Rapamycin prevents cyclophosphamide-induced ovarian follicular loss and potentially inhibits tumour proliferation in a breast cancer xenograft mouse model.

STUDY QUESTION: To what extent and via what mechanism does the concomitant administration of rapamycin (a follicle activation pathway inhibitor and antitumour agent) and cyclophosphamide (a highly toxic ovarian anticancer agent) prevent cyclophosphamide-induced ovarian reserve loss and inhibit tumour proliferation in a breast cancer xenograft mouse model? SUMMARY ANSWER: Daily concomitant administration of rapamycin and a cyclic regimen of cyclophosphamide, which has sufficient antitumour effects as a single agent, suppressed cyclophosphamide-induced primordial follicle loss by inhibiting primordial follicle activation in a breast cancer xenograft mouse model, suggesting the potential of an additive inhibitory effect against tumour proliferation. WHAT IS KNOWN ALREADY: Cyclophosphamide stimulates primordial follicles by activating the mammalian target of the rapamycin (mTOR) pathway, resulting in the accumulation of primary follicles, most of which undergo apoptosis. Rapamycin, an mTOR inhibitor, regulates primordial follicle activation and exhibits potential inhibitory effects against breast cancer cell proliferation. STUDY DESIGN, SIZE, DURATION: To assess ovarian follicular apoptosis, 3 weeks after administering breast cancer cells, 8-week-old mice were randomized into three treatment groups: control, cyclophosphamide, and cyclophosphamide + rapamycin (Cy + Rap) (n = 5 or 6 mice/group). Mice were treated with rapamycin or vehicle control for 1 week, followed by a single dose of cyclophosphamide or vehicle control. Subsequently, the ovaries were resected 24 h after cyclophosphamide administration (short-term treatment groups). To evaluate follicle abundance and the mTOR pathway in ovaries, as well as the antitumour effects and impact on the mTOR pathway in tumours, 8-week-old xenograft breast cancer transplanted mice were randomized into three treatment groups: vehicle control, Cy, and Cy + Rap (n = 6 or 7 mice/group). Rapamycin (5 mg/kg) or the vehicle was administered daily for 29 days. Cyclophosphamide (120 mg/kg) or the vehicle was administered thrice weekly (long-term treatment groups). The tumour diameter was measured weekly. Seven days after the last cyclophosphamide treatment, the ovaries were harvested, fixed, and sectioned (for follicle counting) or frozen (for further analysis). Similarly, the tumours were resected and fixed or frozen. PARTICIPANTS/MATERIALS, SETTING, METHODS: Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed to examine ovarian follicular apoptosis in the short-term treatment groups. All subsequent experiments were conducted in the long-term treatment groups. Tumour growth was evaluated using the tumour volume index. The tumour volume index indicates the relative volume, compared to the volume 3 weeks after tumour cell injection (at treatment initiation) set to 100%. Tumour cell proliferation was evaluated by Ki-67 immunostaining. Activation of the mTOR pathway in tumours was assessed using the protein extracts from tumours and analysed by western blotting. Haematoxylin and eosin staining of ovaries was used to perform differential follicle counts for primordial, primary, secondary, antral, and atretic follicles. Activation of the mTOR pathway in ovaries was assessed using protein extracts from whole ovaries and analysed by western blotting. Localization of mTOR pathway activation within ovaries was assessed by performing anti-phospho-S6 kinase (downstream of mTOR pathway) immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Ovaries of the short-term treatment groups were resected 24 h after cyclophosphamide administration and subjected to TUNEL staining of apoptotic cells. No TUNEL-positive primordial follicles were detected in the control, Cy, and Cy + Rap groups. Conversely, many granulosa cells of growing follicles were TUNEL positive in the Cy group but negative in the control and Cy + Rap groups. All subsequent experimental results were obtained from the long-term treatment groups. The tumour volume index stabilized at a mean of 160-200% in the Cy group and 130% in the Cy + Rap group throughout the treatment period. In contrast, tumours in the vehicle control group grew continuously with a mean tumour volume index of 600%, significantly greater than that of the two treatment groups. Based on the western blot analysis of tumours, the mTOR pathway was activated in the vehicle control group and downregulated in the Cy + Rap group when compared with the control and Cy groups. Ki-67 immunostaining of tumours showed significant inhibition of cell proliferation in the Cy + Rap group when compared with that in the control and Cy groups. The ovarian follicle count revealed that the Cy group had significantly fewer primordial follicles (P < 0.001) than the control group, whereas the Cy + Rap group had significantly higher number of primordial follicles (P < 0.001, 2.5 times) than the Cy group. The ratio of primary to primordial follicles was twice as high in the Cy group than in the control group; however, no significant difference was observed between the control group and the Cy + Rap group. Western blot analysis of ovaries revealed that the mTOR pathway was activated by cyclophosphamide and inhibited by rapamycin. The phospho-S6 kinase (pS6K)-positive primordial follicle rate was 2.7 times higher in the Cy group than in the control group. However, this effect was suppressed to a level similar to the control group in the Cy + Rap group. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The combinatorial treatment of breast cancer tumours with rapamycin and cyclophosphamide elicited inhibitory effects on cell proliferative potential compared to cyclophosphamide monotherapy. However, no statistically significant additive effect was observed on tumour volume. Thus, the beneficial antitumour effect afforded by rapamycin administration on breast cancer could not be definitively proven. Although rapamycin has ovarian-protective effects, it does not fully counteract the ovarian toxicity of cyclophosphamide. Nevertheless, rapamycin is advantageous as an ovarian protective agent as it can be used in combination with other ovarian protective agents, such as hormonal therapy. Hence, in combination with other agents, mTOR inhibitors may be sufficiently ovario-protective against high-dose and cyclic cyclophosphamide regimens. WIDER IMPLICATIONS OF THE FINDINGS: Compared with a cyclic cyclophosphamide regimen that replicates human clinical practice under breast cancer-bearing conditions, the combination with rapamycin mitigates the ovarian follicle loss of cyclophosphamide without interfering with the anticipated antitumour effects. Hence, rapamycin may represent a new non-invasive treatment option for cyclophosphamide-induced ovarian dysfunction in breast cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This work was not financially supported. The authors declare that they have no conflict of interest.

Locally advanced endometrial cancer with multiple immune-related adverse events coinciding with the complete response to radiotherapy after immune checkpoint inhibitor therapy: A case report.

We report a case of a 70-year-old female patient with locally advanced endometrial cancer with primary empty sella who developed multiple immune-related adverse events (irAEs), including hypopituitarism coinciding with the complete response to radiotherapy after receiving immune checkpoint inhibitors. A computed tomography scan acquired after a traffic accident led to the discovery of endometrial cancer that had invaded the vulva and primary empty sella. Following adriamycin and cisplatin, pembrolizumab was administered for three cycles. No irAEs were observed during treatment, but the tumor was progressive. The patient underwent radiotherapy for the residual tumor. Four months after the last dose of pembrolizumab, hypopituitarism caused secondary adrenal insufficiency, primary hypothyroidism, and pseudogout at the end of radiotherapy. The tumor later achieved a complete response. In conclusion, radiotherapy after immune checkpoint inhibitor (ICI) therapy is expected to have an antitumor effect by stimulating tumor-specific immunity. However, proper management of irAEs is necessary. Patients with primary empty sella may be prone to pituitary insufficiency induced by ICIs.

Evaluation of lens opacity due to inhibition of cholesterol biosynthesis using rat lens explant cultures.

Drug-induced lens opacity has the potential to cause blindness and is of concern in drug development. Inhibition of cholesterol biosynthesis is one of the causes of lens opacity. Lens opacity is only observed after chronic administration in in vivo nonclinical studies in drug development. Thus, to save resources (e.g., time and cost) and to reduce burden on animals, it is required to develop in vitro evaluation systems that can predict and avoid the risk of lens opacity earlier and easier. In this study, we investigated whether rat lens explant cultures could be useful for the evaluation of drug-induced lens opacity via inhibition of cholesterol biosynthesis. Nineteen drugs, including statins, allylamine, thiocarbamate, azole, and morpholine, which inhibit cholesterol biosynthesis, as well as a negative control (acetaminophen, rosiglitazone and troglitazone), were used. Rat lens explants were treated with drugs for 13 days at concentrations close to IC50 values or higher against cholesterol biosynthesis, and lens opacity (severity and region) was evaluated. In most cases, region-specific lens opacity limited in the equator to posterior pole, as observed in vivo was observed at IC50 values or higher concentrations. The severity of opacity was likely to be related to the inhibitory potency toward cholesterol biosynthesis, concentration of drugs distributed in the lens, or time of exposure. Furthermore, GSH levels were also involved in the deterioration of lens opacity. In conclusion, we demonstrated that rat lens explant cultures can be useful to assess the potential drug-induced lens opacity associated with inhibition of cholesterol biosynthesis and to elucidate the mechanisms of lens opacity.

Primary malignant melanoma of uterine cervix treated with pembrolizumab as adjuvant immunotherapy.

This is the case report of primary malignant melanoma (MM) of uterine cervix treated by immune checkpoint inhibitor: the Pembrolizumab. Despite the merge of the novel drugs that has been strikingly improving prognosis of MM, we still struggle treatment of MM of uterine cervix that has aggressive characteristics with unknown etiology. We present our case to contribute its rarity of the disease case report, the primary MM of the uterine cervix that had poor response to pembrolizumab and had OS of 6 months. The treatment ineffectiveness is mainly considered for mucosal MM of low tumor mutation burden and its unusual type of pathology. Accumulation of retrospective studies exclusively on cervical melanoma needs to be proceeded to investigate on characteristics between poor and long survival to establish standardized treatment.

[A Case of Lymphoepithelioma-Like Carcinoma of the Ureter].

Lymphoepithelioma-like carcinoma (LELC) of the ureter is very rare and only 14 previous cases have been reported. Here, we report a case of LELC of the ureter. A 76-year-old woman was admitted to our hospital complaining of gross hematuria. Left ureteral cancer was suspected by the imaging examination, and laparoscopic left total nephroureterectomy was performed. Histopathological examination showed pure type of LELC in the ureter. She is alive without disease recurrence at fifteen months after surgery.

Antioxidant vitamins and lysophospholipids are critical for inducing mouse spermatogenesis under organ culture conditions.

In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.

In vitro spermatogenesis in two-dimensionally spread mouse testis tissues.

PURPOSE: Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. METHODS: Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr-Gfp, which reflects the progression of spermatogenesis. RESULTS: Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. CONCLUSIONS: The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

Oral administration of EP4-selective agonist KAG-308 suppresses mouse knee osteoarthritis development through reduction of chondrocyte hypertrophy and TNF secretion.

Osteoarthritis (OA) is one of the world's most common degenerative diseases, but there is no disease-modifying treatment available. Previous studies have shown that prostaglandin E2 (PGE2) and PGE2 receptor 4 (EP4) are involved in OA pathogenesis; however, their roles are not fully understood. Here, we examined the efficacy of oral administration of KAG-308, an EP4-selective agonist, in surgically induced mouse knee OA. Cartilage degeneration and synovitis were significantly inhibited by the KAG-308 treatment. Chondrocyte hypertrophy and expression of tumor necrosis factor alpha (TNF) and matrix metalloproteinase 13 (Mmp13) in the synovium were suppressed in the KAG-308-treated mice. In cultured chondrocytes, hypertrophic differentiation was inhibited by KAG-308 and intranuclear translocation of histone deacetylase 4 (Hdac4) was enhanced. In cultured synoviocytes, lipopolysaccharide (LPS)-induced expression of TNF and Mmp13 was also suppressed by KAG-308. KAG-308 was detected in the synovium and cartilage of orally treated mice. TNF secretion from the synovia of KAG-308-treated mice was significantly lower than control mice. Thus, we conclude that oral administration of KAG-308 suppresses OA development through suppression of chondrocyte hypertrophy and synovitis. KAG-308 may be a potent candidate for OA drug development.

Neonatal testis growth recreated in vitro by two-dimensional organ spreading.

Organ culture experiments can be hampered by central degeneration or necrosis due to the inadequate permeation of oxygen and nutrients, which deteriorates the function and growth of cultured tissues. In the current study, we aimed to overcome this limitation of organ culture through spreading the tissue two dimensionally on an agarose gel stand and molding into a disc shape by placing a ceiling of polydimethylsiloxane (PDMS) chip, which is highly oxygen permeable. By this, every part of the tissue can receive a sufficient supply of oxygen through PDMS as well as nutrients through the agarose gel below. This method not only prevented central necrosis of tissues, but also supported the tissue growth over time. In addition, such growth, as volume enlargement, could be easily measured. Under these conditions, we examined the effect of several factors on the growth of neonatal mouse testis, and found that follicle stimulating hormone (FSH) and insulin significantly promoted the growth. These results are in good agreement with previous in vivo reports. Notably, the growth achieved over 7 days in our in vitro system is almost comparable to, about 80% of, that observed in vivo. Thus, we successfully monitored the promotion of tissue growth beyond the limits of the conventional organ culture method. This extremely simple method could offer a unique platform to evaluate the growth as well as functional properties of organs, not only the testis but also others as well.

A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility.

In our previous study, we produced a microfluidic device (MFD) which successfully maintained spermatogenesis for over 6 months in mouse testis tissues loaded in the device. In the present study, we developed a new MFD, a monolayer device (ML-D) with a barrier structure consisting of pillars and slits, which is simpler in design and easier to make. This ML-D was also effective for inducing mouse spermatogenesis and maintained it for a longer period than the conventional culture method. In addition, we devised a way of introducing sample tissue into the device during its production, just before bonding the upper layer of polydimethylsiloxane (PDMS) and bottom glass slide. The tissue can obtain nutrients horizontally from the medium running beside it and oxygen vertically from above through PDMS. In addition, the glass slide set at the bottom improved the visibility of the sample tissue with an inverted microscope. When we took photos of cultured tissue of the Acr-Gfp transgenic mouse testis in ML-D sequentially every day, morphological changes of the acrosome during spermiogenesis were successfully recorded. The ML-D is simple in design and useful for culturing testis tissue for inducing and maintaining spermatogenesis with clearer visibility. Due to the new method of sample loading, tissues other than testis should also be applicable.

In vitro mouse spermatogenesis with an organ culture method in chemically defined medium.

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

Pumpless microfluidic system driven by hydrostatic pressure induces and maintains mouse spermatogenesis in vitro.

Three-dimensional aggregation and organ culture methods are critical for recreating in vivo cellular phenomena outside the body. Previously, we used the conventional gas liquid interphase organ culture method to induce complete mouse spermatogenesis. After incorporating microfluidic systems, we achieved a significant increase in efficiency and duration of spermatogenesis. One of the major drawbacks preventing the popularization of microfluidics, however, is the use of a power-pump to generate medium flow. In this study, we produced a pumpless microfluidic device using hydrostatic pressure and a resistance circuit to facilitate slow, longer lasting medium flow. During three months of culture, results in induction and maintenance of spermatogenesis showed no difference between pumpless and pump-driven devices. Correspondingly, the spermatogonial population was favorably maintained in the pumpless device compared to the conventional method. These results show the advantage of using microfluidic systems for organ culture experiments. Our pumpless device could be applied to a variety of other tissues and organs, and may revolutionize organ culture methods as a whole.

Inverse correlation between reactive oxygen species in unwashed semen and sperm motion parameters as measured by a computer-assisted semen analyzer.

This study investigated the correlation between sperm motion parameters obtained by a computer-assisted semen analyzer and levels of reactive oxygen species in unwashed semen. In total, 847 patients, except for azoospermic patients were investigated. At the time of each patient's first consultation, semen parameters were measured using SMAS™ or CellSoft 3000™, and production of reactive oxygen species was measured using a computer-driven LKB Wallac Luminometer 1251 Analyzer. The patients were divided into two groups: reactive oxygen species - positive and negative. The semen parameters within each group were measured using one of the two computer-assisted semen analyzer systems and then compared. Correlations between reactive oxygen species levels and sperm motion parameters in semen from the reactive oxygen species - positive group were also investigated. Reactive oxygen species were detected in semen samples of 282 cases (33.3%). Sperm concentration (P < 0.01; P < 0.01), motility (P < 0.01; P < 0.05), and progressive motility (P < 0.01; P < 0.01) were markedly lower in the reactive oxygen species - positive group than in the reactive oxygen species - negative group. Among the sperm motion parameters in the reactive oxygen species - positive group, sperm concentration (P < 0.01; P < 0.01), motility (P < 0.05; P < 0.01), mALH (P < 0.05; P < 0.01), and progressive motility (P < 0.05; P < 0.01) also showed inverse correlations with the logarithmic transformed reactive oxygen species levels. Therefore, this study demonstrated that excessive reactive oxygen species in semen damage sperm concentration, motility, and other sperm motion parameters.

Comparison of the performance of experienced and novice surgeons: measurement of gripping force during laparoscopic surgery performed on pigs using forceps with pressure sensors.

BACKGROUND: Laparoscopic surgical techniques are difficult to learn, and developing such skills involves a steep learning curve. To ensure surgeons achieve a high skill level, it is important to be able to measure and assess their skills. Therefore, it is necessary to understand the performance differences between experienced and novice surgeons, as such information could be used to help surgeons learn laparoscopic skills. We examined the differences in gripping and reaction force between experienced and novice surgeons during laparoscopic surgery. METHODS: We measured the gripping force generated during laparoscopic surgery performed on pigs using forceps with pressure sensors. Several sensors, including strain gauges, accelerometers, and a potentiometer, were attached to the forceps. This study included 4 experienced and 4 novice surgeons. Each subject was asked to elevate the kidney in order to approach the renal hilus using the forceps. Throughout the experiment, we measured the gripping force and reaction force generated during the movement of the forceps in real time. RESULTS: The experienced and novice surgeons exhibited similar reaction force levels. Conversely, gripping force differed significantly between the groups. The experienced and novice surgeons exhibited mean gripping force levels of 3.06 and 7.15 N, respectively. The gripping force standard deviation values for the experienced and novice surgeons were 1.43 and 3.54 N, respectively. The mean and standard deviation gripping force values of the experienced surgeons were significantly lower than those of the novice surgeons (P = 0.015 and P = 0.011, respectively). CONCLUSIONS: This study indicated that experienced surgeons generate weaker but more stable gripping force than novice surgeons during laparoscopic procedures.

Usefulness of Measuring the Serum Elastin Fragment Level in the Diagnosis of an Acute Aortic Dissection.

Previous reports have shown that serum elastin fragments (SEFs) may be a useful biomarker for the diagnosis of an acute aortic dissection (AAD). However, because the reference interval of SEFs has not been established, it has not been determined whether SEFs are really useful for the diagnosis of AAD. The purpose of this study was to determine the usefulness of measuring SEFs for the diagnosis of AAD. A total of 42 consecutive patients aged 68 ± 18 years who were diagnosed with an AAD were studied. Patient background and SEF levels were examined on admission. SEF levels were also measured in patients undergoing a medical examination (n = 531, age 54 ± 17 years) to compare with those with an AAD. In the control group, SEF levels increased with age (R = 0.725, p <0.001). Then, we defined the upper limit of the reference interval of SEF levels as the 97.5th percentile of control SEF grouped by decade of life from the sixth to ninth decade. The overall risk of AAD exceeding the upper limit of the reference interval at each decade was 10% (4 of 42). For patients in their 60s and 70s, median SEF levels in the AAD group (89 [77 to 104], 93 [60 to 123] ng/ml, respectively) were not significantly higher than those in the control group (79 [68 to 92], 90 [79 to 106] ng/ml, respectively; p = 0.081 and 0.990, respectively). Our data suggest that measuring SEF levels may not be useful in the diagnosis of an AAD as the upper limit of the reference interval of the SEF level was unexpectedly higher.

Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFN-γ production in iNKT cells.

Invariant natural killer T (iNKT) cells are a subset of innate-like T cells that act as important mediators of immune responses. In particular, iNKT cells have the ability to immediately produce large amounts of IFN-γ upon activation and thus initiate immune responses in various pathological conditions. However, molecular mechanisms that control IFN-γ production in iNKT cells are not fully understood. Here, we report that basic helix-loop-helix transcription factor family, member e40 (Bhlhe40), is an important regulator for IFN-γ production in iNKT cells. Bhlhe40 is highly expressed in stage 3 thymic iNKT cells and iNKT1 subsets, and the level of Bhlhe40 mRNA expression is correlated with Ifng mRNA expression in the resting state. Although Bhlhe40-deficient mice show normal iNKT cell development, Bhlhe40-deficient iNKT cells show significant impairment of IFN-γ production and antitumor effects. Bhlhe40 alone shows no significant effects on Ifng promoter activities but contributes to enhance T-box transcription factor Tbx21 (T-bet)-mediated Ifng promoter activation. Chromatin immunoprecipitation analysis revealed that Bhlhe40 accumulates in the T-box region of the Ifng locus and contributes to histone H3-lysine 9 acetylation of the Ifng locus, which is impaired without T-bet conditions. These results indicate that Bhlhe40 works as a cofactor of T-bet for enhancing IFN-γ production in iNKT cells.

Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device.

In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

Design of Enzyme-Encapsulated Protein Containers by In Vivo Crystal Engineering.

Crystalline protein assemblies of polyhedra crystal (PhC) can be utilized as solid enzyme containers for long-term storage of enzymes with retention of their enzymatic activity. The enzymes can be released from the crystals at the optimum pH for the enzymatic activity by dissolution of the crystals using in vivo crystal engineering.

Invariant natural killer T cell deficiency leads to the development of spontaneous liver inflammation dependent on γδT cells in mice.

BACKGROUND: Immune tolerance is maintained in the liver, and perturbation of tolerance can lead to immune-mediated liver diseases such as autoimmune hepatitis (AIH). Invariant natural killer T (iNKT) cells and γδT cells have been shown to maintain immune homeostasis as regulatory cells and to play pathogenic roles in immune-mediated diseases as effector cells. We hypothesized that iNKT cells and γδT cells are involved in the maintenance of hepatic immune tolerance and immune-mediated liver disease. METHODS: We measured liver inflammation and the cytokine profiles of liver mononuclear cells in BALB/c wild-type (WT) mice and BALB/c Jα18-deficient (KO) mice lacking iNKT cells. We also examined the role of γδT cells in AIH using liver tissue from AIH patients and control subjects. RESULTS: Spontaneous liver inflammation, hepatocyte damage, and anti-nuclear-antibody production occurred in Jα18 KO mice but not in WT mice. Furthermore, liver mononuclear cells from Jα18 KO mice, but not those from WT mice, produced interleukin-17 (IL-17). γδT cells were the primary producers of the cytokine, and they were more abundant in the livers of Jα18 KO mice than in those of WT mice. In Jα18 KO mice, the administration of anti-γδT-cell-receptor antibody abolished liver inflammation, hepatocyte damage, and IL-17 production. γδT cells accumulated in the livers of AIH patients but not in those of the control subjects. CONCLUSIONS: Our results suggest a protective role for iNKT cells, a pathologic role for γδT cells, and an association between these cells in the pathogenesis of AIH.