Studies on neurosteroids XV. Development of enzyme-linked immunosorbent assay for examining whether pregnenolone sulfate is a veritable neurosteroid

An enzyme-linked immunosorbent assay (ELISA) of pregnenolone sulfate (PREGS) has been developed for examining whether it is a veritable neurosteroid. 11-Hemiglutaryloxy-PREGS was newly synthesized and conjugated with bovine serum albumin (BSA), which was injected to rabbits for the production of anti-PREGS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 30 pg. Although the antibody showed some cross-reactivity with PREG (4.4%), it easily discriminated other related steroids reported to exist in the mammalian brain. The rat brain homogenate was treated with hexane and subjected to an OASIS HLB cartridge, which was washed with AcOEt to remove the unconjugated steroids, and then the desired sulfate was eluted with EtOH. The recovery rate of PREGS through the pretreatment was satisfactory, but its brain levels in the preliminary experiments were much lower than those previously measured by gas chromatography (GC)–mass spectrometry (MS) after solvolysis. These results practically agreed with our previous results by liquid chromatography (LC)–electrospray ionization (ESI)–MS without deconjugation.     

Repeated hepatocyte injury promotes hepatic tumorigenesis in hepatitis C virus transgenic mice

Although hepatitis C virus (HCV) is a well-known causative agent of hepatocellular carcinoma (HCC), the mechanism by which HCV induces HCC remains obscure. To elucidate the role of HCV in hepatocarcinogenesis, a model of hepatocyte injury was established using HCV core transgenic mice, which were developed using C57BL/6 mice transfected with the HCV core gene under control of the serum amyloid P component promoter. After 18–24 months, neither steatosis nor hepatic tumors were found in transgenic mice. The extent of hepatocyte injury and tumorigenesis were then examined in transgenic mice following repeated administration of carbon tetrachloride (CCl₄) using various protocols (20%, 1/week; 10%, 2/week and 20%, 2/week). Serum alanine aminotransferase (ALT) levels did not differ among HCV core transgenic mice and non-transgenic littermates; however, after 40 weeks, hepatic adenomas preferentially developed in transgenic mice receiving 20% CCl₄ once weekly. Moreover, HCC was observed in transgenic mice receiving 2 weekly injections of a 20% solution of CCl₄, and was not observed in the non-transgenic control mice. In conclusion, the HCV core protein did not promote hepatic steatosis or tumor development in the absence of hepatotoxicity. However, the HCV core protein promoted adenoma and HCC development in transgenic mice following repeated CCl₄ administration. These results suggest that hepatotoxicity resulting in an increased rate of hepatocyte regeneration enhances hepatocarcinogenesis in HCV-infected livers. Furthermore, this experimental mouse model provides a valuable method with which to investigate hepatocarcinogenesis.     

Follow-up studies of anorectal malformations after posterior sagittal anorectoplasty

Background/Purpose: There are few follow-up studies comparing posterior sagittal anorectoplasty (PSARP) with conventional procedures for patients with anorectal malformations (ARM). The authors have examined retrospectively postoperative anorectal function of patients with ARM treated with PSARP compared with those treated with conventional methods. Methods: Anorectal function in 23 patients with high and intermediate type anorectal malformations (PSARP group), who underwent PSARP more than 4 years previously, were assessed by Kelly's clinical scoring system and objective studies. These results were compared with those in 14 cases (5 high and 9 intermediate type cases; control group), who underwent other conventional surgical procedures. Results: Using Kelly's clinical scoring system, scores of the PSARP group compared with the control group were good in 48% versus 21%, fair in 48% versus 58%, and poor in 4% versus 21%, respectively. Barium enema studies suggested better anorectal sphincteric function in patients with high anorectal malformation in the PSARP group. Magnetic resonance imaging (MRI) studies showed more correct placement of the rectum through the striated muscle complex in the PSARP group at the I-line level. Manometric studies showed no difference in maximum resting pressure, anal canal length, and the incidence of anorectal reflex between the two groups. Conclusions: The favorable results of MRI and barium enema studies can be explained by direct visualization of the striated muscle complex with the aid of electrical stimulation as well as no harmful effects of amputation of the sphincter muscle in PSARP. However, manometric studies suggest anorectal function in patients with high and intermediate anorectal malformations is limited even after PSARP. Long-term postoperative follow-up with adequate bowel management is required for all patients with high or intermediate anorectal malformation.     

Inhaled nitric oxide with early surgery improves the outcome of antenatally diagnosed congenital diaphragmatic hernia

Background/Purpose: The outcome of antenatally diagnosed congenital diaphragmatic hernia (CDH) has remained poor despite aggressive therapeutic strategies. Since 1996, the authors have used a new approach including early surgery and inhaled nitric oxide (iNO). The aim of this study is to determine whether early surgery in combination with iNO improves the clinical outcome of antenatally diagnosed CDH. Methods: From 1988, 40 consecutive neonates with antenatally diagnosed CDH were admitted to the authors' hospital. Ten cases of fatal chromosomal anomalies or major cardiac anomalies were excluded from this study. From 1988 through 1995 (period 1: n = 13), delayed surgery was used in high-risk CDH. From 1996 through 2000 (period 2: n = 17), early surgery in combination with iNO was used. The severity of lung hypoplasia was evaluated using the fetal lung/thorax transverse area ratio (L/T). High-frequency oscillatory ventilation (HFOV) was used routinely during the study periods, and extracorporeal membrane oxygenation (ECMO) was used on basis of conventional entry criteria. The authors compared the clinical outcome, use of ECMO, and the L/T between the 2 periods retrospectively. Results: Patients in the 2 periods were comparable in terms of birth weight, gestational age, and the L/T. The mean age at surgery was 3.1 [plusmn] 4.9 days in period 1, and 0.8 [plusmn] 1.1 days in period 2. Fewer infants in period 2 compared with period 1 were treated with ECMO (period 1, 62% v period 2, 6%; P [lt ].01). There was significant difference in the survival rate between the 2 periods (period 1, 38% v period 2, 94%; P [lt ] .01). Conclusion: Our data suggest that early surgery and iNO improves the outcome and reduces the requirement of ECMO in the treatment of antenatally diagnosed CDH. J Pediatr Surg 37:1188-1190.     

IP telephony--new horizon for telemedicine and e-health

Internet Protocol (IP) based networks are increasingly being used as alternatives to the traditional circuit-switched networks for carrying voice traffic. IP telephony is taking a firm hold in the world telecommunication market for a wide range of applications including telemedicine. The merits of IP telephony for telemedicine lie in cost-savings and new applications and functionalities it may render. In this paper, we try to discuss and analyze the implications of IP telephony on the evolution and deployment of telemedicine and e-health.     

Early attachment of uncultured retinal pigment epithelium from aged donors onto Bruch's membrane explants

Retinal pigment epithelium (RPE) transplantation might replace cells lost as a consequence of choroidal neovascular membrane excision in patients with age-related macular degeneration (AMD). Autologous transplantation of RPE cells harvested from a peripheral biopsy may overcome problems of immune rejection. To study the feasibility of autologous RPE cell transplantation, the authors examined the attachment of freshly harvested RPE cells from aged donors onto Bruch's membrane explants, debrided to (1) remove or (2) preserve the RPE basement membrane. Human retinal pigment epithelial sheets were harvested from adult donor eyes (N = 12, mean age 79.00 +/- 9.40 years) and, following incubation in collagenase, were mechanically fragmented into microaggregates. Microaggregates (approximately 120 000 cells) were seeded onto the paired explants (7 mm diameter) and incubated for 20 min, 1, 4, or 24 hr at 37 degrees C. The percent coverage of the debrided surface by microaggregates was determined by sampling the center of the explants with scanning electron microscopy. RPE microaggregate attachment to Bruch's membrane was significantly greater at all time points analysed in samples with intact basement membrane versus those with an exposed inner collagenous layer. Coverage of debridements retaining intact RPE basement membrane was 1.83 +/- 1.10% at 20 min, 3.54 +/- 2.14% at 1 hr, and 8.68 +/- 2.63% at 4 hr. Coverage of debridements lacking basement membrane was 0.10 +/- 0.04% at 20 min, 0.39 +/- 0.25% at 1 hr, and 0.63 +/- 0.42% at 4 hr. Based on their morphologic appearance, many cells were dying as early as 1 hr following seeding. To increase surface coverage, the authors seeded four times the above number of cells and incubated the specimens for 1 hr. Coverage on explants lacking RPE basement membrane showed no increase in the number of cells attached to the inner collagenous layer. There was a significant approximately three-fold increase in the number of cells attached in the presence of basement membrane. These results indicate that if RPE cells from aged human donors are used for transplantation, some modification of the Bruch's membrane surface or the cells must be considered for cell attachment and eventual cell survival.     

Association between a polymorphism in the promoter region of the dopamine D2 receptor gene and schizophrenia in Japanese subjects: replication and evaluation for antipsychotic-related features

To replicate a previously found negative association between the Del allele of the -141C Ins/Del polymorphism in the 5'-promoter region of the dopamine D2 receptor gene (DRD2) and schizophrenia in Japanese subjects and to examine whether this polymorphism is related to the features of antipsychotic drug treatment, we genotyped 94 control subjects and 234 schizophrenic patients. The schizophrenic patients had a significantly lower frequency of the Del allele (p < 0.05). We found a non-significant trend towards a higher frequency of the Del allele in schizophrenic patients susceptible to neuroleptic-induced extrapyramidal symptoms. The daily dosage of haloperidol, the steady-state concentration of serum haloperidol per daily dosage, and the recent 1-yr cumulative neuroleptic dosage were lower in patients with the Del/Del genotype than in the other patients. These findings support the view that the polymorphism is associated with schizophrenia in Japanese subjects and provide hints for further attempts to establish the relationship between this polymorphism and the features of antipsychotic drug treatment.     

New treatment of dry eye: the effect of calcium ointment through eyelid skin delivery

AIM: To demonstrate the efficacy of a petrolatum based calcium ointment applied to the lower lid skin in the management of dry eye. METHODS: In a controlled double masked study, the effects of water free petrolatum ointment containing calcium carbonate (10% w/w) on tear functional factors and ocular surface vital staining in dry eye patients were observed. Petrolatum without calcium carbonate served as control. Patients were instructed to place ointment to the lower lid skin twice a day. Evaluation of subjective complaints, fluorescein and rose bengal staining patterns, blink rate, tear evaporation and tear break up time (BUT) were performed before and 3 months after treatment. In order to demonstrate the movement of petrolatum from the skin to the tear film, petrolatum containing 1% sodium fluorescein was placed on the lower lid of four healthy volunteers, and the concentration of fluorescein in the tear film was followed up to 6 hours using an anterior fluorometer. RESULTS: Subjective symptoms significantly improved in both the calcium group (p=0.001) and control (p=0.012), while only the calcium group demonstrated a significant improvement in fluorescein (p=0.043), rose bengal (p=0.021) scores, and blink rate (p=0.004). Tear evaporation also significantly decreased in both the calcium group (p=0.0004) and control (0.043). BUT did not improve in either group. CONCLUSION: Petrolatum based calcium ointment significantly improved symptoms, tear dynamics, and ocular surface staining in dry eye patients. However, some of the therapeutic effects may be due to lipids in the petrolatum vehicle. Petrolatum applied to the lower lid skin is an effective drug delivery system for slowly releasing drugs to the ocular surface.