Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.     

An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss

A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.     

Cyclosporine A inhibits acetylcholinesterase activity in selected parts of the rat brain

Cyclosporine A (CsA) is the major immunosuppressive drug used for organ and neural transplantation and the therapy of selected autoimmune diseases. We investigated the effect of CsA on the activity of acetylcholinesterase (AChE) in the frontal cortex, hippocampus, septum, and basal ganglia. AChE was determined spectrophotometrically with acetylthiocholine as substrate and 5,5-bis-2-nitrobenzoic acid as chromogen. CsA was administered in single doses of 20 or 45 mg/kg perorally; in the case of the higher dose we also performed a repeated administration of CsA in three consecutive doses separated by 24 h intervals. Both lower and higher doses of CsA decreased AChE activity in the frontal cortex and hippocampus to practically the same extent. On the contrary, AChE activity was more diminished in the case of the higher dose of CsA used in the septum and basal ganglia. Repeated administration of the higher dose of CsA did not lead, with the exception of the hippocampus, to a further decrease in AChE activity in the brain structures observed. These findings contribute to rare evidence concerning the interaction of CsA and the cholinergic system in the brain.     

Malaria vectors in the Bioko Island (Equatorial Guinea): estimation of vector dynamics and transmission intensities

The current study was performed on the Bioko Island (Equatorial Guinea) with the aim of establishing a rapid assessment technique for mapping malaria risk and measuring vector densities. Human bait collection, tent traps, light traps, indoor resting collection, and window exit traps were used to collect Anopheles gambiae s.s. and Anopheles funestus, the two anopheline species involved in malaria transmission in this island. Capture data were used to compare differences in the behavior and vectorial capacity of An. gambiae s.s. and An. funestus. Differences in the two species of mosquitoes were found in relation to the season and trapping methods used. Entomological inoculation rates (EIR) for Plasmodium falciparum were calculated using a polymerase chain reaction (PCR) test with individual anopheline mosquitoes from human bait collections in two villages during the dry and rainy seasons. P. falciparum sporozoites were detected from both dissected heads/thorax and abdomens of both species.     

A dinucleotide mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome (LWFS); a horse variant of Hirschsprung disease

Lethal white foal syndrome (LWFS) is a congenital anomaly of horses characterized by a white coat colour and aganglionosis of the bowel, which is similar to Hirschsprung disease (HSCR). We decided to investigate possible mutations of the endothelin-B receptor gene ( EDNRB ) in LWFS as recent studies in mutant rodents and some patients have demonstrated EDNRB defects. First, we identified a full-length cDNA for horse EDNRB . This cDNA fragment contained a 1329 bp open reading frame which encoded 443 amino acid residues. The predicted amino acid sequence was 89, 91 and 85% identical to human, bovine and mouse as well as rat EDNRB respectively, but only 55% identical to the human, bovine and rat endothelin A receptor (EDNRA). Secondly, sequence analysis, together with allele-specific PCR and the amplification- created restriction site (ACRS) technique, revealed a dinucleotide TC-- >AG mutation, which changed isoleucine to lysine in the predicted first transmembrane domain of the EDNRB protein. This was associated with LWFS when homozygous and with the overo phenotype when heterozygous.      

Malignant mesothelioma of the tunica vaginalis

Case report of a new case of malignant mesothelioma of the tunica vaginalis testis. A mediterranean male age 62, presented with enlargement and swelling of the scrotum, treated as orchiepydidymitis within the previous 3 months. Physical and ultrasonography examination showed a left scrotal mass with extension to the skin of the scrotum. After surgery (orchiectomy and partial hemiscrotectomy) and histopathology diagnosis of locally advanced malignant mesothelioma, CT showed metastasis in retroperitoneum, Lung and Liver. The patient undergoes chemotherapy and radiotheraphy with a poor prognose. We review the diagnosis, histopathology and therapeutical approach for this uncommon kind of paratesticular tumor (less than 80 cases reported in the last 30 years).     

Informed consent, parental awareness, and reasons for participating in a randomised controlled study

BACKGROUND: The informed consent procedure plays a central role in randomised controlled trials but has only been explored in a few studies on children. AIM: To assess the quality of the informed consent process in a paediatric setting. METHODS: A questionnaire was sent to parents who volunteered their child (230 children) for a randomised, double blind, placebo controlled trial of ibuprofen syrup to prevent recurrent febrile seizures. RESULTS: 181 (79%) parents responded. On average, 73% of parents were aware of the major study characteristics. A few had difficulty understanding the information provided. Major factors in parents granting approval were the contribution to clinical science (51%) and benefit to the child (32%). Sociodemographic status did not influence initial participation but west European origin of the father was associated with willingness to participate in future trials. 89% of participants felt positive about the informed consent procedure; however, 25% stated that they felt obliged to participate. Although their reasons for granting approval and their evaluation of the informed consent procedure did not differ, relatively more were hesitant about participating in future. Parents appreciated the investigator being on call 24 hours a day (38%) and the extra medical care and information provided (37%) as advantages of participation. Disadvantages were mainly the time consuming aspects and the work involved (23%). CONCLUSIONS: Parents' understanding of trial characteristics might be improved by designing less difficult informed consent forms and by the investigator giving extra attention and information to non-west European parents. Adequate measures should be taken to avoid parents feeling obliged to participate, rather than giving true informed consent.