Deregulation of the G1/S-phase control in human testicular germ cell tumours

Deregulated cell cycle and defective genome-integrity checkpoints are among the hallmarks of cancer.Here we summarize our recent studies of key components of the GI/S machinery in normal human spermatogenesis, and their abnormalities in testicular germ cell tumours (TGCTs), with special emphasis on carcinoma in situ lesions (CIS). Our combined immunohistochemical and immunoblotting analyses of normal human adult and fetal testes, CIS, seminomas, embryonal carcinomas, and teratomas, revealed an 'unorthodox' spectrum of defects within the so-called RB pathway in TGCTs. The early aberrations included lack of expression of the retinoblastoma tumour suppressor (pRB) and the CDK inhibitor pl9ink4d, and overexpression of cyclin D2. Progression from CIS to invasive TGCTswas associated with loss of another two CDK inhibitors and tumour suppressors: pl6ink4a and pl8ink4c. We also found the lack of pRB and pl9ink4d in fetal gonocytes, the candidate target cell for all types of TGCTs. These findings, together with the status of the Chk2-p53 DNA-integrity checkpoint, are considered in relation to the origin, biology and pathogenesis of TGCTs, and potential implications of the GI/S defects for the curability of these tumours.     

Different modulation by histamine of IL-4 and interferon-gamma (IFN-γ) release according to the phenotype of human Th0, Th1 and Th2 clones

Histamine, an important inflammatory mediator in allergic diseases and asthma, has been reported to have modulator effects on T cells, suggesting that the bronchial microenvironment may regulate the function of resident T cells. We examined the effect of histamine on the release of the Th2-associated cytokines IL-4 and IL-5 and the Th1-associated cytokine IFN-γ by 30 CD4⁺ T cell clones from peripheral blood or bronchial biopsy of one atopic subject. Based on the IL-4/IFN-γ ratio, the clones were ascribed to the Th2 (ratio >1), Th0 (ratio 0.1 and 1) or Th1 (ratio <0.1) phenotype. Histamine inhibited IFN-γ production by Th1-like cells (P<0.02, Kruskall–Wallis), especially from bronchial biopsy, but had no effect on IL-4 release. Regarding Th0 clones, histamine inhibited IL-4 production (P<0.02) in a dose-dependent manner and slightly inhibited IFN-γ production, but had no effect on Th2-like cells. Histamine had a heterogeneous and insignificant effect on IL-5 production. The H₂-receptor antagonist ranitidine completely reversed the inhibition of IL-4 and IFN-γ production, whereas the agonist dimaprit mimicked this effect. In contrast, H₁- and H₃-receptor agonists and antagonists had no significant effect. These data demonstrate that histamine has different effects on IL-4 and IFN-γ release by T helper cells according to their phenotype via H₂-receptors. This study extends the immunomodulatory effects of histamine which may contribute to the perpetuation of airway inflammation in asthma.     

Frequent polymorphism of the mitochondrial DNA polymerase gamma gene (POLG) in patients with normal spermiograms and unexplained subfertility

BACKGROUND: Male fertility largely depends on the quality of sperm production, which may be affected by environmental and genetic factors. In this study, we explored a possible role of the polymerase gamma (POLG) gene polymorphism, recently reported to be associated with male infertility in some populations. METHODS: The polymorphic CAG repeat (usually 10 codons long) in the POLG gene was studied in 1298 male subjects: 429 patients with infertility/subfertility, and 869 controls (495 men from the general population with unknown fertility and 374 recent fathers). In all subjects, the POLG polymorphism was assessed in relation to their semen quality, and--in the fertile controls--with biological fecundity measured as waiting time-to-pregnancy (TTP) for the couples. In the patients lacking the common POLG allele, the outcome of the assisted reproductive techniques (ART) for the couples was evaluated. RESULTS: The absence of one (10/ not equal to 10) or both common POLG alleles (not equal to 10/not equal to 10) was more frequent among the subfertile patients than among fertile controls (P=0.021 and P=0.04 respectively). The estimated predictive value for infertility in a man homozygous for the POLG polymorphism was 15.5% (95% CI: 4.8-51%). There was a positive association with sperm concentration: 14.3% of the normospermic subfertile patients were homozygous for the absence of the common POLG allele (not equal to 10/not equal to 10), in comparison with 2.3% of unselected controls (P=0.001) and 0.9% of the fertile men (P=0.0001). No association with sperm motility, morphology and TTP was found. Spermatozoa of the three not equal to 10/not equal to 10 patients treated with IVF retained the ability to penetrate the egg, but the fertilization rate was low. Nine homozygous not equal to 10/ not equal to 10 patients were treated with ICSI, resulting in pregnancy in seven couples. CONCLUSIONS: The POLG gene polymorphism should be considered as a possible contributing factor in patients with unexplained subfertility and normal spermiograms. The oocyte penetration ability of sperm may be partially impaired in the not equal to 10/not equal to 10 patients but most of them can be successfully treated with ICSI.     

A study of finger lengths, semen quality and sex hormones in 360 young men from the general Danish population

BACKGROUND: It has been suggested that finger length may correlate with function or disorders of the male reproductive system. This is based on the HOXA and HOXD genes' common embryological control of finger development and differentiation of the genital bud. The objective of this study was to explore the association between the ratio of 2nd to 4th finger length (2D:4D ratio) and testis function in a sample of young Danish men from the general population. METHODS: Semen samples and finger measurements were obtained from a total of 360 young Danish men in addition to blood samples for sex hormone analysis to describe the possible association between 2D:4D and semen and sex-hormone parameters. RESULTS: A statistically significant inverse association with the 2D:4D was found only in relation to hormone levels of FSH in the group of young men with a 2D:4D >1 (P = 0.036) and a direct association with the total sperm count in the group of young men with a 2D:4D < or = 1 (P = 0.045). CONCLUSION: The statistically significant results may be 'false positives' (type I error) rather than representing true associations. This relatively large study of young, normal Danish men shows no reliable association between 2D:4D finger ratio and testicular function. Measurements of finger lengths do not have the power to predict the testicular function of adult men.     

The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission

Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increased in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60) repeats for FRAXA, 31-60 for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.      

Semen quality and reproductive hormones before and after orchiectomy in men with testicular cancer

PURPOSE: We clarify the impact of removal of the tumor bearing testis on semen quality and reproductive hormones in men with testicular cancer. MATERIALS AND METHODS: Semen quality and levels of reproductive hormones were investigated in 48 men before and after orchiectomy for testicular cancer. Semen analysis was done in 35 of these men and hormone analyses were done in 47. The hormone data of patients with (14) or without (33) elevated values of human chorionic gonadotropin (HCG) were analyzed separately. RESULTS: Median sperm concentration and total sperm count decreased from 17 x 10(6)/ml. (range 0 to 117) and 39 x 10(6) (0 to 433), respectively, before to 7 x 10(6)/ml. (0 to 69) and 30 x 10(6) (0 to 200), respectively, after orchiectomy. After orchiectomy sperm concentration was decreased in 30 of 35 men (p = 0.001) and azoospermia developed in 3 (9%). In men without detectable HCG median follicle-stimulating hormone levels increased (p <0.001) from 5.7 IU/l. (range 0.01 to 30) before to 10.0 IU/l. (4.6 to 48) after orchiectomy in 33 of 33 patients. Median inhibin B significantly decreased (p = 0.003) from 108 pg./l. (range 60 to 193) before to 95 pg./l. (less than 20 to 141) after orchiectomy. Median luteinizing hormone increased significantly from 3.1 IU/l. (range 1.1 to 9.9) before to 5.2 IU/l. (2.1 to 27) after treatment (p <0.001). Testosterone and sex hormone-binding globulin did not change significantly after orchiectomy. Patients with detectable serum HCG before orchiectomy had a considerable increase in follicle-stimulating hormone after orchiectomy, and a concomitant decrease in testosterone and estradiol. CONCLUSIONS: Semen quality was poor at diagnosis and deteriorated further after orchiectomy compared with pretreatment values. Our findings indicate that in some patients the most appropriate time for cryopreservation of semen is before orchiectomy. Androgen production was maintained by increased luteinizing hormone stimulation after orchiectomy.     

Nosocomial respiratory syncytial virus infection in Canadian pediatric hospitals: a Pediatric Investigators Collaborative Network on Infections in Canada Study

OBJECTIVE: To determine nosocomial transmission of respiratory syncytial virus (RSV) in Canadian pediatric hospitals, outcomes associated with nosocomial disease, and infection control practices. DESIGN: A prospective cohort study in the 1992 to 1994 winter respiratory seasons. SETTING: Nine Canadian pediatric university-affiliated hospitals. PARTICIPANTS: Hospitalized children with symptoms of lower respiratory tract infection (at least one of cough, wheezing, dyspnea, tachypnea, and apnea) and RSV antigen identified in a nasopharyngeal aspirate. RESULTS: Of 1516 children, 91 (6%) had nosocomial RSV (NRSV), defined as symptoms of lower respiratory tract infection and RSV antigen beginning >72 hours after admission. The nosocomial ratio (NRSV/[com-munity-acquired RSV {CARSV})] + NRSV) varied by site from 2.8% to 13%. The median length of stay attributable to RSV for community-acquired illness was 5 days, but 10 days for nosocomial illness. Four children with NRSV (4. 4%) died within 2 weeks of infection, compared with 6 (0.42%) with CARSV (relative risk = 10.4, 95% confidence interval: 3.0, 36.4). All sites isolated RSV-positive patients in single rooms or cohorted them. In a multivariate model, no particular isolation policy was associated with decreased nosocomial ratio, but gowning to enter the room was associated with increased risk of RSV transmission (incidence rate ratio 2.81; confidence interval: 1.65, 4.77). CONCLUSIONS: RSV transmission risk in Canadian pediatric hospitals is generally low. Although use of barrier methods varies, all sites cohort or isolate RSV-positive patients in single rooms. Children with risk factors for severe disease who acquire infection nosocomially have prolonged stays and excess mortality.     

A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation

P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG₁) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M_r) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIbα). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of ¹²⁵I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825±2886, mean ±SD) (K_d=1.5±0.5 nm ) compared to resting platelets (2801±1278, mean ±SD) (K_d=1.5±0.6 nm ), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG₁, F(ab′)₂ or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf ) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell–cell contact, may play an active role in platelet–platelet interactions.     

Preserved male fertility despite decreased androgen sensitivity caused by a mutation in the ligand-binding domain of the androgen receptor gene

Mutations in the androgen receptor gene are considered as incompatible with preservation of fertility and have been suggested as a cause of male infertility. Two adult brothers, referred because of gynecomastia and hormonal levels in serum indicating androgen insensitivity (high sex hormone-binding globulin, and LH levels, despite extremely high testosterone concentration), turned out to be relatives to a third young man, referred independently of the two others and exhibiting identical clinical and hormonal stigmata. In all three men, we found a C-->A substitution at position 2470 (exon 7) in the androgen receptor gene, leading to a Gln824Lys mutation in the ligand-binding domain of the receptor. Exploring the family history revealed that their grandfathers, on their mothers' side, were brothers; and the Gln824Lys mutation was also found in the one of them who was still alive. Binding studies with the mutant receptor in transfected COS-7 cells, with mibolerone as ligand, exhibited equal Kd (0.7 vs. 1.0 nmol/ L), IC50 (0.8 vs. 1.1 nmol/L), and maximum binding (7.1 vs. 8.9 fmol/ 10(6) cells), as compared with the wild-type (WT) receptor. In a chloramphenicol acetyl transferase trans-activation assay, the activity of the mutant receptor was identical to that of the WT, when the synthetic androgen R1881 was'used as a ligand; but with dihydrotestosterone, in concentrations up to 10 nmol/L, the activity of Gln824Lys mutated receptor was 10-62% of the WT variant. Thus, Gln824Lys mutation was found, both in vivo and in vitro, to cause slight impairment of receptor function but was compatible with preservation of male fertility. The patients inherited the mutation from their grandfathers through their mothers, and one of the young men possessing the mutation has fathered a daughter.