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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose: Epidermal growth factor receptor (EGFR) is commonly overexpressed in lung cancer. Cetuximab is a chimeric mouse-human antibody
targeted against EGFR. Compared with its inhibitory properties, ...its immunologic mechanisms have not been well studied. In
this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab against lung cancer
cell lines.
Experimental Design: We studied the correlation between EGFR expression in lung cancer cell lines and the ADCC activity of cetuximab as well as
the influence of interleukin-2 and chemotherapy on the ADCC activity. EGFR expression was measured by a quantitative flow
cytometric analysis and immunohistochemistry. The ADCC activity was assessed by a 4-h 51 Cr release assay. Peripheral blood mononuclear cells, purified T cells, natural killer (NK) cells, and monocytes from healthy
donors or lung cancer patients were used as effector cells.
Results: Fresh peripheral blood mononuclear cells exhibited cetuximab-mediated ADCC activity against lung cancer cell lines at a low
concentration of cetuximab (0.25 μg/mL). A logarithmic correlation was observed between the number of EGFRs and ADCC activity.
Even low EGFR expression, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity, and
further increases in EGFR expression on the target cells had no further effect on the ADCC activity. In addition, ADCC activity
was enhanced by interleukin-2 mainly through activation of NK cells and was less susceptible to immunosuppression by chemotherapy
than NK activity in lung cancer patients.
Conclusions: These observations suggest the importance of ADCC activity as an immunologic mechanism of cetuximab in biological therapy
for lung cancer patients.
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 Nd
2
Fe
14
B 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.
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.
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 51Cr 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. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02330.x, 2012)
The Gibbs energy functions of the phases in the Al-Cu binary system are taken from the CALPHAD-type thermodynamic assessment (Witusiewicz et al., 2004; Ansara et al., 1998) 1,2, where the effect of ...the monovacancy (Va), divacancy (VaVa) and Va-solute atom pair are taken into account based on the formulation (Abe et al., In press). The divacancy is modeled as an associate, VaVa, in the FCC solid solution. The contributions from the Va-solute pair are included through the ternary excess Gibbs energy term. Using the Gibbs energy functions provided in this data article, the fractions of the monovacancies and divacancies, even in various metastable conditions, can be calculated. Since the Gibbs energy functions and phase descriptions are written in the TDB (Thermodynamic DataBase) format, one can use this file with various thermodynamic software packages, such as OpenCalphad 3 etc.
Abstract Background/Purpose Increased glycolysis is among the biochemical characteristics of cancerous tissue. The glucose transporter isoform 1 ( GLUT1 ) gene encodes a key factor for glucose ...transport into cancerous tissue. However, the expression and functional significance of GLUT1 in neuroblastoma have not been fully characterized. Therefore, we investigated the association of GLUT1 expression with clinical outcomes in patients with neuroblastoma using immunohistochemical staining for GLUT1 in neuroblastoma tissues. We also assessed the efficacy of glycolysis inhibition as an anticancer treatment in neuroblastoma cell lines with altered expression of GLUT1. Methods We obtained total RNA from cancerous tissue by microdissection in 47 patients with neuroblastoma. GLUT1 expression levels were evaluated by quantitative real-time polymerase chain reaction. We analyzed the association of GLUT1 expression levels with clinical outcomes. We also examined changes in GLUT1 expression and proliferative responses in vitro using 4 neuroblastoma cell lines treated with a glycolysis inhibitor, 3-Bromopyruvate acid. Results Elevated GLUT1 expression was associated with poor prognosis. Moreover, elevated GLUT1 expression independently predicted overall survival. Immunohistochemical analysis showed that GLUT1 expression tended to be localized to the centers of neuroblastoma cell nests. Our in vitro studies showed that 3-Bromopyruvate acid significantly suppressed the proliferation of neuroblastoma cells with high GLUT1 gene expression compared with those with low expression. Conclusion Glycolysis inhibitors are a potential therapeutic option for treating aggressive tumors expressing GLUT1.
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