Comparative development of<Emphasis Type="Italic"> Eimeria tenella</Emphasis> (Apicomplexa) in host cells in vitro

Attempts to propagate Eimeria tenella in cell culture over the years have met with limited success. The host cell type is an important parameter in such cultures. This study assessed the ability of different host cell lines to support E. tenella infection in vitro. The initial development in cell lines and the effects of incubation at 37 degrees C and 41 degrees C on the host-parasite relationship was investigated. Eleven cell lines were seeded into 96 well plates and incubated at 37 degrees C in 5% CO(2) to reach confluency. Sporozoites of E. tenella were inoculated into wells and allowed to invade at 37 degrees C and 41 degrees C. Intracellular parasite development was quantified using (3)H-uracil incorporation. All cell lines facilitated parasite invasion and development. The MDBK cell line supported the highest degree of E. tenella development. A time-course study was undertaken to look at this host cell-parasite relationship during infection.     

New Enhancements of the Scrotal One-Incision Technique for Placement of Artificial Urinary Sphincter Allow Proximal Cuff Placement

IntroductionUrinary incontinence impairs sexual functioning and sexual satisfaction. Traditional artificial urinary sphincter (AUS) implantation requires perineal incision for cuff placement and a second inguinal incision for reservoir and pump placement. We believed AUS could be placed easier and quicker through one scrotal incision.AimIn an effort to effect more proximal placement of the cuff while keeping the advantages of the one scrotal incision technique, we report enhancements to the original surgical technique.MethodsThirty patients have been operated upon using the enhanced technique. A modification of the SKW retractor system (AMS) facilitates deep bulbar exposure. Twenty patients were first time implantations and 10 were revisions with five of the revisions having had the original AUS placed by traditional two-incision technique. Two of the first time AUS patients received an inflatable penile prosthesis through the same incision.Main Outcome MeasuresWe evaluated site of cuff placement, sizes of cuffs used, postoperative continence status.ResultsAll of the virgin AUS required dissection of the bulbocavernosus muscle prior to cuff placement. In scrotally placed revisions, replacement cuffs were situated considerably proximal (4.5–7.5 cm) to the original cuff site. The perineal placed revisions were accomplished through a scrotal incision with replacement of two cuffs in the same site and the three other patients immediately distal. No intraoperative complications were seen. One patient developed scrotal hematoma requiring drainage. Only 15 patients are available for follow-up and all are socially continent (one pad or less).ConclusionsTransscrotal approach is used safely and efficiently for penile implants and AUS implantation. The new enhancements to the one-scrotal incision technique allow more proximal cuff placement as evidenced by the bulbocavernosus muscle dissection and use of larger cuffs. Continence rate is similar to rates achieved with perineal placement of cuff found in the literature. Wilson SK, Aliotta PJ, Salem EA, and Mulcahy JJ. New enhancements of the scrotal one incision technique for placement of artificial urinary sphincter allow proximal cuff placement.     

Hepatic Abscess After Yttrium-90 Radioembolization for Islet-Cell Tumor Hepatic Metastasis

Infectious complications after yttrium-90 (y-90) radioembolization of hepatic tumors are rare. Most reports describe hepatic abscesses as complications of other locoregional therapies, such as transcatheter arterial embolization or chemoembolization. These usually occur in patients with a history of biliary intervention and present several weeks after treatment. We report a case of hepatic abscess formed immediately after y-90 radioembolization of a hepatic metastasis in a patient who had no history of previous biliary instrumentation.     

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIV(KU-1bMC33)) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques

Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5' to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIV(Vpenv)) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIV(Vpenv)-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV(Vpenv)-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIV(KU-1bMC33). Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIV(Vpenv)-infected cells compared to cultures inoculated with parental SHIV(KU-1bMC33). Furthermore, virus was observed maturing into intracellular vesicles of SHIV(Vpenv)-infected cells. To assess the pathogenicity of SHIV(Vpenv), three pig-tailed macaques were inoculated with the SHIV(Vpenv) and monitored for 6 months for CD4(+) T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIV(Vpenv) caused a severe CD4(+) T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4(+) T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIV(Vpenv) with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.     

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

Novel placental ultrasound assessment: Potential role in pre-gestational diabetic pregnancy

Objectives

Management of women with pre-gestational diabetes continues to be challenging for clinicians. This study aims to determine if 3D power Doppler (3DPD) analysis of placental volume and flow, and calculation of placental calcification using a novel software method, differ between pregnancies with type 1 or type 2 diabetes and normal controls, and if there is a relationship between these ultrasound placental parameters and clinical measures in diabetics.

Methods

This was a prospective cohort study of 50 women with diabetes and 250 controls (12–40 weeks gestation). 3DPD ultrasound was used to evaluate placental volume, vascularisation index (VI), flow index (FI) and vascularisation-flow index (VFI). Placental calcification was calculated by computer analysis. Results in diabetics were compared with control values, and correlated with early pregnancy HbA1c, Doppler results and placental histology.

Results

Placental calcification and volume increased with advancing gestation in pre-gestational diabetic placentae. Volume was also found to be significantly higher than in normal placentae. VI and VFI were significantly lower in diabetic pregnancies between 35 and 40 weeks gestation. A strong relationship was seen between a larger placental volume and both increasing umbilical artery pulsatility index and decreasing middle cerebral artery pulsatility index. FI was significantly lower in cases which had a booking HbA1c level ≥6.5%. Ultrasound assessed placental calcification was reduced with a histology finding of delayed villous maturation. No other correlation with placental histology was found.

Conclusions

This study shows a potential role for 3D placental evaluation, and computer analysis of calcification, in monitoring pre-gestational diabetic pregnancies.     

Post-operative abdominal CT scanning in extrahepatic biliary atresia

A retrospective review of the abdominal CT scans of 26 children with extrahepatic biliary atresia was performed, and the results were correlated with available surgical and pathologic data. Associated congenital anomalies or acquired abnormalities were identified in these patients. Congenital anomalies included polysplenia, venous anomalies and bowel stenosis. Acquired abnormalities developed secondary to cirrhosis, portal hypertension, intrahepatic biliary duct dilatation, and hepatic ischemia. Despite frequent episodes of ascending cholangitis in these children, no hepatic abscesses were identified by CT or by pathologic examination. In conclusion, abdominal CT scanning of children with extrahepatic biliary atresia can define congenital and acquired abnormalities and provide important anatomic data for the surgeons before liver transplantation.     

Is phenytoin contraindicated in patients receiving cranial irradiation ?

Three recent publications have reported the development of erythema multiforme and Stevens-Johnson syndrome in patients receiving cranial irradiation and sodium phenytoin. Some authors have recommended that patients receiving whole brain radiation therapy and who have had seizures should not be prescribed phenytoin but an alternative anticonvulsant. This article reviews the current literature pertaining to the development of this potentially lethal complication in patients receiving whole brain radiation and phenytoin, with reference to the single recorded case of Stevens-Johnson syndrome in a patient receiving cranial irradiation and phenytoin in Auckland, New Zealand. While the clinical picture in the 16 patients reported in the literature and the current case report differed from the classical form of erythema multiforme, a similar pattern of presentation and outcome appeared in all patients reviewed, suggesting that the combination of phenytoin, cranial irradiation and the gradual reduction of concomitant steroids seem to lead to the development of erythema multiforme and/or Stevens-Johnson syndrome. The data presented, although sparse, suggest that phenytoin should not be prescribed in patients receiving cranial irradiation.