prognostic marker in intrahepatic cholangiocarcinoma (IHCC). Previous studies have shown a link between hypoxia and CA19-9 in cancer, and we have previously demonstrated a correlation between HDAC1 and HIF-1α in IHCC. Here, we evaluated the expression and correlation of CA19-9 with HIF-1 and HDAC in IHCC. Patients and methods This study included 62 patients with IHCC who underwent primary hepatectomy at our department. Clinicopathological characteristics were examined. Immunohistochemical expression of HIF-1 and HDAC1 in specimens was quantitatively evaluated. Results Patients with high preoperative serum CA19-9 levels showed clinicopathological characteristics associated with tumour progression. High CA19-9 levels were associated with worse overall and recurrence-free survival. Univariate and multivariate analysis detected high CA19-9 levels as an independent poor prognostic factor for IHCC. Serum CA19-9 was significantly correlated with both HIF-1α and HDAC1 expression. Conclusion High serum CA19-9 level is a poor prognostic factor for overall survival in IHCC and correlates with HIF-1α and HDAC1 expression.

The concept of 'diversity and inclusion' is being adopted worldwide, but it is not yet understood well in Japan. We conducted this study to evaluate the impact of a lecture aimed at increasing awareness of academic careers and the benefits of having diversity and inclusion.Two female surgeons delivered a 120-min lecture on "diversity and inclusion" to third-year medical students at Tokushima University. To assess the impact of the lecture, a questionnaire was distributed, for participants to complete anonymously before and after the lecture.Eighty-two students participated in the study (39 men, 38 women, and 5 unknown). Based on the questionnaire responses, 57.1% of the students had already perceived inequality in conduct because of gender. A comparison of pre- and post-lecture responses revealed a significant increase in confidence to succeed in their medical career (56.5% vs. 77.5%, p 0.01). Learners were more likely to believe that gender would not become a barrier to career development (42.4% vs. 66.7%, p 0.01). Moreover, 90.4% of the students felt positively about a career in surgery following the lecture.The lecture promoted awareness about diversity, self-awareness, and career development and motivated students to consider specializing in surgery later in their career.

To clarify whether the preoperative lymphocyte/C-reactive protein (CRP) ratio (LCR) is a prognostic factor for patients with intrahepatic cholangiocarcinoma (IHCC), and investigate its mechanism via tumor-infiltrating lymphocytes.The subjects of this retrospective study were 42 patients who had undergone hepatectomy for IHCC. We divided the patients into low LCR and high LCR groups (cutoff value: 8780) and analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated the levels of stromal tumor-infiltrating lymphocytes (TILs) and CD8A low LCR was identified in 21 patients and was significantly correlated with older age, a high CRP-albumin ratio, and advanced disease stage, and was a prognostic factor for OS and DFS. Multivariate analysis revealed that a low LCR was an independent prognostic factor for worse OS (HR 10.40, P = 0.0077). Although the LCR and levels of stromal TILs were not significantly related, LCR and levels of CD8The preoperative LCR may predict the postsurgical prognosis of patients with IHCC and reflect the CD8

Background Lymphocyte/C-reactive protein (CRP) ratio (LCR), is a promising prognostic factor that can reflect tumor inflammation and systemic patient condition. The aim of this study was to investigate whether preoperative LCR can be a prognostic factor for post-surgical outcomes among patients with intrahepatic cholangiocarcinoma (IHCC). We also investigated the relationship between LCR and tumor infiltrating lymphocytes (TILs) to clarify whether systemic host immune parameters reflect local tumor immunity.Methods We enrolled 45 patients who had undergone hepatectomy for IHCC. Patients were divided into low LCR and high LCR groups, according to reactive operating characteristic curve (Cut-off value: 8780). We analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated stromal TILs and numbers of CD8+ TILs in surgical specimens, and the relationship between LCR and TILs.Results Twenty-one patients (46.7%) were associated with low LCR. Low LCR was significantly correlated with older age, high CRP and advanced disease stage, and was a prognostic factor for OS and DFS. And multivariate analysis revealed that low LCR was an independent prognostic factor for worse OS (HR: 2.81 P+ TILs were significantly related. Conclusions Preoperative LCR levels could predict the post-surgical prognosis of patients with IHCC, and reflected numbers of intra-tumoral CD8+ TILs.Trial registration This study was approved by Tokushima University Hospital ethics committee and with the approval of corresponding regulatory agencies, and all the experiments were carried out in accordance with the approved guidelines (Tokushima Clinical Trial Management System Number; 3215). All the patients involved in this study signed informed consent forms and agreed to participate.

The aim of our study is to determine whether insulin-producing cells (IPCs) differentiated from adipose-tissue-derived stem cells (ADSCs) can be cryopreserved. Human ADSCs were differentiated into IPCs using our two-step protocol encompassing a three-dimensional culture and xenoantigen-free method. Thereafter, IPCs were frozen using three different methods. First, IPCs were immediately frozen at −80°C (−80°C group). Second, IPCs were initially placed into a Bicell freezing container before freezing at −80°C (BICELL group). Third, a vitrification method for oocytes and embryos was used (CRYOTOP group). Cell counting kit-8 (CCK-8) assay showed that cell viability was decreased in all groups after cryopreservation ( P < 0.01). Corroboratively, the amount of adenosine triphosphate was markedly decreased after cryopreservation in all groups ( P < 0.01). Immunofluorescence staining showed a reduced positive staining area for insulin in all cryopreservation groups. Furthermore, 4′,6-diamidino-2-phenylindole and merged immunofluorescence images showed that cryopreserved cells appeared to be randomly reduced in the −80°C group and CRYOTOP group, while only the central region was visibly reduced in the BICELL group. Using immunohistochemical staining, IPCs after cryopreservation were shown to be positive for cleaved caspase-3 antibody in all groups. Finally, insulin secretion following glucose stimulation was significantly reduced in IPCs from all groups after cryopreservation ( P < 0.01). In conclusion, IPCs may be too fragile for cryopreservation with accomplished methods and further investigations for a suitable preservation method are required.

Venous thromboembolism (VTE) is a major complication after abdominal surgery; however, the best strategy for preventing VTE after surgery is not yet established. We performed this study to investigate the incidence of perioperative VTE and evaluate the efficacy of a VTE preventive strategy for patients undergoing surgery for colorectal cancer (CRC). The subjects of this retrospective analysis were 228 patients who underwent curative surgical resection for CRC between 2012 and 2016. The patients were treated with thromboprophylaxis including enoxaparin. We assessed the perioperative VTE occurrence rate and investigated the risk factors for postoperative VTE. Among the 228 patients, 77 had a preoperative D-dimer level of > 1.0 μg/mL and 12 had deep vein thrombosis (DVT) diagnosed by ultrasonography preoperatively. Of the remaining 216 patients, short-term VTE (

The expression of programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) indicate the efficacy of anti-PD-1/PD-L1 therapy in colorectal cancer (CRC), but are less useful for monitoring the efficacy of therapy of CRC liver metastasis (CRLM). This study investigated the effects of immune molecules on the prognosis of CRLM. We enrolled 71 patients with CRLM who underwent curative resection for CRC. We used immunohistochemistry to analyze the expression of PD-1, PD-L1, indoleamine-pyrrole 2,3-dioxygenase (IDO), and CD163 (a marker of tumor-associated macrophages [TAMs]) in metastatic tumors. The immune molecules PD-1, PD-L1, IDO, and TAMs were expressed in 32.3%, 47.8%, 45.0%, and 47.9% of metastatic CRC samples, respectively. The 5-year overall survival rates associated with immune molecule-positive groups were significantly better than in the negative groups (PD-1: 87.7% vs 53.2%, p = 0.023; PD-L1: 82.4% vs 42.3%, p = 0.007; IDO: 80.7% vs 43.5%, p = 0.007; TAMs: 82.6% vs 48.0%, p = 0.005). Multivariate analysis revealed PD-1 expression (p = 0.032, hazard ratio: 0.19), IDO expression (p = 0.049, hazard ratio: 0.37), and tumor differentiation (p<0.001, hazard ratio: 0.02) as independent prognostic indicators. PD-1 and TAMs in metastases were associated with less aggressive features such as smaller tumors. Furthermore, TAMs positively and significantly correlated with PD-1 expression (p = 0.011), PD-L1 expression (p = 0.024), and tended to correlate with IDO expression (p = 0.078). PD-1, PD-L1, IDO, and TAMs in CRLM were associated with less aggressive features and better prognosis of patients with CRC, indicating adaptive antitumor immunity vs immune tolerance. These molecules may therefore serve as prognostic markers for CRLM.

Intrahepatic cholangiocarcinoma (IHCC) has a poor prognosis, and surgery remains the only effective treatment. However, tumor recurrence after primary hepatectomy is common. We examined the significance of repeat surgery for IHCC. We collected data for all patients with IHCC between 1992 and 2018 (n = 67) in our database. Fifty-three (79.1%) of all 67 patients experienced recurrence after primary hepatectomy and we analyzed data for those 53 recurrent patients. We divided recurrent patients into a repeat surgery group (n = 9), chemotherapy group (n = 19), and best supportive care group (n = 25). We analyzed differences in patients’ clinicopathological factors, including prognosis, between the three groups. The IHCC recurrence rate after hepatectomy in our institution was 79.1%. Of the 53 patients with recurrence, nine underwent repeat surgery (17.0%). The characteristics of the patients undergoing repeat surgery was lower stage at primary hepatectomy. Recurrence sites in the repeat surgery group were liver (n = 6), lung (n = 2), and adrenal gland (n = 1), as a single nodule. The period between primary hepatectomy and recurrence was comparatively longer in the repeat surgery group, at 1.8 years. The prognosis in patients undergoing repeat surgery was significantly better compared with the other groups. Not undergoing repeat surgery (hazard ratio: 5.506; p = 0.0077) and positive lymph node metastasis (hazard ratio: 2.207; p = 0.0242) were independent poor prognostic factors. Repeat surgery should be considered in patients with IHCC with a single recurrence site and negative lymph node metastasis at primary surgery and at least more than 6 months of disease-free period after primary surgery.

The post-synthetic modification of an oligonucleotide is a powerful strategy for the synthesis of various analogs of the oligonucleotide, aiming to achieve the desired functions. In this study, we synthesized the thymidine phosphoramidite of 2′-N-pentafluorophenoxycarbonyl-2′-amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2′-N-pentafluorophenoxycarbonyl-2′-amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM K2CO3 in MeOH at room temperature for 4 h). Moreover, by treatment with various amines as a post-synthetic modification, the oligonucleotides were successfully converted into the corresponding 2′-N-alkylaminocarbonyl-2′-amino-LNA (2′-urea-LNA) derivatives. The duplex- and triplex-forming abilities of the synthesized oligonucleotides were evaluated by UV-melting experiments, which showed that 2′-urea-LNAs could stabilize the nucleic acid complexes, similar to the proto-type, 2′-amino-LNA. Thus, 2′-urea-LNAs could be promising units for the modification of oligonucleotides; the design of a substituent on urea may aid the formation of useful oligonucleotides. In addition, pentafluorophenoxycarbonyl, an amino moiety, acted as a precursor of the substituted urea, which may be applicable to the synthesis of oligonucleotide conjugates.

Background Diagnosis of cystic tumor of the pancreas is based on the World Health Organization criteria that classify pancreatic cystadenomas into four types: intra-ductal papillary mucinous neoplasms, mucinous cystic neoplasms (MCNs), serous cystic neoplasms, and solid pseudo-papillary neoplasms depending on their secretion and presence of ovarian-like stroma. Recently, Albores-Saavedra identified non-mucinous cystadenomas of the pancreas with pancreato-biliary phenotype and ovarian-like stroma. This precipitated examination of the proportions of these rare tumors in patients treated at Tokushima University Hospital. Case presentation Case 1 was a 40-year-old woman with a cystic tumor in the tail of the pancreas. Computed tomography (CT) revealed a diffuse and non-enhanced cystic tumor in the tail of the pancreas. This tumor was diagnosed as a simple cyst at this point. However, 2 years later, the tumor had increased in size by 3 cm. Thus, laparoscopic distal pancreatectomy was performed. The content of the cyst was serous. The epithelial cells were lined with a single layer of cuboidal cells and the tumor had ovarian-like stroma pathologically. The final pathological diagnosis was non-mucinous cystadenoma of the pancreas with ovarian-like stroma. In Case 2, a cystic tumor in the pancreas was found by medical examination in a woman in her sixties who presented without symptoms. CT showed a 1.5-cm cystic tumor in the tail and body of the pancreas and a septum in the cyst. Nine years later, the tumor had grown to 2.4 cm in diameter and had a clear septum in the cyst. This tumor was diagnosed preoperatively as MCN. Thus, laparoscopic distal pancreatectomy was performed. The cyst contained serous fluid. Microscopic examination showed no ovarian-like stroma and the epithelial cells were lined by a single layer of cuboidal cells. The final pathological diagnosis was non-mucinous cystadenoma of the pancreas with ovarian-like stroma. Conclusions Accurate preoperative diagnosis of this type of pancreatic cystic tumor may be difficult, although it occurs more often than expected. Non-mucinous cystadenomas of the pancreas with ovarian-like stroma need to be considered as a differential diagnosis.

The aim of this study was to assess the viability of vitrified-warmed in vivo-derived pig embryos after measuring the oxygen consumption rate. Six days after artificial insemination, blastocysts were collected from gilts and vitrified by the micro volume air cooling method. The oxygen consumption rate was measured in 60 vitrified-warmed embryos, which were then cultured for 48h to assess the viability. The survival (re-expansion) rate of embryos after warming was 85.0%. The average oxygen consumption rate of embryos immediately after warming was greater in embryos which could re-expand during subsequent culture (F=0.75±0.04) than that in those which failed to re-expand (F=0.33±0.05). Moreover, the oxygen consumption rate of vitrified-warmed embryos was greater in the hatched (F=0.88±0.06) than that in the not-hatched group (F=0.53±0.04). When the oxygen consumption rate of the vitrified-warmed embryos and the numbers of viable and dead cells in embryos were determined, there was a positive correlation between the oxygen consumption rate and the number of live cells (P

The post-synthetic modification of an oligonucleotide is a powerful strategy for the synthesis of various analogs of the oligonucleotide, aiming to achieve the desired functions. In this study, we synthesized the thymidine phosphoramidite of 2&prime, N-pentafluorophenoxycarbonyl-2&prime, amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2&prime, amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM K2CO3 in MeOH at room temperature for 4 h). Moreover, by treatment with various amines as a post-synthetic modification, the oligonucleotides were successfully converted into the corresponding 2&prime, N-alkylaminocarbonyl-2&prime, amino-LNA (2&prime, urea-LNA) derivatives. The duplex- and triplex-forming abilities of the synthesized oligonucleotides were evaluated by UV-melting experiments, which showed that 2&prime, urea-LNAs could stabilize the nucleic acid complexes, similar to the proto-type, 2&prime, amino-LNA. Thus, 2&prime, urea-LNAs could be promising units for the modification of oligonucleotides, the design of a substituent on urea may aid the formation of useful oligonucleotides. In addition, pentafluorophenoxycarbonyl, an amino moiety, acted as a precursor of the substituted urea, which may be applicable to the synthesis of oligonucleotide conjugates.

The biological functions of recombinant human follicle-stimulating hormone (FSH) and transforming growth factor-α (TGF-α) on in vitro maturation of porcine oocytes were investigated. Cumulus-oocyte complexes were matured in defined porcine oocyte medium containing 0-0.1 IU/ml FSH in the presence or absence of 10 ng/ml TGF-α. The percentage of oocytes reaching metaphase II was significantly higher with the addition of 0.01-0.1 IU/ml FSH compared with no addition, and was further enhanced in the presence of TGF-α. The rates of sperm penetration and blastocyst formation were significantly higher with the addition of 0.05-0.1 IU/ml FSH compared with no addition after in vitro fertilization and embryo culture. There was no beneficial effect of FSH and TGF-α on nuclear maturation of denuded oocytes. The specific EGF receptor inhibitor, AG1478, completely inhibited TGF-α-induced meiotic resumption, but did not completely prevent the stimulatory effect of FSH. Addition of both FSH and TGF-α significantly enhanced cumulus expansion compared with no addition. When cumulus expansion-related genes (HAS2, HAPLN1, and VCAN) mRNA expression in COCs was measured during in vitro maturaiton, addition of both of FSH and TGF-α upregulated the expression of HAS2 mRNA after 20 hr culture and HAPLN1 mRNA after 44 hr culture compared with no addition. Expression of VCAN mRNA was significantly higher in the presence of FSH compared with addition of TGF-α alone. These results suggest that FSH and TGF-α synergistically enhance porcine oocyte maturation via cumulus cells, and act through different signaling pathways.

We examined the effect of different embryo developmental stages, culture periods, and media on the cryotolerance of in vitro-produced porcine blastocysts. All media used for in vitro production, vitrification, and warming were chemically defined. When in vitro-produced embryos at the blastocyst and expanded blastocyst stages were vitrified using the Cryotop method on Day 5 or 6 (Day 0 = the day of IVF), the survival rate and hatching rate of expanded blastocysts after warming were higher than those of blastocysts. The viability after vitrification and warming of Day-6 embryos cultured in porcine blastocyst medium from Day 5 were higher than that of embryos cultured in porcine zygote medium 5. On the other hand, there were no significant differences on the cryotolerance between Day-5 embryos cultured in porcine zygote medium 5 and those replaced with porcine blastocyst medium on Day 4. There were no significant differences in viability between the embryonic ages of 5 and 6 days after vitrification and warming. When expanded blastocysts vitrified on Day 5 or 6 were surgically transferred to recipient gilts, all three recipients became pregnant in the Day-5 group, whereas only one out of three recipients became pregnant in the Day-6 group. These results indicate that the cryotolerance of porcine in vitro-produced blastocysts after vitrification appears to depend on the embryonic stage, culture period, and medium.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-beta superfamily, is known to be expressed specifically in ovaries of various mammalian species and to be important for normal follicular development in mice. In the present study, a cDNA encoding for bovine GDF-9 was isolated and characterized, and expression of GDF-9 mRNA in ovarian oocytes and in-vitro-derived embryos was examined. Isolation of bovine GDF-9 was achieved using the polymerase chain reaction (PCR) with primers based on an ovine GDF-9 cDNA sequence. A 1385 bp cDNA encodes a deduced 453-amino acid sequence which contains the proregion (318 residues) and the mature protein (135 residues) portion. The deduced amino acid sequence of bovine GDF-9 is 98 and 93% identical to ovine GDF-9 and human GDF-9 in the mature portion of the molecule, respectively. Results from reverse transcription (RT)-PCR analysis detected bovine GDF-9 mRNA in preantral follicles (150-200 microm in diameter), early antral follicles (400-800 microm in diameter), and immature oocytes, whereas no detectable PCR signal was observed in cumulus/granulosa cells. In addition, bovine GDF-9 mRNA continued to be expressed in developing embryos up to the 8-cell stage, but was undetectable at the blastocyst stage. These findings give rise to new possibilities regarding an additional physiological role of GDF-9 in early embryonic development as well as in the development of follicles.

The aim of this study was to evaluate whether two completely serum-free media (IVMD101 and IVD101) could improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes. The media were evaluated in the presence (IVMD101) or absence (IVD101) of bovine cumulus/granulosa cell (BCGC) cocultures. The proportion of embryos developing to the blastocyst stage in IVMD101 medium with BCGC cocultures (36.5%) and IVD101 medium without BCGC cocultures (37.1%) was significantly higher than in serum-supplemented medium (TCM199 + 5% calf serum) with BCGC cocultures (25.1%). Furthermore, the mean cell numbers per blastocyst on Day 7 developed in IVMD101 medium (179.5 cells) and IVD101 medium (177.1 cells) were greater than in the serum-supplemented medium (145.7 cells). The survival rates of blastocysts derived in IVMD101 medium (73.3%) and IVD101 medium (60.0%) based on hatching after 72 h of post-thaw culture were superior to that of blastocysts derived in the serum-supplemented medium (48.1%). Under microscopic observation, bovine blastocysts derived in the serum-supplemented medium showed abundant lipid droplets, largely into the trophectoderm cells. This morphological difference may partly explain the sensitivity of serum-derived embryos after freezing and thawing. In conclusion, these new serum-free culture media are useful, not only to study the mechanisms of early embryogenesis, but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

A serum-free culture system was used to allow determination of the fatty acid composition of bovine embryos during in vitro development. The proportion of embryos developing to the blastocyst stage in serum-free medium (TCM199 supplemented with bovine serum albumin, insulin, apotransferrin and transforming growth factor-α; BITTα) was as high as in serum-supplemented medium (TCM199+5% calf serum). Bovine blastocysts grown in serum-supplemented medium contained abundant lipid droplets in the cytoplasm. The fatty acid composition of bovine immature oocytes, embryos at 2-cell and blastocyst stages cultured in serum-free or serum-supplemented medium was determined by capillary column gas chromatography. Additionally, the fatty acid composition of calf serum alone was determined. Myristic acid (59.6%) was the most abundant fatty acid in immature oocytes followed by docosahexaenoic acid (12.3%). The fatty acid profiles of two-cell embryos derived in the serum-free medium were similar to those of immature oocytes. In contrast, two-cell embryos derived in the serum-supplemented medium showed high levels of palmitic (27.6%) and stearic (27.2%) acids and a low level of myristic acid (21.9%). For blastocysts grown in the serum-free medium, myristic acid (35.7%) was high and palmitic (17.0%) and stearic (12.5%) acids were moderately high. On the other hand, blastocysts developed in the serum-supplemented medium retained high levels of palmitic (30.5%) and stearic (24.2%) acid compositions and had considerably increased levels of palmitoleic (16.3%) and oleic (12.1%) acids. High levels of oleic (17.3%), palmitic (16.5%) and stearic (12.3%) acids were detected in calf serum, which were similar to the profile of the 2-cell and blastocyst stage embryos cultured with serum. This study shows that bovine embryos grown in serum-supplemented medium showed different morphology and fatty acid composition when compared to those cultured in serum-free medium.

This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN2). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN2 for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN2.

The matrix metalloproteinases and their inhibitors are thought to play an important role in such biological processes as ovulation, embryo development, implantation, angiogenesis and tumor cell metastasis. Because recent findings indicated that the tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by granulosa cells improved bovine embryo development in vitro, the patterns of gene expression have been determined for TIMP-1 and collagenase during bovine preimplantation development by reverse transcription-polymerase chain reaction (RT-PCR). mRNA transcripts for bovine TIMP-1 and collagenase were detectable in the morula and blastocyst stages of bovine embryos. This data suggested that TIMP-1 and collagenase may be involved in the growth and development of bovine embryos in an autocrine fashion as well as in a paracrine manner.

Embryogenesis-stimulating activity (ESA) was found in serum-free conditioned media (CM) of bovine cumulus/granulosa cells (BGC) and bovine oviductal epithelial cells (BOEC). The CM of BGC (BGC-CM) contained two molecular species of ESA, one with a low molecular weight (Mr 30 000) and another with a high molecular weight (M, 80 000); but only the activity with low molecular weight was detected in CM of BOEC by gel-permeation chromatography. The smaller ESA (embryogenin-1 ) in BGCCM was purified to homogeneity, as a common activity in both CM by a combination of gel-permeation chromatography, ionexchange chromatography, and reverse-phase HPLC. Embryogenin-1 has a molecular weight of 31 100 (reduced) and has been identified as a bovine tissue inhibitor of metalloproteinase-1 by NH,-terminal amino acid sequence analysis, Western blot analysis, anti-proteinase activities against metalloproteinases, and the nucleotide sequence of cDNA isolated from a gtl I cDNA library of the bovine ovary by a polyclonal antibody against embryogenin-1. These data suggest that the tissue inhibitor of metalloproteinase-1 produced by BGC and BOEC is a major ESA for in vitro development of bovine embryos.