Improvements in in vitro spermatogenesis: oxygen concentration, antioxidants, tissue-form design, and space control.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Development of a prototype thermodynamic database for Nd-Fe-B permanent magnets.

For the Nd-Fe-B permanent magnets, a prototype thermodynamic database of the 8-element system (Nd, Fe, B, Al, Co, Cu, Dy, Ga) was constructed based on literature data and assessed parameters in the present work. The magnetic excess Gibbs energy of the Nd2Fe14B compound was reassessed using thoroughly measured heat capacity data. The Dy-Nd binary system was reassessed based on formation energies estimated from ab initio calculations. The constructed database was applied successfully for estimations of phase equilibria during the grain boundary diffusion processes (GBDP) and the reactions in the hydrogenation decomposition desorption recombination (HDDR) processes.

Colonic Histological Criteria Predict Development of Pouchitis after Ileal Pouch: Anal Anastomosis for Patients with Ulcerative Colitis.

BACKGROUND/AIMS: Pouchitis is one of the main complications after ileal pouch-anal anastomosis in patients with ulcerative colitis. The aim of this study was to determine whether the use of colonic histological criteria can predict the development of pouchitis. METHODOLOGY: We retrospectively reviewed 147 patients' clinical data and performed a histological evaluation of the resected total colon using Tanaka's criteria, which comprise the following 6 factors: ulceration (H1), crypt abscesses (H2), degree of mononuclear cell infiltration (MNCI) (H3), segmental distribution of MNCI (H4), eosinophil infiltration (H5), and extent of disease of resected colon (H6). RESULTS: The development of pouchitis and chronic pouchitis within 3 years after restoration of gastrointestinal continuity was recognized in 52 (35.4%) and 26 (17.7%) of the 147 patients, respectively. Using various combinations of each score, the H3 + H4 - H5 scores of patients with pouchitis or chronic pouchitis were significantly higher than those of patients without. A H3 + H4 - H5 score of >0.4 was a statistically significant risk factor for the development of both pouchitis and chronic pouchitis. CONCLUSIONS: The combination of the degree of MNCI, segmental distribution of MNCI, and eosinophil infiltration from histological criteria has utility in predicting the future development of pouchitis, especially chronic pouchitis.

Long-term functional outcome after untethering surgery for a tethered spinal cord in patients with anorectal malformations.

BACKGROUND: Anorectal malformation (ARM) is associated with a tethered spinal cord (TSC). Long-term functional outcome of untethering surgery for TSC in patients with ARM has not been well evaluated. METHODS: Patients aged 7 years and older who underwent repair of ARM and spinal magnetic resonance imaging from January 1995 to December 2008 were reviewed retrospectively. Untethering surgery was performed in all patients who were diagnosed with TSC, regardless of the presence or of neurological symptoms. Clinical symptoms reflecting anorectal, urinary, and lower limb function were compared between patients complicated with TSC (TSC group, n = 17) and those without TSC (non-TSC group, n = 14). RESULTS: The median age at functional evaluation was 11.7 and 12.9 years in the TSC and non-TSC groups, respectively (p = 0.52). Untethering surgery for TSC was performed at a median age of 1.3 years. Preoperative urinary and lower limb dysfunction, except for vesicoureteral reflux in the TSC group in one patient, was improved after surgical detethering. Current anorectal function was comparable between the groups. CONCLUSIONS: Long-term functional outcome in patients with ARM and TSC undergoing untethering surgery is equivalent to that in those without TSC. Prophylactic surgical detethering for patients with ARM and TSC can be a treatment of choice to maximize neurological functional outcome.

Thoracoscopic repair of congenital diaphragmatic hernia with countermeasures against reported complications for safe outcomes comparable to laparotomy.

BACKGROUND: Thoracoscopic repair is the preferred treatment for congenital diaphragmatic hernia (CDH); however, several complications, including visceral injury, hypercapnia, and a high incidence of recurrence, have been reported. The purpose of this study was to evaluate the efficacy of countermeasures against these complications at ensuring safe thoracoscopic repair. METHODS: Between January 2000 and December 2014, 40 patients with Bochdalek-type CDH were treated. Of these, 24 patients met the defined criteria for this study, 8 of whom underwent thoracoscopic repair beginning in January 2010 (TS group) and 16 underwent laparotomy before December 2009 (LT group). Perioperative variables and postoperative complications were compared between the groups. Countermeasures against adverse events in the TS group included an endoscopic surgical spacer to prevent visceral injury, intrapulmonary percussive ventilation to avoid hypercapnia, pausing CO2 insufflation to reduce tension during the repair, and prioritizing patch repair in cases of strong tension at the defect. RESULTS: Primary closure was performed in 4 of 8 cases in the TS and 11 of 16 cases in the LT group. There was no visceral injury or conversion to laparotomy in the TS group. The mean operative duration was significantly longer (212 vs. 115 min, respectively, p = 0.0001), and the mean blood loss was significantly less in the TS than in the LT group (1.0 vs. 10.1 mL, respectively, p = 0.01). The intraoperative minimum arterial pH and maximum pCO2 were similar between the groups. All patients survived, and none experienced recurrence. CONCLUSIONS: Our countermeasures to complications of thoracoscopic repair may contribute to safe outcomes equivalent to those of laparotomy in patients meeting our criteria.

γ-Tocotrienol reduces human airway smooth muscle cell proliferation and migration.

AIMS: Vitamin E is an antioxidant that occurs in 8 different forms (α, ß, γ, and δ tocopherol and tocotrienol). Clinical trials of tocopherol supplementation to assess the impact of antioxidant activity in asthma have yielded equivocal results. Tocotrienol exhibits greater antioxidant activity than tocopherol in several biological phenomena in vivo and in vitro. We tested the effect of tocotrienol on human airway smooth muscle (ASM) cell growth and migration, both of which mediate airway remodeling in asthma. MAIN METHODS: We measured platelet-derived growth factor-BB (PDGF-BB)-induced ASM cell proliferation and migration by colorimetric and Transwell migration assays in the presence and absence of γ-tocotrienol (an isoform of tocotrienol). KEY FINDINGS: PDGF-BB-induced ASM cell proliferation and migration were inhibited by γ-tocotrienol. This effect was associated with inhibition of RhoA activation, but it had no effect on p42/p44 mitogen-activated protein kinase (MAPK) or Akt1 activation. We confirmed that pharmacological inhibition of Rho kinase activity was sufficient to inhibit PDGF-BB-induced ASM cell proliferation and migration. SIGNIFICANCE: γ-Tocotrienol could impart therapeutic benefits for airway remodeling in asthma by inhibiting human ASM cell proliferation and migration.

Infantile Crohn's disease is one of the risk factors for catheter-related bloodstream infection.

BACKGROUND: Central venous catheterization is recognized as a lifeline that is important for chemotherapy or nutritional support in pediatric patients with malignant disease and intestinal failure. This study analyzed the risk of infection with Broviac line use among these patients at a single Japanese center. METHODS: Two hundred and four Broviac lines were inserted in patients in the pediatric ward from January 2003 to October 2011. We analyzed the risk of catheter-related bloodstream infection (CR-BSI) using clinical characteristics including underlying disease, sepsis history, inserted situation, drug use, and laboratory data at the time of Broviac insertion or before CR-BSI. RESULTS: During the study period, data from a total of 15 lines were excluded because of missing blood culture data. In the remaining 189 Broviac lines, 52 lines developed CR-BSI. On univariate analysis, leukemia, infantile Crohn's disease, sepsis history before Broviac insertion, existence of a stoma opening, and immunosuppressant use before CR-BSI were risk factors for CR-BSI. On multivariate logistic regression analysis infantile Crohn's disease, sepsis history before Broviac insertion, and immunosuppressant use before CR-BSI were independently associated with CR-BSI (P = 0.015, P = 0.045, and P = 0.043, respectively). CONCLUSIONS: Infantile Crohn's disease carries a high risk for CR-BSI because of its pathological condition, the therapeutic drugs required, and surgical intervention.

[Three cases of proliferative cystitis causing hydronephrosis].

We report three cases of proliferative cystitis causing hydronephrosis. Three patients presented with a complaint of miction pain, gross hematuria or pollikisuria. Cystoscopic findings revealed papillary sessile tumor from neck to orifice. Transurethral resection of the bladder tumor (TURBT) was performed because the tumor was not responsive to medical treatment. The pathological diagnosis was intestinal type or typical type of cystitis glandularis and no malignant cells were observed. After the operation, although hydronephrosis improved in two cases, the left hydronephrosis did not improve in one case and ureteralileostomy was performed. Five year after the last operation, there is no evidence of recurrence of the tumor. Tumor formation arising from proliferative cystitis is relatively rare. Pathogenesis and management of this rare condition are discussed.

AMP deaminase 3 plays a critical role in remote reperfusion lung injury.

Remote reperfusion lung injury following skeletal muscle ischemia and reperfusion accounts for high morbidity and mortality. AMP deaminase (AMPD), a key enzyme for nucleotide cycle, has been implicated in the regulation of this phenomenon. However, the function of Ampd2 and Ampd3 subtype has not been elucidated in remote reperfusion rodent lung injury. We utilized AMPD3 and AMPD2-deficient mice. The two types of AMPD-deficient mice and wild-type (WT) littermates were subjected to ischemia-reperfusion injury. After 3h bilateral hind-limb ischemia and reperfusion, AMPD3 mRNA, AMPD activity and inosine monophosphate (IMP) increased significantly in WT and AMPD2-deficient mice lungs, while they did not show significant alterations in AMPD3-deficient mice lungs. Genetic inactivation of Ampd3 resulted in markedly accelerated myeloperoxidase (MPO) activity along with exaggerated neutrophils infiltration and hemorrhage in the lungs compared to WT and AMPD2-deficient mice, furthermore, IMP treatment significantly attenuated MPO activity and neutrophils infiltration in WT and the two types of AMPD-deficient mice lungs after 3h reperfusion. These findings demonstrate for the first time in AMP-deficient mice models that AMPD3 plays a critical role in remote reperfusion lung injury via generation of IMP and validate the potential to use IMP into the clinical arena to attenuate remote ischemia-reperfusion lung injury.

Identification of patients with developing ulcerative colitis-associated neoplasia by nitrative DNA damage marker 8-nitroguanin expression in rectal mucosa.

GOAL: To clarify whether the expression of nitrative and oxidative DNA damage markers in the rectal mucosa of patients with ulcerative colitis (UC) could be used to predict UC-associated neoplasia. BACKGROUND: A longer duration of UC can increase the risk of developing UC-associated cancer (UCAC). Effective diagnostic markers are being sought to provide more selective screening and treatment strategies for patients with long-standing UC. STUDY: A total of 141 patients with UC who underwent a proctocolectomy were enrolled in this study. The expression of 8-nitroguanine (8-NG), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and inducible nitric oxide synthase (iNOS) in the rectal mucosa were evaluated using immunohistochemistry (IHC) and assessed relative to the pathogenesis of UC-associated neoplasia. RESULTS: Eighteen patients (12.8%) had UC-associated neoplasia including low-grade or high-grade dysplasia and UCAC. IHC scores of 8-NG in UC-associated neoplasia group was significantly higher than in non-neoplasia group (P<0.0001). In contrast, IHC score of 8-oxodG in non-neoplasia group was significantly decreased compared with UC-associated neoplasia group (P=0.0028). In logistic regression analysis, duration of disease >8 years, high IHC scores of 8-NG, and low 8-oxodG in the rectal mucosa were significantly associated with the development of UC-associated neoplasia (P<0.01). The expression of 8-NG was more frequently observed in patients with UCAC than in patients with sporadic colorectal cancer (P<0.01). CONCLUSION: These results suggest that evaluating the expression levels of 8-NG in the rectal mucosa may be a useful biomarker for detecting patients with UC-associated neoplasia.

Predictive factors for development of chronic pouchitis after ileal pouch-anal anastomosis in ulcerative colitis.

BACKGROUND: No previous studies have examined predictive factors for chronic pouchitis after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) with consideration of changes in pouchitis subtypes during follow-up. This study evaluated the independent predictive factors for chronic pouchitis. METHODS: A total of 244 consecutive patients who underwent IPAA were enrolled. We assessed the possible associations between pouchitis and clinical factors using Cox proportional hazard regression. RESULTS: 231 patients met the inclusion criteria. 66 (28.5%) patients developed pouchitis. In 9 of 44 (20.4%) patients, antibiotic-responsive pouchitis at the first episode changed into chronic pouchitis after the occurrence of a subsequent episode. The median duration from occurrence of antibiotic-responsive pouchitis to alteration into chronic pouchitis was 502 (range 147-1,697) days. Overall pouchitis was finally classified into 35 acute pouchitis and 31 chronic pouchitis cases. Multivariate analysis revealed that a ≥ 7.5-g cumulative steroid dose before colectomy and a ≥ 500-mg monthly steroid dose just before colectomy were significant predictive factors for chronic pouchitis (p = 0.0001 and 0.0095, respectively). CONCLUSION: Patients with UC and a higher cumulative steroid dose before colectomy or higher monthly steroid dose just before colectomy may have a predictive factor for developing chronic pouchitis.

Low NDRG1 mRNA expression predicts a poor prognosis in neuroblastoma patients.

PURPOSE: N-myc downstream regulated gene 1 (NDRG1) markedly reduces metastasis of numerous tumors. However, NDRG1's function in malignant tumors has not been fully determined. Therefore, we investigated the association of NDRG1 expression with clinical outcomes in neuroblastoma (NB) patients. METHODS: We obtained total RNA from residual cancer cells using microdissection from NB patients. Furthermore, we examined the expression of NDRG1 in NB patients using immunohistochemical staining. RESULTS: Of the 48 patients observed, low NDRG1 expression was associated with poor prognostic factors such as primary tumor size and MYCN amplification. Low expression of NDRG1 was associated with a poor prognosis (p = 0.001) and multivariate analysis identified low expression of NDRG1 as an independent risk factor for predicting poor prognosis in NB patients. Furthermore, in the MYCN non-amplification group (n = 33), low expression of NDRG1 was associated with a poor prognosis (p = 0.001). Immunohistochemical analysis showed NDRG1 expression at the plasma membranes of NB cells. NDRG1 expression levels were also correlated with expression of NDRG1 mRNA. CONCLUSION: We confirmed that low NDRG1 expression is a significant and independent prognostic indicator in NB by multivariate analysis. Furthermore, NDRG1 may be a novel prognostic marker in MYCN non-amplification NB patients.

Self-reported assessment of health-related quality of life in children who underwent restorative proctocolectomy with ileal J-pouch anal anastomosis for ulcerative colitis.

PURPOSE: To clarify health-related quality of life (HRQOL) by self-evaluation after restorative proctocolectomy with ileal J-pouch anal anastomosis (IPAA) in children with ulcerative colitis, a questionnaire using the Pediatric Quality of Life Inventory™ 4.0 (PedsQL) was administered. METHODS: The PedsQL was administered to 13 consecutive children (mean age 14.5 years) who underwent IPAA between 2005 and 2010 in our hospital and age-matched healthy controls. The mean duration after IPAA was 2.5 years (range 0.08-6 years) at the time of this study. Healthy children completed the same questionnaire by retrospective imaging during the past 1 month by the PedsQL evaluation policy. RESULTS: Patients' total score and each functioning score after IPAA reached the same levels as those in healthy controls. Soiling, pouchitis occurrence, and bowel movements had no significant relationship to the PedsQL total score and each functioning score. CONCLUSIONS: Interference of physical activity, emotional status, and social life caused by refractory ulcerative colitis (UC) worsens patients' HRQOL. IPAA could resolve these problems in children with UC and result in an HRQOL comparable with that in healthy children.

Culture-space control is effective in promoting haploid cell formation and spermiogenesis in vitro in neonatal mice.

The classical organ culture method, in which tissue is placed at the gas‒liquid interphase, is effective at inducing mouse spermatogenesis. However, due to reginal variations in the supply of oxygen and nutrients within a tissue, the progress of spermatogenesis was observed only in limited areas of a tissue. In addition, haploid cell formation and its differentiation to spermatozoon, i.e. spermiogenesis, were infrequent and inefficient. Here, we show that the polydimethylsiloxane (PDMS)-chip ceiling (PC) method, which ensures a uniform supply of nutrients and oxygen throughout the tissue by pressing it into a thin, flat shape, can provide control over the culture space. We used this method to culture testis tissue from neonatal mice, aged 1 to 4 days, and found that modulating the culture space during the experiment by replacing one chip with another that had a higher ceiling effectively increased tissue growth. This adjustment also induced more efficient spermatogenesis, with the process of spermiogenesis being particularly promoted. Meiotic cells were observed from culture day 14 onward, and haploid cells were confirmed at the end of each experiment. This technique was also shown to be a sensitive assay for testicular toxicity. Culture-space control will be a critical regulation parameter for sophisticated tissue culture experiments.

In vitro spermatogenesis in isolated seminiferous tubules of immature mice.

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

Small Bowel Obstruction Caused by Migration of Fractured Metal Stent in Patients with Malignant Gastric Outlet Obstruction: A Report of Two Cases and Review of the Literature.

Gastroduodenal stenting (GDS) is a less invasive alternative to gastrojejunostomy for the management of malignant gastric outlet obstruction (mGOO). GDS is a minimally invasive treatment with good technical and clinical success, and severe complications that require surgical intervention are rare. Stent fracture is an uncommon complication associated with GDS; however, migration of the fractured distal segment can result in small bowel obstruction. Adverse effects of stent fractures in patients with mGOO have rarely been reported. We herein report two surgical cases of small bowel obstruction caused by the migration of fractured metal stent in patients with mGOO.

Generation of rat offspring using spermatids produced through in vitro spermatogenesis.

An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.

Diagnostic and prognostic impact of serum-soluble UL16-binding protein 2 in lung cancer patients.

UL16-binding protein 2 (ULBP2) is one of the ligands for NKG2D (NKG2DL). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Soluble forms of NKG2DL have been reported in the serum of patients with several types of cancer. The present study investigated the diagnostic and prognostic significance of serum-soluble ULBP2 (sULBP2) in lung cancer patients. We used flow cytometry to evaluate the surface expression of NKG2DL by various lung cancer cells, while sULBP2 was measured using our original ELISA. In addition, the immunological effect of sULBP2 on peripheral blood mononuclear cells (PBMC) was examined by the (51) Cr release assay. We found that ULBP2 was highly expressed and that the sULBP2 level was elevated in supernatants of cultured non-small-cell lung cancer (NSCLC) cells as well as in the serum of NSCLC patients. ULBP2 levels were especially high in squamous cell carcinoma (SQ) patients. Clinical stage IIIB and IV NSCLC patients with a sULBP2 level ≥ 8.7 pg/mL showed significantly shorter survival than patients with sULBP2 <8.7 pg/mL. In multivariate analysis, a sULBP2 level ≥ 8.7 pg/mL (hazard ratio [HR], 2.13; P = 0.038) and clinical stage IV (HR, 2.65; P = 0.019) were independent determinants of a poor outcome. As a possible mechanism, we demonstrated that sULBP2 directly suppresses the cytolytic activity of PBMC. In conclusion, ULBP2 is the most significant NKG2DL for lung cancer, and sULBP2 is useful in the diagnosis of SQ and as a prognostic indicator for patients with advanced NSCLC.