Activation of stress response axis as a key process in environment-induced sex plasticity in fish.

The determination of sex is an important hallmark in the life cycle of organisms, in which the fate of gonads and then the individual sex are defined. In gonochoristic teleost fish, this process is characterized by a high plasticity, considering that in spite of genotypic sex many environmental factors can cause shifts from one to another molecular pathway, resulting in organisms with mismatching genotypic and phenotypic sexes. Interestingly, in most instances, both female-to-male or male-to-female sex-reversed individuals develop functional gonads with normal gametogenesis and respective progenies with full viability. The study of these mechanisms is being spread to other non-model species or to those inhabiting more extreme environmental conditions. Although water temperature is an important mechanism involved in sex determination, there are other environmental stressors affected by the climate change which are also implicated in stress response-induced masculinization in fish. In this regard, the brain has emerged as the transducer of the environment input that can influence the gonadal fate. Furthermore, the evaluation of other environmental stressors or their synergic effect on sex determination at conditions that simulate the natural environments is growing gradually. Within such scope, the concerns related to climate change impacts rely on the fact that many of biotic and abiotic parameters reported to affect sex ratios are expected to increase concomitantly as a result of increased greenhouse gas emissions and, particularly worrying, many of them are related to male bias in the populations, such as high temperature, hypoxia, and acidity. These environmental changes can also generate epigenetic changes in sex-related genes affecting their expression, with implications on sex differentiation not only of exposed individuals but also in following generations. The co-analysis of multi-stressors with potential inter- and transgenerational effects is essential to allow researchers to perform long-term predictions on climate change impacts in wild populations and for establishing highly accurate monitoring tools and suitable mitigation strategies.

Thermal and endocrine regulation of gonadal apoptosis during sex differentiation in pejerrey Odontesthes bonariensis.

The fate of the differentiating gonads in pejerrey Odontesthes bonariensis is determined by the environmental water temperature experienced by the larvae during the critical period of sex determination. We previously reported a link between apoptosis, temperature and sex differentiation in this species. To clarify this link, we subjected larvae to thermal and endocrine treatments between hatching and the onset of histological sex differentiation of the gonads and assessed the patterns of gonadal development and apoptosis by light microscopic histology and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, respectively. Apoptotic labeling was widespread among somatic cells of the anterior region of the right gonads of fish reared at the male-producing temperature (MPT) and part of the fish at the mixed sex-producing temperature prior to sex differentiation. In contrast, TUNEL-positive cells were rarely observed in gonads at the female-producing temperature (FPT). Administration of exogenous estrogen completely prevented MPT-induced masculinization, induced feminization and reduced gonadal apoptosis, whereas an aromatase inhibitor (fadrozole) induced TUNEL signals in the gonads of FPT-reared larvae. These results provide strong evidence that apoptosis in somatic cells in the right lobe of the gonads might play a key role in testicular differentiation in pejerrey and that estrogens are involved in the regulation of this process.

Correlation between treated hypertension in prepregnancy and transplanted kidney function deterioration during pregnancy even if within pregnancy permission criteria.

Neither pregnancy nor birth is easy in female patients with chronic renal failure, but after kidney transplantation, childbirth is possible when the graft function is good. There are few guidelines for pregnancy permission and multiple reports of decreased transplanted kidney function after pregnancy. In this study, we analyzed factors that influenced transplanted kidney function deterioration during pregnancy. Twenty-one women among 33 total pregnancies have given birth in our institution. Factors analyzed were donor and recipient age at transplantation, birth age of recipient, living or cadaveric donor, hemodialysis period before transplantation, delivery method, presence of hypertension and protein urea at the beginning of pregnancy, and period between pregnancy and transplantation. Maternal graft function at the beginning of the pregnancy was 1.16 ± 0.39 mg/dL (range = 0.5-2.1). A rise in serum creatinine (S-Cr) before delivery was observed in 10/21 cases: six cases showed a rise in S-Cr levels at 1 or more years after delivery. From the analysis, graft function at the beginning of pregnancy became a significant factor correlating with the elevation of S-Cr levels during pregnancy (P = .002). Patients were divided into two groups by S-Cr levels at the beginning of pregnancy: group A was S-Cr ≤ 1; group B was S-Cr 1-2 mg/dL. All group A cases showed stable graft function before and after delivery. Some individuals in group B experienced deterioration of graft function during pregnancy; the others had stable graft function. The presence of treated hypertension at the beginning of pregnancy in group B significantly impacted renal dysfunction during pregnancy (P < .05). In conclusion, the presence of treated hypertension at the beginning of pregnancy was a significant risk factor for functional deterioration of the transplanted kidney during pregnancy even if the individual was initially within pregnancy permission criteria.

(99m)Tc-mercaptoacetyl triglycine renography to monitor renal transplant function among kidneys from donors after cardiac death.

PURPOSE: Delayed graft function usually occurs after kidney transplantation from donors after cardiac death, It is important to monitor graft function during the anuric period, but there have been few useful tools. Consequently, we evaluated the availability of (99m)-Tc mercaptoacetyltriglycine (MAG3) renography. METHODS: Thirty-two patients underwent renal transplantation from donors after cardiac death between June 2, 2005, and April 14, 2011. One patient was excluded due to an acute rejection episode which developed during the dialysis period. The first (99m)Tc-MAG3 renogram was performed as early as possible after the operation and repeated until the patient was weaned from dialysis. The corrected tubular extraction rate (cTER; mL/min/1.73 m(2)) was calculated; it represents the MAG3 clearance corrected by body surface area. RESULTS: cTER was low immediately after transplantation, but increased gradually until the patient was weaned from dialysis. A significant correlation was observed between early cTER and the period of dialysis-dependence (r = -0.677, P < .001) as well as the short-term best corrected creatinine clearance (r = 0.526, P = .002). CONCLUSION: We observed that graft function can be monitored by routinely performing (99m)Tc-MAG3 renography after transplantation of kidneys from donors after cardiac death.

Renal impairment after laparoscopic radical nephrectomy affects hypoglycaemic therapy.

WHAT IS KNOWN AND OBJECTIVE: Renal impairment is unavoidable after laparoscopic radical nephrectomy (LRN) and is an important consideration for drug therapy. It is possible that the renal impairment after LRN causes adverse reactions following reduced elimination of some renally excreted drugs, such as hypoglycaemic drugs. However, there are few studies of renal function in patients with diabetes mellitus (DM) in the first week after LRN. The purpose of this study was to examine whether renal impairment after LRN affected glycaemic control. We assessed pre- and postoperative renal function of DM patients and examined whether re-administration of hypoglycaemic drugs in the first week after LRN causes episodes of hypoglycaemia. METHODS: Renal carcinoma patients undergoing LRN in Nagoya University Hospital from January 2007 to December 2009 were identified in a retrospective cohort study design. Patients were divided into non-DM (n = 60) and DM (n = 14) groups. RESULTS AND DISCUSSION: There were significant differences in postoperative estimated glomerular filtration rate values between the non-DM and DM groups. Four of nine patients (44%) experienced hypoglycaemia induced by re-administration of hypoglycaemic drugs, namely, sulfonylureas. WHAT IS NEW AND CONCLUSION: In the present study, we found the first evidence that renal impairment in the first week after LRN was a risk factor of hypoglycaemia. To prevent hypoglycaemia after LRN, assessment of renal function and the use of insulin therapy are important.

Analysis of sexually dimorphic expression of genes at early gonadogenesis of pejerrey Odontesthes bonariensis using a heterologous microarray.

The process of morphological development of a differentiated gonad from an undifferentiated primordium is a very important step of gonadogenesis. Studies on sexually dimorphic gene expression are important to increase our understanding of this process and to investigate how environmental factors such as temperature can regulate gonadal development. The aim of this study was to identify putative genes involved in sex differentiation in pejerrey (Odontesthes bonariensis) reared at male- and female-producing temperatures (MPT and FPT, respectively) using a microarray heterologous from the medaka (Oryzias latipes), a closely phylogenetic species. Genes related to numerous processes presented higher expression at MPT, including those involved in muscular contraction, metabolic pathways, developmental processes, and reproduction. Genes induced by FPT were classified under the gene ontology terms of response to stimulus, transport and proteolysis. From genes selected for validation, at MPT ndrg3 expression was observed in the somatic cells, whereas pen-2 was detected in germ cells in the caudal portion of the gonads, where no apoptotic signals were observed. Finally, hsp90 was highly expressed in somatic cells of the gonads at the FPT. The results suggest that the interplay of pro-apoptotic and anti-apoptotic genes is important during the masculinization process and for the prevention of sterility following exposure to warm temperatures.

Is estimated donor glomerular filtration rate before death a better predictor of decreased-donor kidney function?

The worldwide shortage of deceased-donor kidneys for transplantation has become a serious issue in the past decade, leading to study of marginal donors. However, both the availability and the utility of kidneys from deceased donors are still unclear. The aim of the present study was to evaluate another method to estimate donor kidney function rather than using donor creatinine (Cr). We studied 129 recipients of deceased-donor kidneys from Maastriche donor categories III and IV. We analyzed donor Cr levels before death and recipient Cr levels at 1 year after transplant, as well as estimated glomerular filtration rates (eGFR). There was no significant difference in donor Cr levels at admission to the hospital and before death according to eGFR at 1 year after transplantation: <30 mL/min/1.73 m(2) versus ≧30 mL/min/1.73 m(2). However, recipients whose donors showed lower average eGFR levels on admission displayed better renal function at 1 year after transplant (P = .025). In conclusion, donor Cr levels before death was a less useful measurement to relate to recipient renal function; eGFR provided a better index.

[Solitary fibrous tumor of the seminal vesicle]. / Solitärer fibröser Tumor der Samenblase.

A 56-year-old man presented to our hospital with a pelvic mass. The tumor was diagnosed to occur from right seminal vesicle and to be a benign solitary fibrous tumor by transrectal tumor biopsy. The tumor enlarged during follow up, and he under-went resection of the tumor.

Establishment of a strain inheriting a sex-linked SNP marker in Patagonian pejerrey (Odontesthes hatcheri), a species with both genotypic and temperature-dependent sex determination.

The Patagonian pejerrey Odontesthes hatcheri is an atherinopsid species presenting genotypic sex determination (GSD) at intermediate temperatures and temperature-dependent sex determination at the low and high ranges of thermal tolerance. A recent study revealed the presence of a sex-linked SNP marker in some males of this species, but a strain which inherits the marker faithfully has not been established. This research was conducted to develop such a strain, for use as a tool to study the molecular mechanisms of gonadal sex differentiation and sexual dimorphism, and to obtain basic information on the GSD mode in this species. For these purposes, we performed backcrosses and full-sibling crosses using males and females whose presumptive genotypic sex was inferred from the presence of the sex-linked SNP marker. Four backcrosses between SNP(-) daughters and their SNP(+) father generated balanced sex ratios with the phenotypic sex matching the genotypic sex in most cases (98.21%) at an intermediate, sexually neutral temperature (21 degrees C). Full-sibling crosses between these four SNP(-) females and their SNP(+) brothers produced three progenies with balanced sex ratios and one with 94.4% males. The results of this study confirm that a strain inheriting the sex-linked SNP marker was successfully developed. Moreover, the inheritance pattern of the marker and the sex ratios of the progenies provide strong evidence that the GSD mode in O. hatcheri is the XX-XY system.

Nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy in 2007: general view of the pathogens' antibacterial susceptibility.

For the purpose of a nationwide surveillance of the antimicrobial susceptibility of bacterial respiratory pathogens in patients in Japan, the Japanese Society of Chemotherapy conducted their second year survey, during the period from January to August, 2007. A total of 1178 strains were collected from clinical specimens obtained from adult patients with well-diagnosed respiratory tract infections. Susceptibility testing was evaluable for 1108 strains (226 Staphylococcus aureus, 257 Streptococcus pneumoniae, 6 Streptococcus pyogenes, 206 Haemophilus influenzae, 120 Moraxella catarrhalis, 122 Klebsiella pneumoniae, and 171 Pseudomonas aeruginosa). A total of 44 antibacterial agents, including 26 beta-lactams (four penicillins, three penicillins in combination with beta-lactamase inhibitors, four oral cephems, eight parenteral cephems, one monobactam, five carbapenems, and one penem), three aminoglycosides, four macrolides (including ketolide), one lincosamide, one tetracycline, two glycopeptides, six fluoroquinolones, and one oxazolidinone were used for the study. Analysis was conducted at the central reference laboratory according to the method recommended by the Clinical and Laboratory Standards Institute (CLSI). The incidence of methicillinresistant Staphylococcus aureus (MRSA) was high, at 59.7%, and the incidences of penicillin-intermediateresistant and -resistant Streptococcus pneumoniae (PISP and PRSP) were 30.4% and 5.1%, respectively. Among Haemophilus influenzae strains, 19.9% of them were found to be beta-lactamase-non-producing ampicillin (ABPC)-intermediately-resistant (BLNAI), 29.1% to be beta-lactamasenon-producing ABPC-resistant (BLNAR), and 6.7% to be beta-lactamase-producing ABPC-resistant (BLPAR) strains. Extended-spectrum beta-lactamase-producing Klebsiella pneumoniae was not isolated. Two isolates (1.2%) of Pseudomonas aeruginosa were found to be metallo-beta-lactamase-producing strains, including one (0.6%) suspected multidrug-resistant strain showing resistance to imipenem, amikacin, and ciprofloxacin. These data will be a useful reference for future periodic surveillance studies and for investigations to control resistant infections as well. Continued surveillance is required to prevent the further spread of these antimicrobial resistances.

Ewing's sarcoma / primitive neuroectodermal tumor of the kidney.

A 42-year-old female presented with right back pain. The CT scan revealed a 72-mm space-occupying lesion in the middle portion of the right kidney. No metastasis was proven. She underwent laparoscopic radical nephrectomy and lymph node disection. The histopathological examination revealed a high-grade primitive small round tumor the cells of which were strongly positive for CD99 and vimentin. Fluorescence in situ hybridization analysis using a DNA probe for the Ewing sarcoma breakpoint region 1 (EWSR 1) on chromosome 22g12 revealed a rearrangement of the EWSR 1 locus. The diagnosis was Ewing's sarcoma / primitive neuroectodermal tumor of the kidney. She underwent 13 cycles of chemotherapy, and has no evidence of recurrence 19 months after surgery.

Successful renal transplantation for a patient with pyoderma gangrenosum.

Pyoderma gangrenosum (PG), a rare skin disease of unknown etiology, forms intractable skin ulcers at surgical or traumatic sites. This case is a 40-year-old woman with PG who experienced end-stage renal disease due to type 1 diabetes mellitus. Arteriovenous fistula (AVF) creation and peritoneal dialysis introduction were considered to be difficult, because this patient had a history of developing intractable aseptic ulcers at surgical sites. Therefore, she continued hemodialysis via a temporary catheter. With frequent catheter exchange, there was stenosis of both the femoral veins and the internal jugular vein. Therefore, a hemodialysis catheter that could be used for the long term was inserted into the left jugular vein as a final site. To prevent the patient not being able to continue hemodialysis, we performed a kidney transplantation to save her life. We performed a blood type-compatible, living donor kidney transplantation after confirming the absence of active skin lesions. The 69-year-old donor was her mother. Induction immunotherapy started with tacrolimus, mycophenolate mofetil, steroids, and basiliximab. Intravenous pulses of methylprednisolone were performed to prevent ulceration of the surgical site on days 0-2 (500 mg/d). The postoperative course was excellent. After the operation, ulceration of the surgical site was never observed. The serum creatinine value was 0.87 mg/dL at 6 months. To our knowledge, renal transplantations for a patient with PG has not been previously reported.

Experimentally induced depletion of germ cells in sub-adult Patagonian pejerrey (Odontesthes hatcheri).

Germ cell (GC) transplantation (GCT) is a novel reproductive technology with application in seed production and conservation of endangered species. This study examined the suitability of treatment with Busulfan, a cytotoxic agent, and warm water, known to cause GC degeneration, for depletion of endogenous GCs in sub-adult Patagonia pejerrey Odontesthes hatcheri intended as hosts in GCT. In two experiments, fish were treated with six combinations of temperature (intermediate and high, 20 and 25 degrees C, respectively) and Busulfan (0, 20, and 40 mg/kg body weight), given intraperitoneally (ip) as a single (0 week) or repeated (0 and 4 week) dose. The effectiveness of the treatments was assessed by gonado-somatic index, histology, and (germ cell-specific) vasa gene expression after 8 weeks. Fish were allowed to recover at 17 degrees C for 4-8 weeks after the treatments to ascertain the permanency of the effects. The high temperature (25 degrees C) alone induced only incipient gonadal degeneration and germ cell loss, but was highly effective in combination with double administration of 40 mg/kg Busulfan. Males tolerated Busulfan better and were more easily depleted of germ cells than females. Animals treated for 8 weeks were severely devoid of germ cells, but were still capable of gametogenesis. Thus, the combination of Busulfan and high water temperature appeared to be efficient for depletion of GCs in adult fish; and the treated gonads retained the ability to support GC proliferation and differentiation. Furthermore, quantitative analysis of vasa transcript levels was found to be an useful to monitor the degree of gonad sterility during treatment.

Efficiency of laparoscopic-assisted renal biopsy.

BACKGROUND: This study was made to present our experience and results with transperitoneal laparoscopic-assisted renal biopsy (LARB) in Nagoya University Hospital as a good alternative for open renal biopsy. METHODS: 21 patients (14 male, 7 female, mean age 58 years, range 21-83 years) were unsuitable for percutaneous renal biopsy. Therefore, they underwent laparoscopic-assisted renal biopsy. The kidney was approached transperitoneally via three ports, cortical tissue was obtained using a 16-gauge gun-mounted semiautomatic biopsy needle. Hemostasis was obtained by applying pressure on the renal puncture using gauze until bleeding had been stopped (range 5-20 min). RESULTS: Adequate cortical tissue and accurate diagnoses were obtained in all patients. Mean operative time was 83 min (range 65-120 min) and mean estimated blood loss was 5.5 ml (range 1-10 ml). There were no intraoperative complications: no open conversion, blood transfusions or gross hematuria. All patients walked about freely and could tolerate regular food on the first postoperative day. The only postoperative complication was a hernia formation at the place of trocar insertion 3 months after the operation in one patient who previously underwent multiple surgery for 3 arterial grafts and appendicitis. CONCLUSIONS: LARB is a safe and accurate procedure for obtaining cortical biopsies with minimal blood loss. Although LARB remains a surgical procedure which requires general anesthesia, LARB to date may be considered as a good alternative to open renal biopsy for patients in whom a closed percutaneous approach is either a relative or absolute contraindication.

Conversion from mycophenolate mofetil to mizoribine for patients with positive polyomavirus type BK in urine.

It is known that administration of mycophenolate mofetile (MMF) is associated with BK virus (BKV) nephropathy in renal transplant recipients. To determine any inhibitory effect of mizoribine for BKV, seven patients with positive BKV in their urine who took MMF as immunosuppressive therapy were evaluated after MMF was changed to mizoribine. Baseline BKV DNA in urine, which ranged from 2.2 x 10(2) to 5.5 x 10(6) copies per milliliter, decreased in all cases (mean = 1.9 x 10(-1) times; median 2.8 x 10(-3) times). Four cases turned negative within 6 months and one within 12 months. No acute rejection or deterioration of graft function occurred during the administration of mizoribine. An inhibitory effect of mizoribine on BKV was suggested.

The first nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy. Part 1: a general view of antibacterial susceptibility.

The Japanese Society of Chemotherapy (JSC) conducted the first nationwide surveillance of bacterial respiratory pathogens during the period from January to August 2006. With the cooperation of 32 medical institutions throughout Japan, a total of 924 strains belonging to seven clinically relevant bacterial species were collected from adult patients with well-diagnosed respiratory tract infections (RTIs). Antimicrobial susceptibility testing of the 887 evaluable strains (205 Staphylococcus aureus, 200 Streptococcus pneumoniae, 9 Streptococcus pyogenes, 165 Haemophilus influenzae, 91 Moraxella catarrhalis, 74 Klebsiella pneumoniae, and 143 Pseudomonas aeruginosa) to 42 antibacterial agents was conducted at the Central Laboratory of the Research Center for Anti-infective Drugs of the Kitasato Institute, according to recommendations issued by the Clinical and Laboratory Standards Institute (CLSI). The antibacterial agents employed were 25 beta-lactams, three aminoglycosides, four macrolides (including one azalide and one ketolide), one lincosamide, one tetracycline, two glycopeptides, five fluoroquinolones, and one oxazolidinone. The incidence of methicillin-resistant S. aureus (MRSA) was 63.4%, and the incidences of penicillin-intermediately resistant S. pneumoniae (PISP) and penicillin-resistant S. pneumoniae (PRSP) were 35.0% and 4.0%, respectively. Among H. influenzae, 21.2% of the strains were found to be beta-lactamase-nonproducing ampicillin (ABPC)-intermediately resistant (BLNAI), 29.1% to be beta-lactamase-nonproducing ABPC-resistant (BLNAR), and 4.8% to be beta-lactamaseproducing ABPC-resistant (BLPAR) strains. The incidence of extended-spectrum beta-lactamase-producing K. pneumoniae was 2.7% (2 of 74 strains). Three (2.1%) of the 143 P. aeruginosa strains were found to be metallo-beta-lactamaseproducing, including 1 (0.7%) multidrug-resistant strain. Through the nationwide surveillance, we obtained fundamental antimicrobial susceptibility data of clinically relevant bacterial pathogens in adult RTI to various antibacterial agents. These data will be a useful reference for future periodic surveillance studies, as well as for investigations to control antimicrobial-resistant pathogens.

Dimorphic expression of dmrt1 and cyp19a1 (ovarian aromatase) during early gonadal development in pejerrey, Odontesthes bonariensis.

The pejerrey (Odontesthes bonariensis) is a teleost fish with strong temperature-dependent sex determination (TSD). Several studies have shown that dmrt1 and gonadal aromatase (cyp19a1) are implicated in the sex differentiation process in teleosts but little is known on the expression balance and endocrine regulation of these two genes during TSD. This study was designed to clarify the expression patterns of both genes during gonadal sex differentiation of pejerrey reared at female-, male- and mixed-sex-producing temperatures (FPT, MPT, and MixPT, respectively). The expression of dmrt1 was found to be significantly higher during gonadal sex differentiation at MPT compared to FPT. Conversely, cyp19a1 expression clearly increased during differentiation at FPT but not at MPT. The expression of both genes at MixPT showed a dimorphic profile with individual values resembling either those at the MPT or FPT. Administration of exogenous 17beta-estradiol down- and up-regulated the expression of dmrt1 and cyp19a1, respectively, regardless of temperature, and rescued the female phenotype at the MPT. However, treatment with the aromatase inhibitor Fadrozole caused masculinization without changing the pattern of gene expression. These results are strong evidence of the involvement of both genes in the gonadal differentiation process of pejerrey. The involvement of estradiol is discussed.

Autonomous catheter insertion system using magnetic motion capture sensor for endovascular surgery.

BACKGROUND: In order to reduce fluoroscope usage in endovascular surgery, there is a need to develop autonomous catheter insertion systems. METHODS: We propose a system for tracking the position and speed of a catheter using a magnetic motion capture sensor to provide feedback to a catheter-driving mechanism, to perform autonomous catheter insertion in major vasculature. Catheter insertion speed control and path reconstruction experiments were performed with the system inside a silicone model of major vasculature to simulate surgery. RESULTS: The system controlled the catheter for speeds of 6.14 mm/s and reproduced a two-dimensional path inside the silicone blood vessel phantom with less than 7 mm of error. CONCLUSIONS: We found that error in speed control rises as a result of friction between the catheter and the model wall. Path reconstruction error depends on the model's cross-sectional diameter, the properties of the catheter insertion mechanism, the magnetic sensor and the system guidance technique.

Temperature-dependent sex determination in Hd-rR medaka Oryzias latipes: gender sensitivity, thermal threshold, critical period, and DMRT1 expression profile.

The developmental time and thermal threshold for temperature-dependent sex determination (TSD), gender differences in temperature sensitivity, the fertility of thermally sex reversed fish, and the effect of temperature on the expression of two major sex determination/differentiation genes (DMY/DMRT1bY and DMRT1) were examined in the Hd-rR strain of medaka, Oryzias latipes. Fertilized eggs were exposed from either shortly after fertilization (8-16 cells; embryonic stages 5-6) or from middle embryogenesis (heart development stage; stage 36) until hatching to temperatures ranging from 17 degrees C to 34 degrees C. Secondary sexual characteristics, gonadal histology, progeny testing, sex-linked body coloration and gene expression were used to determine phenotypic and genotypic sex. Sex determination was unaffected by low or high temperatures in genotypic (XY) males. In contrast, genotypic (XX) females treated from stages 5-6 showed increasing rates of sex reversal into phenotypic males at temperatures above 27 degrees C up to 100% at 34 degrees C. Thermal manipulation of sex was ineffective after stage 36, indicating that gonadal fate in medaka is determined considerably earlier than histological differentiation (stage 39). High temperature induced DMRT1 expression in genotypic females, which was observed already from stage 36. Sex-reversed males had histologically normal testes, were capable of sexual courtship and, with the exception of fish from 34 degrees C, sired viable progeny when mating with fertile females. These results clarify the pattern of TSD in medaka and provide important clues to understand the mechanism of sex determination in this species. They also suggest that a brief exposure to high temperature early in life could impair the fertility of medaka as adults.

Long time follow up of CD28- CD4+ T cells in living kidney transplant patients.

We previously reported that the CD28(-) CD4(+) T cell subpopulation was expanded in the kidney allograft patients with long graft survival, although these T cells were rarely found in patients with graft survival <5 yr. To understand the CD28(-) CD4(+) T cells in the long-term acceptance of kidney allografts, we examined functions of this population and performed a 4 yr follow up study. Peripheral blood mononuclear cells (PBMC) were obtained from 47 long-term living related kidney allograft recipients. CD28(+) CD4(+) and CD28(-) CD4(+) T cells purified by cell sorting were analyzed for expression of V(beta) repertoire. Donor-specific response was examined in mixed lymphocyte reaction (MLR). A follow up study with long-term kidney allograft patients was performed for 4 yr about the rate of CD28(-) CD4(+) T cells. Eleven patients were examined by MLRs against donors and third party. Four patients with a marked increase of CD28(-) CD4(+) T cells showed the donor-specific responses appeared to be lower when compared with third party-specific responses. Freshly sorted CD28(-) CD4(+) T cells showed a restricted V(beta) repertoire, whereas the V(beta) usage of CD28(+) CD4(+) T cells from the same patients was much diversified. Such difference in V(beta) repertoire was not evident between the two populations from healthy control. A follow up study showed the ratio of CD28(-) CD4(+) T cells appeared to be lower in patients who were suspected of chronic rejection. These unusual CD4(+) T cells might be related to the long-term acceptance of human transplant allografts.