The white rot basidiomycete Gelatoporia subvermispora produces fatty aldehydes that enable fungal manganese peroxidases to degrade recalcitrant lignin structures.

The ability of some white rot basidiomycetes to remove lignin selectively from wood indicates that low molecular weight oxidants have a role in ligninolysis. These oxidants are likely free radicals generated by fungal peroxidases from compounds in the biodegrading wood. Past work supports a role for manganese peroxidases (MnPs) in the production of ligninolytic oxidants from fungal membrane lipids. However, the fatty acid alkylperoxyl radicals initially formed during this process are not reactive enough to attack the major structures in lignin. Here, we evaluate the hypothesis that the peroxidation of fatty aldehydes might provide a source of more reactive acylperoxyl radicals. We found that Gelatoporia subvermispora produced trans-2-nonenal, trans-2-octenal, and n-hexanal (a likely metabolite of trans-2,4-decadienal) during the incipient decay of aspen wood. Fungal fatty aldehydes supported the in vitro oxidation by MnPs of a nonphenolic lignin model dimer, and also of the monomeric model veratryl alcohol. Experiments with the latter compound showed that the reactions were partially inhibited by oxalate, the chelator that white rot fungi employ to detach Mn3+ from the MnP active site, but nevertheless proceeded at its physiological concentration of 1 mM. The addition of catalase was inhibitory, which suggests that the standard MnP catalytic cycle is involved in the oxidation of aldehydes. MnP oxidized trans-2-nonenal quantitatively to trans-2-nonenoic acid with the consumption of one O2 equivalent. The data suggest that when Mn3+ remains associated with MnP, it can oxidize aldehydes to their acyl radicals, and the latter subsequently add O2 to become ligninolytic acylperoxyl radicals.IMPORTANCEThe biodegradation of lignin by white rot fungi is essential for the natural recycling of plant biomass and has useful applications in lignocellulose bioprocessing. Although fungal peroxidases have a key role in ligninolysis, past work indicates that biodegradation is initiated by smaller, as yet unidentified oxidants that can infiltrate the substrate. Here, we present evidence that the peroxidase-catalyzed oxidation of naturally occurring fungal aldehydes may provide a source of ligninolytic free radical oxidants.

Gender differences on preoperative psychologic factors affecting acute postoperative pain in patients with lumbar spinal disorders.

BACKGROUND: Psychosexual factors are one of the preoperative factors influencing acute postoperative pain. Because of gender differences in pain, the preoperative factors that influence acute postoperative pain may also differ between males and females. However, there have been no reports on such factors in patients with spinal disorders that focused on gender differences. Therefore, the purpose of this study was to examine the preoperative factors that influence acute postoperative pain, focusing on gender differences. METHODS: The subjects were 75 males and 60 females admitted for surgery for lumbar spinal disorders. Preoperatively, the following were assessed: low back pain using the Numeric Rating Scale (NRS); anxiety and depression using the Japanese version of the Hospital Anxiety and Depression Scale (HADS); catastrophic thinking using the Pain Catastrophizing Scale (PCS); psychiatric problems using the Brief Scale for Psychiatric Problems in Orthopaedic Patients (BS-POP); and neurological assessments. Acute postoperative pain was also assessed using the NRS within 48 h, postoperatively. Based on these data, we analyzed gender differences in preoperative factors affecting acute postoperative pain in patients with lumbar spinal disorders. RESULTS: Postoperative NRS and preoperative PCS scores were higher in females compared to males. In the males, the coefficient of determination of the multiple regression equation was 0.088, and PCS (ß = 0.323, p = 0.015) was extracted as a significant factor. In the females, the coefficient of determination of the multiple regression equation was 0.075, and BS-POP (ß = 0.300, p = 0.019) was extracted as a significant factor. CONCLUSION: Preoperative factors influencing acute postoperative pain for patients with lumbar spinal disorders vary by gender. It was suggested that males should be screened using PCS. In females, on the other hand, PCS alone is not sufficient for evaluation. It was suggested that evaluation using BS-POP should be considered in addition to PCS.

A Rare Pancreatic Tumor with Adenomatoid Tumor-like Findings.

A 74-year-old woman was referred to our hospital for the evaluation of slightly elevated tumor marker levels. Computed tomography revealed a well-demarcated tumor, approximately 15 mm in diameter, in the pancreatic tail. Endoscopic ultrasound-guided fine-needle aspiration findings suggested poorly differentiated cancer. The tumor was surgically resected, but postoperative pathologic confirmation was not possible. After one year without treatment and no recurrence, an evaluation by a specialized facility was requested for a definitive diagnosis. Adenomatoid tumor was deemed most likely based on the histopathology and immunostaining findings; however, a definitive diagnosis was difficult because of atypical findings. The patient was recurrence-free for 36 months at the last follow-up.

Usefulness of combined NGS and QF-PCR analysis for product of conception karyotyping.

Purpose: Since chromosomal abnormalities can be detected in more than half of miscarriages, cytogenetic testing of the product of conception (POC) can provide important information when preparing for a subsequent pregnancy. Conventional karyotyping is the common diagnostic method for a POC but can be problematic due to the need for cell culture. Methods: We here conducted shallow whole-genome sequencing (sWGS) using next-generation sequencing (NGS) for alternative POC cytogenomic analysis. Since female euploidy samples can include 69,XXX triploidy, additional QF-PCR was performed in these cases. Results: We here analyzed POC samples from miscarriages in 300 assisted reproductive technology (ART) pregnancies and detected chromosomal abnormalities in 201 instances (67.0%). Autosomal aneuploidy (151 cases, 50.3%) was the most frequent abnormality, consistent with prior conventional karyotyping data. Mosaic aneuploidy was detected in seven cases (2.0%). Notably, the frequency of triploidy was 2.3%, 10-fold lower than the reported frequency in non-ART pregnancies. Structural rearrangements were identified in nine samples (3%), but there was no case of segmental mosaicism. Conclusions: These data suggest that NGS-based sWGS, with the aid of QF-PCR, is a viable alternative karyotyping procedure that does not require cell culture. This method could also assist with genetic counseling for couples who undergoes embryo selection based on PGT-A data.

Antimicrobial Drug-resistance Profile of Vibrio Parahaemolyticus isolated from Japanese Horse Mackerel (Trachurus Japonicus).

This study aimed at investigating antimicrobial resistance (AMR) profile of Vibrio parahaemolyticus (V. parahaemolyticus). The bacteria were isolated from wild-caught and farmed Japanese horse mackerel (Trachurus japonicus), and examined for the antimicrobial drug resistance. Furthermore, the serotype, and the genes of thermostable direct hemolysin (tdh) and cholera toxin transcriptional activator (toxR) of the isolates were investigated by using a serotype testing kit and PCR method. Eighty-eight and 126 V. parahaemolyticus strains were isolated from wild-caught and farmed Japanese horse mackerel, respectively. Ten and 18 distinct serotypes were detected from wild-caught and farmed Japanese horse mackerel. All strains were negative for tdh genes but positive for toxR genes. Resistances to ampicillin (ABP) and to both ABP and fosfomycin (FOM) were observed in 54 and 23 strains from the wild-caught fish, while those resistant strains from farm fish were 112 and 7 strains. Multidrug-resistance to three or four drugs including ABP was observed in one or two strains from the wild-caught fish. These results strongly suggest that the environmental exposure of antimicrobial drugs results in the spread of resistant genes in Japanese horse mackerel. This study highlights the need for monitoring the spread of resistance genes to the human intestinal flora as well as to other bacteria in the environment.

Clenbuterol accelerates recovery after immobilization-induced atrophy of rat hindlimb muscle.

Using immunohistochemistry, we investigated whether daily administration of clenbuterol (CLE; 1 mg/kg body weight per day) accelerates recovery after casted immobilization(IMM)-induced atrophy of fast-twitch plantaris and slow-twitch soleus muscles. Adult male Sprague-Dawley rats were assigned to the control (CON), casted immobilization (IMM), casted immobilization following recovery control (RCON), and casted immobilization following recovery with CLE administration (RCLE) groups. Casted immobilization and recovery periods were 9 and 14days, respectively. Rats of the CON group were subjected to the experiment simultaneously with the IMM group. Nine days of immobilization induced muscle fiber atrophy, which was greater in the soleus muscle than in the plantaris muscle. After the 2-week recovery period, the cross-sectional areas of each fiber type in both muscles were higher in the RCON group than in the IMM group. The cross-sectional areas of each fiber type in both muscles in the RCLE group were larger than those in the RCON group. The myonuclear number of each fiber type of the plantaris muscle in the RCON and RCLE groups was higher than that in the CON group. In contrast, the myonuclear number per fiber of the soleus muscle was not affected by hindlimb immobilization, reloading, and clenbuterol administration regardless of muscle fiber type. These results suggest that CLE accelerates the recovery of atrophied plantaris and soleus muscles fibers and that their mechanisms of responses to CLE in both muscles may be different during recovery period after muscle atrophy.

Prognostic significance of neutrophil-lymphocyte ratio in resectable pancreatic neuroendocrine tumors with special reference to tumor-associated macrophages.

BACKGROUND: Recent studies have shown that the systemic inflammatory response induced by cancer leads to cancer progression. Neutrophil-to-lymphocyte ratio (NLR) is the most reliable marker to detect systemic inflammation. In this study, we investigated the significance of NLR in patients with well-differentiated pancreatic neuroendocrine tumors (PanNETs) according to the World Health Organization 2017 classification. METHODS: We retrospectively collected data for patients with PanNET who underwent pancreatic resection with curative intent between January 2008 and December 2017 at six institutions. Clinicopathological factors, recurrence, and immunohistochemical staining of tumor-associated macrophages (TAMs) were analyzed in a total of 55 patients in this study. RESULTS: High NLR (>3.41) in patients was significantly associated with higher white blood cell count, higher Ki-67 index, higher mitotic count, higher grade, higher incidence of lymph node metastasis, higher incidence of lymphatic and neural invasion, massive blood loss, and a large number of CD163-expressing TAMs. Recurrence-free survival of patients with high NLR was significantly poorer than that of patients with low NLR. Multivariate analysis identified high NLR, NET Grade 2 (G2) or Grade 3 (G3), and synchronous hepatic resection as independent risk factors for recurrence after curative resection. CONCLUSIONS: NLR is a promising predictor of recurrence after pancreatectomy that needs to be further investigated and that accumulation of TAMs in the tumor could be one of the causes of NLR elevation.

Effect of a lower limb strength training programme on physical activity during the snowy season among community-dwelling elderly individuals.

Background: Promoting continued physical activity during winter in snowy-cold regions would be of benefit as a health promotion strategy among elderly individuals. Aim: To evaluate the effect of a lower limb strength training programme for elderly individuals on the daily step counts during the snowy season. Subjects and methods: Fourteen community-dwelling elderly individuals were recruited from a snowy-cold region of northern Japan. Methods: Daily step counts were measured during the snowy season in 2018 (with no exercise intervention) and 2019 (after the exercise intervention). Physical function was measured before and after the intervention, including body mass index, 30-s chair test, grip strength, normal gait speed, short-test battery for locomotive syndrome, Kihon checklist and 25-question geriatric locomotive function scale. Results: The study group included 11 women and three men (78.6 ± 5.2 years old). The exercise intervention improved physical function, with a significant increase in step count among individuals with lower baseline strength. Improved lower limb strength positively correlated with an increase in step count. Conclusion: Lower limb muscle strengthening is an effective intervention to improve physical activity during the snowy season among community-dwelling elderly.

Significance of Lymph Node Metastasis in Resectable Well-differentiated Pancreatic Neuroendocrine Tumor.

OBJECTIVES: Understanding the effect of lymph node metastasis (LNM) on prognosis in pancreatic neuroendocrine neoplasm is helpful for surgery and follow-up. In this study, we investigated the significance of LNM in well-differentiated pancreatic neuroendocrine tumors (PanNETs) according to the World Health Organization 2017 classification. METHODS: We retrospectively collected data for 95 consecutive patients with PanNET who underwent pancreatic resection with curative intent between January 2008 and December 2017 at 6 institutions. The clinicopathological factors were compared in patients with and without LNM, and prognostic factors were analyzed. RESULTS: Lymph node metastasis was significantly associated with malignant potential of PanNET, such as larger tumor size, higher Ki-67 index, higher tumor grade, and higher incidence of lymphatic, vessel, and neural invasion. Lymph node metastasis was also associated with disease-free but not overall survival. Multivariate analysis identified NET grade 2 (G2) and G3 as independent risk factors for recurrence after curative resection. CONCLUSIONS: World Health Organization 2017 classification was the most independent prognostic factor in patients with resectable well-differentiated PanNETs. Patients with G2 and higher-grade tumors require lymph node dissection to improve prognosis.

Impact of season on the association between muscle strength/volume and physical activity among community-dwelling elderly people living in snowy-cold regions.

BACKGROUND: Although the benefits of physical activity are well-known, levels of physical inactivity are increasing in many countries. Physical activity, particularly for preventive care of the elderly, must be encouraged. The level of physical activity undertaken by people is influenced by season; however, little is known about seasonal fluctuations of physical activity and its relation to muscle strength/mass. Consequently, we clarified the association between physical activity levels and muscle strength/skeletal muscle mass during non-snowy and snowy seasons in northern Japan. METHODS: Participants were community-dwelling elderly people aged 65 years or older living in Tobetsu, northern Japan. A 30-s chair-stand test (CS-30) and body composition measurements using bioelectrical impedance analysis were conducted prior to physical activity measurement using a three-dimensional acceleration sensor in both non-snowy and snowy seasons. Daily steps for the non-snowy and snowy seasons were compared using Welch's t test. The association between the CS-30/skeletal muscle index and daily steps in both seasons was estimated by fitting multiple linear regression models, with age and sex as covariates. RESULTS: Average daily step counts were significantly lower during the snowy season, compared to the non-snowy season (P < .01). The CS-30 in the snowy season alone was significantly associated with daily step counts. Multiple linear regression analyses results revealed that, for the same muscle strength in both seasons, the daily step counts during the snowy season were fewer than those during the non-snowy season. CONCLUSIONS: The muscle strength required to perform adequate physical activity depended on season. This study obtained basic knowledge to ensure health promotion for elderly people living in snowy-cold regions.

A synthetic polymeric biolubricant imparts chondroprotection in a rat meniscal tear model.

Intra-articular injection of hyaluronic acid (HA) is used to treat osteoarthritis (OA) as a viscosupplement, yet it only provides short-term benefit because HA is cleaved by hyaluronidase and cleared out of the joint after several days. Therefore, we developed a new polymer biolubricant based on poly-oxanorbornane carboxylate to enhance joint lubrication for a prolonged time. Rheological and biotribological studies of the biolubricant reveal viscoelastic properties and coefficient of friction equivalent and superior to that of healthy synovial fluid, respectively. Furthermore, in an ex vivo bovine cartilage plug model, the biolubricant exhibits superior long-term reduction of friction and wear prevention compared to saline and healthy synovial fluid. ISO 10993 biocompatibility tests demonstrate that the biolubricant polymer is non-toxic. In an in vivo rat medial meniscal tear OA model, where the performance of the leading HA viscosupplement (Synvisc-one®) is comparable to the saline control, treatment with the biolubricant affords significant chondroprotection compared to the saline control.

Adult intussusceptions induced by a terminal ileum diverticulum: a case report.

We herein report a case of adult intussusceptions induced by a terminal ileum diverticulum. Histological examination confirmed a terminal ileum diverticulum full of feces, and it was considered an infiltrated region. The clinical characteristics of previously reported adult intussusceptions are also discussed, including jejunoileal diverticulum and surgical management.

Risk factors for an additional port in single-incision laparoscopic cholecystectomy in patients with cholecystitis.

Although single-incision laparoscopic cholecystectomy is now widely performed in patients with cholecystitis, some cases require an additional port to complete the procedure. In this study, we focused on risk factor of additional port in this surgery. We performed single-incision cholecystectomy in 75 patients with acute cholecystitis or after cholecystitis between 2010 and 2014 at Gunma University Hospital. Surgical indications followed the TG13 guidelines. Our standard procedure for single-incision cholecystectomy routinely uses two needlescopic devices. We used logistic regression analysis to identify the risk factors associated with use of an additional full-size port (5 or 10 mm). Surgical outcome was acceptable without biliary injury. Nine patients (12.0%) required an additional port, and one patient (1.3%) required conversion to open cholecystectomy because of severe adhesions around the cystic duct and common bile duct. In multivariate analysis, high C-reactive protein (CRP) values (>7.0 mg/dl) during cholecystitis attacks were significantly correlated with the need for an additional port (P = 0.009), with a sensitivity of 55.6%, specificity of 98.5%, and accuracy of 93.3%. This study indicated that the severe inflammation indicated by high CRP values during cholecystitis attacks predicts the need for an additional port. J. Med. Invest. 64: 245-249, August, 2017.

Enhanced karyopherin-α2 expression is associated with carcinogenesis in patients with intraductal papillary mucinous neoplasms.

BACKGROUND/OBJECTIVES: Intraductal papillary mucinous neoplasms (IPMN) can become malignant. Karyopherin-α2 (KPNA2) plays a central role in nucleocytoplasmic transport and is associated with various types of cancer. The current study examined pancreatic KPNA2 expression in cancer patients and evaluated its association with clinicopathological factors, cancer cell proliferation. METHODS: KPNA2 expression was investigated by immunohistochemistry in 40 surgically resected IPMN samples and its association with clinicopathological factors and Ki-67 expression were examined. RESULTS: Eighteen IPMN samples (45% of patients) showed positive KPNA2 expression. KPNA2 expression levels in IPMN tissue with invasive carcinoma were significantly higher than those in adjacent normal tissues and in IPMN tissue with low-to high-grade dysplasia. KPNA2 expression correlated with pathological malignancy and Ki-67 labeling index and KPNA2 and Ki-67 expression was co-localized in nuclei. E2F were co-localized with KPNA2 in the IPMN tissues with high expression of KPNA2. KPNA2 expression was enhanced in the invasion front and in proliferating Ki-67-positive cells. In addition, KPNA2 expression in IPMN tissues was associated with older age, dilation of main pancreatic duct diameter, the presence of nodules, and histological type. CONCLUSION: KPNA2 expression is associated with carcinogenesis of IPMN through the adenoma-carcinoma sequence.

Poor prognosis in cholangiocarcinoma patients with low FBXW7 expression is improved by chemotherapy.

The tumor suppressor FBXW7 has been demonstrated to degrade several oncoproteins, including c-Myc. Although low FBXW7 expression levels are suggested to be a poor prognostic factor in a number of types of solid tumor, the role of FBXW7 in chemosensitivity is controversial. The purpose of the present study was to determine whether FBXW7 expression may be used as a marker for poor prognosis and chemosensitivity in patients with cholangiocarcinoma (CC). FBXW7 expression was investigated by immunohistochemistry in 100 surgically resected CC samples, and the association between FBXW7 expression, clinicopathological factors and prognosis was evaluated. Nuclear FBXW7 expression tended to be lower compared with normal tissues. A total of 54 patients exhibited high expression levels of FBXW7, and 46 patients exhibited low expression levels. Patients with low FBXW7 expression possessed significantly larger tumors (P=0.049), enhanced expression of c-Myc and Ki-67 and significantly poorer prognoses compared with those with high FBXW7 expression (P=0.016). Multivariate analysis revealed that low FBXW7 expression was an independent negative prognostic factor in CC (P=0.043). In patients with high FBXW7 expression levels, the cancer-specific survival times were not significantly different between patients with or without chemotherapy. However, in patients with low FBXW7 expression levels, the cancer-specific survival times were significantly longer in subjects who underwent chemotherapy compared with those who did not (P=0.001). These data suggest that FBXW7 status in CC is a useful predictor of poor prognosis and cancer progression. Additionally, FBXW7 may be a surrogate marker to predict the efficacy of chemotherapy in CC.

Stathmin 1 promotes the proliferation and malignant transformation of pancreatic intraductal papillary mucinous neoplasms.

Pancreatic intraductal papillary mucinous neoplasms (IPMNs) are a type of pancreatic tumor, which have been identified following improvements in diagnostic imaging. However, the malignant transformation of IPMN has been difficult to diagnose clinically. To date, the mechanisms driving the progression of IPMN to cancer remain to be fully elucidated. The present study focused on Stathmin 1 (STMN1), a protein that is associated with the development of various types of cancer. The expression of STMN1 was examined immunohistochemically in tissues from cases of IPMN. The correlation between the STMN1 staining and clinical pathological factors was evaluated, and the expression of STMN1, p27 and S-phase kinase-associated protein 2 (SKP2) were compared. High expression levels of STMN1 were significantly correlated with regions of malignancy, and was associated with high expression of SKP2, low expression of nuclear p27 and a high Ki-67 index. High expression levels of STMN1 and SKP2 were significantly correlated with the transformation of IPMN to carcinoma. In addition, within the regions of carcinoma, the expression of STMN1 was weak in regions of adenoma and high in the cancerous regions. It was concluded that the high expression of STMN1 contributed to tumor proliferation and malignant transformation in the patients with IPMN. These results suggested that characterization of the expression of STMN1 may be a promising approach for predicting malignant transformation of pancreatic intraductal papillary mucinous adenoma.

Overexpression of karyopherin-α2 in cholangiocarcinoma correlates with poor prognosis and gemcitabine sensitivity via nuclear translocation of DNA repair proteins.

Cholangiocarcinoma is a highly malignant tumor, and the development of new therapeutic strategies is critical. Karyopherin-α2 (KPNA2) functions as an adaptor that mediates nucleocytoplasmic transport. Specifically, KPNA2 transports one of the important DNA repair machineries, the MRE11-RAD50-NBS1 (MRN) complex, to the nucleus. In this study, we clarified the significance of KPNA2 in cholangiocarcinoma. KPNA2 expression evaluated by immunohistochemical analysis was common in malignant tissue but rare in adjacent noncancerous tissues. KPNA2 overexpression was significantly correlated with poor prognosis and was an independent prognostic factor after surgery. In patients with cholangiocarcinoma who received gemcitabine after surgery, KPNA2 overexpression tended to be a prognostic indicator of poor overall survival. In KPNA2-depleted cholangiocarcinoma cells, proliferation was significantly decreased and gemcitabine sensitivity was enhanced in vitro and in vivo. Expression of KPNA2 and the MRN complex displayed colocalization in the nucleus. In addition, nuclear localization of the MRN complex was regulated by KPNA2 in vitro. These results suggest that KPNA2 expression may be a useful prognostic and predictive marker of gemcitabine sensitivity and survival. The regulation of KPNA2 expression may be a new therapeutic strategy for cholangiocarcinoma.

Bone turnover markers and the factors associated with atypical femur fractures among Japanese patients.

Many previous reports have indicated that atypical femur fractures (AFFs) are associated with the administration of bisphosphonates (BPs). A number of risk factors and hypotheses regarding the pathogenesis of AFFs have been reported to date. The purpose of the present study was to identify the factors associated with AFFs in Japanese individuals and to elucidate the association between bone metabolism and AFFs by evaluating bone turnover markers (BTMs). We prospectively reviewed all patients with femur fractures and identified the patients with AFFs and typical femur fractures (TFFs). We collected the demographic and clinical data that were relevant to the present study, namely age, gender, affected side, affected site, concomitant medical history, and comorbid conditions, and measured the levels of BTMs within 24h after trauma. Welch's test and Fisher's exact probability test were used for the statistical analyses. A total of 338 patients, including 10 patients with AFFs and 328 patients with TFFs, were analyzed under the inclusion criteria. The use of BPs (p<0.001) and collagen disease and chronic granulomatous disease (CD/CGD) (p=0.025) were more frequently observed in patients with AFFs than in patients with TFFs, while the levels of BTMs, including N-terminal propeptides of type 1 procollagen (P1NP), isoform 5b of tartrate-resistant acid phosphatase (TRACP-5b) and undercarboxylated osteocalcin (ucOC) were significantly lower in patients with AFFs than in patients with TFFs. Furthermore, the level of TRACP-5b was found to be significantly lower in patients with atypical subtrochanteric fractures than in atypical diaphyseal fractures (p=0.025). Moreover, the levels of P1NP (p=0.016) and TRACP-5b (p=0.015) were found to be significantly lower in patients with AFFs than in patients with TFFs in a subgroup analysis of BPs users. The use of BPs was considered to be a factor associated with AFFs. Our comparison of the BTMs in patients with AFFs and TFFs indicated that the severe suppression of bone turnover was associated with the pathogenesis of AFFs. The extent of the influence of suppressed turnover on the pathogenesis of AFFs may differ depending on the fracture site.

Overexpression of natural killer group 2 member D ligands predicts favorable prognosis in cholangiocarcinoma.

The natural killer group 2 member D (NKG2D) receptor and its ligands are important mediators of immune responses to tumors. NKG2D ligands are overexpressed in several malignant tumor types; however, the prognostic value of these ligands is unclear. Here, we aimed to elucidate the role of NKG2D ligands in extrahepatic cholangiocarcinoma (EHCC). We therefore investigated the expression of the NKG2D receptor and its ligands MHC class I chain-related proteins A and B (MICA/B), unique long 16 binding protein (ULBP) 1, and ULBP2/5/6 in resected specimens from 82 patients with EHCC. All NKG2D ligands were highly expressed in EHCC. High expression of MICA/B or ULBP2/5/6 correlated with overall and disease-free survival. In contrast, high expression of ULBP1 was significantly associated with improved overall survival, but not disease-free survival. Concurrent high expression of multiple NKG2D ligands revealed significantly better overall and disease-free survival than that observed with the overexpression of any one NKG2D ligand. Co-expression of multiple NKG2D ligands was an independent prognostic indicator of improved survival. Furthermore, co-overexpression of multiple NKG2D ligands was significantly correlated with high expression of the NKG2D receptor. Inhibiting interactions between multiple NKG2D ligands and the NKG2D receptor might be a promising approach for controlling cancer progression and improving patient prognosis in EHCC.