Is the protein expression window during testicular development affected in patients at risk for stem cell loss?

STUDY QUESTION: Is the protein expression window during testicular development affected in prepubertal patients at risk for stem cell loss? SUMMARY ANSWER: Nuclear ubiquitin carboxyl-terminal esterase L1 (UCHL1) expression in Sertoli cells and interstitial expression of inhibin α (INHA), sex-determining region Y-box 9 (SOX9) and steroidogenic acute regulatory protein (STAR) was affected in patients with Klinefelter syndrome. WHAT IS KNOWN ALREADY: Some patients undergoing testicular tissue banking have already been treated before the testis biopsy is taken. These treatments include chemotherapy or hydroxyurea, which can have an influence on the stem cell number and function. A germinal loss occurs in Klinefelter patients, but its cause is currently unknown. STUDY DESIGN, SIZE, DURATION: Parrafin-embedded testicular tissue from 5 fetuses, 25 prepubertal patients and 5 adults was used to characterize the spatial and temporal distribution of different testicular marker proteins during testicular development. Expression of the markers was evaluated in germ cells, Sertoli cell and interstitial cells. The integrity of this time window was analyzed in patients at risk for germ cell loss: patients treated with hydroxyurea (n = 7), patients treated with chemotherapy (n = 6) and patients affected by Klinefelter syndrome (n = 5). PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry was performed in normal fetal, prepubertal and adult testicular tissue to set up a timeline for the expression of melanoma antigen family A4 (MAGE-A4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4), homeobox protein NANOG, INHA, anti-Müllerian hormone, androgen receptor (AR), SOX9 and STAR. The established timeline was used to evaluate whether the expression of these markers was altered in patients at risk for germ cell loss (patients treated for sickle cell disease (hydroxyurea) or cancer (chemotherapy) and patients with Klinefelter syndrome). MAIN RESULTS AND THE ROLE OF CHANCE: A protein expression timeline was created using different markers expressed in different testicular cell types. Less positive tubules and less positive cells per tubule were observed for MAGE-A4 and UCHL1 expression in the KS compared with the non-treated group (P < 0.01). Higher nuclear UCHL1 Sertoli cell expression was observed in the KS group compared with the non-treated group (P < 0.05). Higher interstitial expression of INHA (P < 0.05), SOX9 (P < 0.01) and STAR (P < 0.05) was observed in KS compared with the non-treated group. LIMITATIONS, REASONS FOR CAUTION: Important age variations exist in the prepubertal groups. Therefore, data were represented in three age groups. However, owing to the limited access to prepubertal tissue, no statistical comparison was possible between these groups. For the Klinefelter group, tissue was only available from patients older than 12 years. WIDER IMPLICATIONS OF THE FINDINGS: The expression timeline can add knowledge to the process of spermatogenesis and be used to evaluate altered protein patterns in patients undergoing potentially gonadotoxic treatments, to monitor spermatogenesis established in vitro and to unravel causes of germ cell loss in Klinefelter patients.

Asparaginase-induced pseudohyponatremia, a case-driven working strategy in pediatric patients.

OBJECTIVES: When using indirect ion-selective electrode (ISE) methods, hypertriglyceridemia leads to pseudohyponatremia due to water displacement artifacts. Multiple strategies exist to minimize this interference. Our objective was to create a patient-friendly one-tube-fits-all testing setup without compromising the method robustness. METHODS: Four strategies were evaluated in a single patient with hypertriglyceridemia. Additionally, the interchangeability between the Cobas 8000 and ABL Flex was evaluated on samples (n = 2274) with different total protein (TP) concentrations. Finally, a proof-of-concept (n = 40) was performed by re-measuring the routine sample with the ABL90 Flex. RESULTS: ABL90 flex results and calculated sodium did not suffer from the presence of high triglyceride levels. We did not observe any significant differences between the three groups (P > 0.05) of sample types (arterial vs. venous plasma vs. venous whole blood after mixing up) nor for the analysers (Roche vs. ABL90 Flex). Passing-Bablok and Bland-Altman tests revealed interchangeability. CONCLUSION: In future cases of hypertriglyceridemia, 1500 mg/dL will be used as a preliminary threshold for reliable sodium determination. Routine Li-heparin samples can be used for accurate sodium determination without any need for extra arterial or venous blood gas tubes, offering a patient-friendly test setup for similar cases.

Screening, identification, and functional analysis of three novel missense mutations in the TRADD gene in children with ALL and ALPS.

BACKGROUND: Apoptosis is known to be a crucial process involved in embryogenesis, development and homeostasis of the immune system. Impaired apoptosis causes dysfunction of lymphocyte homeostasis, growth advantage of tumor cells as well as resistance to current treatment protocols. To investigate the role of the apoptosis adaptor molecules TRADD and FADD in the development of hematological diseases, patient samples were screened for mutations in these genes. PROCEDURE: Genomic DNA from 51 children suffering from B-lineage-ALL (n = 17), T-lineage-ALL (n = 24), ALPS Type Ia (n = 3) and ALPS Type III (n = 7) were analyzed. Genomic DNA from 50 unrelated donors without hematological diseases served as controls. Identified mutations were cloned and their influence on cell viability and NFkappaB activation was analyzed by flow cytometry and luciferase assay, respectively. RESULTS: In the FADD gene no genetic alteration could be detected. However, three novel missense mutations in the TRADD gene could be detected. They are located within a region of TRADD known to exert mainly anti-apoptotic effects for example through the activation of the NFkappaB pathway. Functional analysis of cells overexpressing mutant TRADD cDNA demonstrated a reduced NFkappaB activity and consequently increased cell death compared to wild-type TRADD. CONCLUSION: Mutations in the TRADD gene may contribute to the development of different hematological diseases. The identified mutations demonstrate a putative impact on TRADD signaling and cell survival but may not mainly explain the pathology of the diseases investigated.

Value of intravenous 6-mercaptopurine during continuation treatment in childhood acute lymphoblastic leukemia and non-Hodgkin's lymphoma: final results of a randomized phase III trial (58881) of the EORTC CLG.

Between November 1990 and November 1996, EORTC Children Leukemia Group conducted a randomized trial in de novo acute lymphoblastic leukemia and lymphoblastic non-Hodgkin's lymphoma patients using a Berlin-Frankfurt-Munster protocol to evaluate the monthly addition of intravenous 6-mercaptopurine (i.v. 6-MP) (1 g/m(2)) to conventional continuation therapy comprising per oral MTX weekly and 6-MP daily. Only during the first 18 months of the randomization period, 6-MP p.o. was interrupted for 1 week after each i.v. 6-MP. A total of 877 patients was randomized to either no i.v. 6-MP (Arm A) or additional i.v. 6-MP (Arm B). A total of 217 relapses (91 in Group A vs 128 in Group B) and 13 deaths in CR (5 vs 8) were reported; a total of 134 patients (55 vs 79) died. The median follow-up was 7.6 years. At 8 years, the disease-free survival rate was lower (P=0.005) in Arm B (69.1% (s.e.=2.2%)) than in Arm A (77.9% (s.e.=2.0%)), and the hazard ratio was 1.45 (95% CI 1.12-1.89). In conclusion, as delivered in this study, i.v. 6-MP was detrimental to event-free survival.

Results of 58872 and 58921 trials in acute myeloblastic leukemia and relative value of chemotherapy vs allogeneic bone marrow transplantation in first complete remission: the EORTC Children Leukemia Group report.

The first EORTC (European Organization of Research and Treatment of Cancer) acute myeloblastic leukemia (AML) pilot study (58872) was conducted between January 1988 and December 1991. Out of 108 patients, 78% achieved complete remission (CR), and event-free survival (EFS) and survival rates (s.e., %) at 7 years were 40 (5) and 51% (6%), respectively. It indicated that mitoxantrone could be substituted for conventional anthracyclines in the treatment of childhood AML without inducing cardiotoxicity. The aim of the next EORTC 58921 trial was to compare the efficacy and toxicity of idarubicin vs mitoxantrone in initial chemotherapy courses, further therapy consisting of allogeneic bone marrow transplantation (alloBMT) in patients with an HLA-compatible sibling donor or chemotherapy in patients without a donor. Out of 177 patients, recruited between October 1992 and December 2002, 81% reached CR. Overall 7-year EFS and survival rates were 49 (4) and 62% (4%), respectively. Out of 145 patients who received the first intensification, 39 had a sibling donor. In patients with or without a donor, the 7-year disease-free survival (DFS) rate was 63 (8) and 57% (5%) and the 7-year survival rate was 78 (7) and 65% (5%), respectively. Patients with favorable, intermediate and unfavorable cytogenetic features had a 5-year EFS rate of 57, 45 and 45% and a 5-year survival rate of 89, 67 and 53%, respectively.

Immunodeficiency in a Child with Rapadilino Syndrome: A Case Report and Review of the Literature.

Rapadilino syndrome is a genetic disease characterized by a characteristic clinical tableau. It is caused by mutations in RECQL4 gene. Immunodeficiency is not described as a classical feature of the disease. We present a 2-year-old girl with Rapadilino syndrome with important lymphadenopathies and pneumonia due to disseminated Mycobacterium lentiflavum infection. An immunological work-up showed several unexpected abnormalities. Repeated blood samples showed severe lymphopenia. Immunophenotyping showed low T, B, and NK cells. No Treg cells were seen. T cell responses to stimulations were insufficient. The IL12/IL23 interferon gamma pathway was normal. Gamma globulin levels and vaccination responses were low. With this report, we aim to stress the importance of screening immunodeficiency in patients with RECQL4 mutations for immunodeficiency and the need to further research into its physiopathology.

Androgens in the fetal guinea-pig after maternal infusion of radioactive testosterone.

Testosterone was measured in plasma pools from male and female fetal guinea-pigs between the ages of 30 and 55 days of pregnancy. Between days 33 and 36 the testosterone concentration in the plasma of males (1.4 ng/ml) was several times higher than that found at other ages or that measured in female fetuses. After infusion of tritiated testosterone for 2 h into pregnant guinea-pigs at day 36 of pregnancy, high levels of testosterone and androstenedione were found in maternal plasma. Nevertheless, tritiated testosterone and androstenedione could hardly be shown in the fetuses. Similar large differences in plasma progesterone levels appeared to exist between the maternal and the fetal circulation. Therefore, only a very small fraction of these steroids can penetrate from the maternal circulation into that of the fetus. This finding might be explained by the large difference in androgen-binding capacity between maternal and fetal plasma, as was shown by equilibrium dialysis.

Social factors and puberty in female rats.

The effects of postweaning social conditions on the age of puberty were studied in female rats from an early- and a late-maturing strain. The postweaning living conditions consisted of the presence or absence of an adult rat (intact or gonadectomized male, or intact female), or of bedding soiled by adult males. The infantile females exposed to these conditions were caged either singly or with three peers. Puberty was studied in terms of first ovulation, verified always by ovarian histology. Females of the late-maturing strain living without peers reached puberty about 2 days earlier than females that grew up in groups of four, irrespective of the presence or absence of an adult animal. In neither strain was puberty accelerated by the presence of intact males or the soiled bedding of such males. These results do not support the view that in the rat pheromones from adult males enhance puberty in females, contrary to what is known to happen in the mouse.

Sexual arousability and the menstrual cycle.

We have repeated a study of the objective (labium minus temperature) and subjective (semantic scales) changes in sexual arousability over the menstrual cycle to erotic video stimulation in 20 women with natural cycles. We extended the study with concomitant vibrotactile stimulation of the clitoral region. In an earlier study women tested for the first time in their follicular phase were sexually more aroused (as indicated by greater increase in labium minus temperature) than women tested for the first time during their luteal phase. When these women were retested in their alternative phase of the menstrual cycle, those who were then follicular did not score higher than those who were then luteal. We then postulated the involvement of a cognitive or conditioned phenomenon which was responsible for the women's repeating their initial phase-dependent response when tested the second time in their alternative phase of the cycle. Identical results were obtained in the present study as far as sexual arousability is concerned during the first test, i.e. follicular women generally scored higher than luteal women; this difference in response persisted during the second test. Concomitant vibrotactile stimulation had no effect on labium minus temperature change but raised subjective sexual arousal and caused a 'more pleasurable' response to the erotic video. Subjective sexual and subjective genital arousal were positively correlated, but neither was significantly correlated with labium minus temperature change. The relationship between menstrual cycle phase and sexual arousal was further substantiated by the reported greater increase in sexual desire after the first erotic video in follicular women than in luteal women. This increased desire, as well as more erotic fantasies, persisted during the next 24 h. In conclusion, studies into effects of menstrual cycle phase on sexual arousability in the laboratory should seriously consider the possible learning and conditioning effects as suggested by the present investigation.

Anterior hypothalamic lesions and pubertal development in female rhesus monkeys.

Electrolytic lesions were made in the anterior hypothalamus of 8 prepubertal female rhesus monkeys, aged 1.1-1.7 years. Six unoperated females served as controls. No effects were found of the lesions upon age and body weight at menarche or at first ovulation, as estimated by blood levels of progesterone and laparoscopic observations. From these findings it appears that the neural control of puberty in the female rhesus may not be exerted through the anterior hypothalamus, in contrast with the rat and ferret. Further, an attempt was made to identify biometric correlates of hormonal changes during puberty. Firstly, the well known dip in growth rate, about 0.4 years before menarche, was observed. Secondly, there was a marked spurt in growth of the nipples starting at 0.2 years before menarche. The close temporal association between accelerated nipple growth and menarche suggests that both of these developmental characteristics result from changes in (presumably ovarian) steroid hormone secretion.

An investigation into the management of patients with erection problems in general practice.

In a study on the diagnosis and treatment of erectile dysfunction (ED) in general practice, a self-selected sample of 90 general practitioners (GPs) (76 males, 14 females) from a predominantly urban population of 525 GPs were interviewed. It appeared that 59% would not actively interrogate a patient when an erection problem was suspected, either because of lack of time or lack of competence. A specific strategy for the management of erection problems was reported by 60% of the GPs. In practice, most GPs treated their own patients. About 60% of the GPs tried to keep up to date through reading and postgraduate courses. Impediments acknowledged in the interaction with patients were the experience of lack of knowledge and skills (63%) and emotional inhibitions to detailed questioning (49%). None of the GPs in this study had as yet utilized more recent treatment methods such as intracavernous drug injections and vacuum constriction devices. It is recommended that in postgraduate training courses GPs receive practical training in proper history taking and in the application of modern treatment methods.

The patient with erection problems and his general practitioner.

Ninety general practitioners (GPs) interviewed about 136 of their latest patients and 38 of these patients participated in a study on the diagnosis and treatment of erectile dysfunction (ED). On average one GP saw 4 patients with ED in one year. About half of these patients were 60 years or over, nearly all had steady relationships, and their level of education seemed to be above average. There was a median patient delay of about one year between the onset and presentation of the complaint. Doctors and patients differed markedly in their reports on the diagnosis and the treatment outcome. Doctors favoured a psychosocial cause, patients a somatic cause. In general, doctors had a more positive view on the results of treatment, while patients were often disappointed. Nevertheless, a much higher proportion of the patients than was expected by the doctors wished to renew contact with their doctor.

The limited practical value of color Doppler sonography in the differential diagnosis of men with erectile dysfunction.

From the files of the outpatient urology department 44 men with ED had undergone both psychophysiological diagnostic screening (PDS, VSS, VSS+VIB, ICI+VSS+VIB) and color Doppler sonography testing (CDS, including VSS). PDS was carried out by one medical physiologist, CDS by one urologist. The diagnoses reached could be compared. This study revealed that CDS in ED-patients often resulted in an incorrect diagnosis, that is a presumed vascular abnormality while many such patients demonstrated firm erections under PDS-laboratory test conditions. Thus, it was concluded that CDS as a (first) screening test in ED-patients is of limited value. It was further emphasized that PDS, although giving quite relevant information about possible etiology and therapeutic treatment, is not a necessary first screen. Good history taking, preferably of the man and his partner, is still the basic first screen and quite often offers enough information to make a treatment plan with reasonable likelihood of success.

Autoimmune lymphoproliferative syndrome type III: an indefinite disorder.

Autoimmune Lymphoproliferative Syndrome (ALPS) is a childhood disorder characterized by chronic nonmalignant lymphoproliferation and autoimmunity. Although the pathogenesis is not fully understood, deficient Fas mediated apoptosis appears to be an important factor. This deficiency can be caused by a mutation of the APT1 gene (ALPS type Ia), of the FasL gene (ALPS type Ib), or of the Caspase-10 gene (ALPS type II). In one sub population of patients, no mutations have been identified as yet (ALPS type III). According to published data, the latter group is much smaller than the group of patients with ALPS type Ia. However, because of the variability of the clinical presentation and the absence of a known genetic defect, this disease is difficult to diagnose, the more so as few data have been reported on these patients. Thus, ALPS type III could be more common than believed until now. In this review we provide evidence for this hypothesis.

Autoimmune lymphoproliferative syndrome type III, an indefinite disorder.

Autoimmune Lymphoproliferative Syndrome (ALPS) is a childhood disorder characterized by chronic nonmalignant lymphoproliferation and autoimmunity. Although the pathogenesis is not fully understood, deficient Fas mediated apoptosis appears to be an important factor. This deficiency can be caused by a mutation of the APTI gene (ALPS type Ia), of the FasL gene (ALPS type Ib), or of the Caspase-10 gene (ALPS type II). In one sub population of patients, no mutations have been identified as yet (ALPS type III). According to published data, the latter group is much smaller than the group of patients with ALPS type Ia. However, because of the variability of the clinical presentation and the absence of a known genetic defect, this disease is difficult to diagnose, the more so as few data have been reported on these patients. Thus, ALPS type III could be more common than believed until now. In this review we provide evidence for this hypothesis.

Attractivity of male rats induced by estradiol and progesterone.

From the present study it appears that castrated male rats can be made attractive to intact males by estradiol benzoate plus progesterone (EB + P). For the determination of attractivity a residential plus-maze was used in which the resident male could choose between the company of the experimental male and that of an ovariectomized female with or without EB + P. In one experiment the males were castrated in adulthood; these animals were less attractive than the ovariectomized females with a similar hormone treatment. In a second experiment it was found that males that had been castrated at birth were as attractive as the ovariectomized females, after treatment with EB + P. These findings indicate that the attractivity that can be induced by EP + P in gonadectomized adult rats is somewhat but not wholly reduced if androgen is present during the neonatal period.

Vaginal bacterial flora partially determines sexual attractivity of female rats.

This study shows that the bacterial flora of the rat's vagina plays a role in the determination of female sexual attractivity. In the experiment described, the change in attractivity of a female rat was monitored during the estrous cycle by using a residential plus-maze. The time that male rats spent in the vicinity of a female rat was used as a measure of the attractivity of that female. The vaginal bacterial flora of one of these females was killed by daily injection in the vagina of an antibiotic solution. It was found that male rats preferred the company of an estrous female with an untreated vagina over the company of an estrous female with a 'sterilized' vagina. This indicates that the vagina produces an olfactory signal which plays a role in her attractivity for a male rat.

The role of the preputial glands in sexual attractivity of the female rat.

This study shows that olfactory cues contribute to attractivity of the female rat but that the preputial glands are a minor source of such olfactory stimuli. In the experiments described, a residential plus-maze was used. The amount of time resident male rats spent in the vicinity of an estrous female rat was used as a measure of female attractivity. In the first experiment it was shown that male rats were attracted to anaesthetized estrous female rats but that these females were less attractive than unanaesthetized estrous females. In the second experiment it was shown that after removal of the preputial glands females were still attractive for male rats. The results of the third experiment demonstrated that estrous preputialectomized female rats remained attractive for male rats when they were anesthetized. When the resident male rats could make a choice between an estrous preputialectomized female and an estrous control female there was a tendency for the latter to be preferred. It can be concluded that olfactory cues enhance the attractivity of an estrous female rat, but that it is not the only sensory modality used by the female to attract a (distant) male. The preputial glands are of minor importance as a source of olfactory signals.

Ontogeny of sex differences in open-field ambulation in the rat.

The effects of age and gonads were studied in rats subjected to open-field tests, during which ambulation behavior was recorded. Subjects were three groups of male and female rats: sham-operation on day 1 and day 21; gonadectomy on day 1 and sham-operation on day 21; and sham-operation on day 1 and gonadectomy on day 21. Half of each group were tested in a circular open field (3 min/day, 3 consecutive days) on days 28-30; the others were tested on days 47-49. Representatives of both batches were tested again in a square open field on days 76-78. There was a sex difference in ambulation at 77 days, but not at earlier ages. In animals gonadectomized on day 1 or day 21 the sex difference in adulthood failed to occur, because castration caused the males to ambulate as much as sham-operated and ovariectomized females. On the basis of our results and reports in the literature it is suggested that testicular secretions around puberty have an organizing effect on ambulation behavior. The intact adult male rat ambulates less than the adult female and this difference persists after castration in adulthood. Castration well before puberty prevents the development of the adult sex difference.