Assessment of neurogenic bowel symptoms with the bowel dysfunction score in children with spina bifida: a prospective case-control study.

AIM: To compare the quality of life (QoL) in children with spina bifida with a control group of their peers using a validated questionnaire, the Neurogenic Bowel Dysfunction Score (NBDS). METHODS: The NBDS questionnaire was prospectively distributed to children attending a multi-disciplinary Spina Bifida clinic and healthy controls attending pediatric urology clinics. A score (out of 41) was assigned to each child based on their responses to the validated questionnaire. A lower score indicates better bowel function-related quality of life. SPSS software (v.25) was used for all statistical analysis. RESULTS: There were 98 respondents to the questionnaire, 48 children with spina bifida and 50 controls. The average age of respondents was 7.88 years (3-16 years). Of those with Spina Bifida, 33 (69%) were on retrograde rectal irrigations, [19 (58%) Peristeen® system, 11 (33%) tube rectal irrigations, and 3 (9%) Willis system], 6 (12%) were on laxatives, and 9 (19%) were on no treatment. The median NBDS for Spina Bifida patients was significantly higher 13.5 (2-32) compared to the control group 2 (0-26, p < 0.001). Amongst Spina Bifida patients, there was no difference in quality of life between the modalities of bowel management (p = 0.203). CONCLUSIONS: Despite active bowel management, children with spina bifida report a worse quality of life compared to the control group. In those with spina bifida, the lack of a difference between various bowel management strategies, including no treatment, indicates the need for a longitudinal study to evaluate the basis for this unexpected finding.

Laparoscopy versus ultrasonography for the evaluation of Mullerian structures in children with complex disorders of sex development.

PURPOSE: The diagnosis of children with disorders of sex development (DSD) requires a karyotype, different biochemical and radiological investigations in the context of a multidisciplinary team. The aim of this study was to compare the diagnostic accuracy of laparoscopy (L) versus ultrasonography (US) in the assessment of children with complex DSD. METHODS: We retrospectively examined the theatre database searching for children with DSD who underwent laparoscopic surgery from 1999 to 2011. The medical and radiological records were reviewed. RESULTS: Eighteen patients were identified. Age at diagnosis ranged from birth to 14 years (mean 2.5 years). There were seven patients with 46XY dysgenetic testicular DSD (4 mosaic Turner, 3 mixed gonadal dysgenesis), seven patients with 46XY non-dysgenetic testicular DSD (4 persistent Mullerian duct syndrome, 2 complete androgen insensitivity syndrome, one unknown), two patients with ovotesticular DSD, one patient with 46XX DSD (congenital adrenal hyperplasia) and one patient with 46XY DSD complete sex reversal. Fifteen underwent ultrasonography prior to laparoscopy. Both modalities identified Mullerian structures in seven (47 %) patients, in one (7 %) patient US and L confirmed the absence of Mullerian structures, while in six (40 %) patients there was discordance, with US failing to visualize pelvic Mullerian structures. In the last patient with 46XY non-dysgenetic testicular DSD, the rectum was thought to be a dilated uterus on ultrasonography. CONCLUSIONS: Pelvic ultrasonography failed to identify Mullerian structures in 40 % of patients with complex DSD. On the contrary, laparoscopy allowed excellent visualization of pelvic structures and gonads in children with complex DSD.

The effect of social media (#SoMe) on journal impact factor and parental awareness in paediatric urology.

BACKGROUND: Social media (SoMe) comprises a number of internet-based applications that have the capability to disseminate multimodal media and allow for unprecedented inter-user connectivity. The role of Twitter has been studied in conferences and education; moreover, there is increasing evidence that patients are more likely to use social media for their own health education. OBJECTIVE: The aim of this study was to assess the impact of social media platforms on the impact factor of both urological and paediatric journals that publish on paediatric urology, and to assess parental awareness of social media in paediatric urology. STUDY DESIGN: A filtered Journal of Citation Reports (JCR) search was performed for the period 2012-16 for journals that published articles on paediatric urology. Journals were ranked according to impact factor, and each individual journal website was accessed to assess for the presence of social media. Parents in paediatric urology clinics and non-paediatric urology patients also filled out a questionnaire to assess for awareness and attitudes to social media. All statistical analysis was performed using Prism 6 software (Prism 6, GraphPad Software, California, USA). RESULTS: Overall, there were 50 urological journals and 39 paediatric journals with a mean impact factor of 2.303 and 1.766, respectively. There was an overall average increase in impact factor across all urological journals between 2012 and 16. The presence of a Twitter feed was statistically significant for a rise in impact factor over the 4 years (P = 0.017). The cohort of parents was statistically more likely to have completed post-secondary education, to have and access to a social media profile, use it for health education, and use it to access journal/physician/hospital social media accounts. DISCUSSION: This study examined, for the first time, the role of social media in paediatric urology, and demonstrated that SoMe use is associated with a positive influence in impact factor, but also a parental appetite for it. Limitations included a non-externally validated questionnaire. There may also have been bias in larger journals that generate and maintain social media platforms such as Twitter, which may then in turn have an influence on impact factor. CONCLUSIONS: Social media use within paediatric urology was associated with a higher impact factor, which remained significant after 4 years of analysis. Parents were more likely to use a wide variety of social media to search for conditions and physicians/healthcare providers; therefore, journals and institutions need to embrace and endorse SoMe as a potential source of important clinical information.

Shorter anogenital and anoscrotal distances correlate with the severity of hypospadias: A prospective study.

INTRODUCTION: Anogenital distance (AGD) is a recognised marker of in utero androgen action. OBJECTIVE: This study aimed to evaluate the relationship between severity of hypospadias and AGD. STUDY DESIGN: Boys undergoing hypospadias repair in a single tertiary centre between May 2012 and February 16 were included in the study. Anogenital distance was measured from the centre of the anus to the base of the penis, and anoscrotal distance (ASD) from the centre of the anus to the junction between the smooth perineal skin and scrotal skin. Trained paediatric urologists made all measurements using digital callipers. RESULTS: Fifty-nine boys with hypospadias and 31 age-matched controls undergoing circumcision (median age 1.37 years, range 1.01-1.96) had AGD and ASD measured under anaesthetic. The patients were divided into two groups, according to hypospadias severity: group 1 - distal penile/subcoronal/glandular (n = 40); and group 2 - perineal/penoscrotal/midshaft (n = 19). The median AGD for controls was 74.0 mm (range 53.2-87.8) and for hypospadias it was 72.3 mm (range 50.7-90.0) (P = 0.816). The median ASD for controls was 42.3 mm (range 31.0-56.1) and for hypospadias it was 39.4 mm (range 20.7-77.0) (P = 0.224). Considering severity of hypospadias, the median AGD for group 1 and group 2 was 73.7 mm (range 50.7-90.0) and 63.3 mm (range 53.6-77.0), respectively (P < 0.001). The median ASD was also higher in group 1, at 41.3 mm (range 20.7-65.0), compared to 35.2 mm (range 23.5-77.0) in group 2 (P = 0.119) (Summary Fig.). DISCUSSION: This study showed that more severe forms of hypospadias are associated with shorter AGD and ASD. These findings agree with two previous studies that identified reduced AGD in boys with hypospadias. However, these studies did not investigate an association with severity of hypospadias. As hypospadias is multifactorial, only a small proportion of cases are thought to be associated with impaired in utero androgen exposure. The shorter AGD in boys with severe hypospadias compared with mild hypospadias would indicate that AGD is a marker of the severity of androgen production. This may also suggest that less severe forms of hypospadias have a different aetiology involving a later stage of development, and that they are not the result of reduced androgen exposure in the male programming window between the 8-14 weeks gestation. CONCLUSION: This study identified that boys with more severe hypospadias are more likely to have a shorter AGD and ASD than boys with mild hypospadias. This may indicate that there is a more profound impairment of in utero androgen action in severe hypospadias.

Y179C, F486L and N550H are BRCA1 variants that may be associated with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Over 600 different pathogenic mutations have been identified in the BRCA1 gene. Nevertheless, numerous missense mutations of unknown biological function still exist. Understanding of biological significance of these mutations should help in genetic counselling to carriers and their families. PATIENTS AND METHODS: A total of 104 patients with breast and/or ovarian cancer whose genetic counselling answered the criteria of the American Society of Clinical Oncology (ASCO 2003), were prospectively screened for mutations in all coding exons of the BRCA1 gene by automatic direct sequencing. RESULTS: During these mutational screening procedures one case presented three mutations classified in the Breast Cancer Information Core Database as unknown variants. These were 655A/G found in exon 8 of BRCA1, 1575T/C and 1767A/C found in exon 11 of the same gene. The identification of the three unknown variants in the proband (16SIRIO) and in her mother and sister indicates that such alterations exist in cis. CONCLUSIONS: Our results suggest that the charge and stechiometry variations determined by the changes in the amino acids Y179C, F486L and N550H might produce an effect on the conformation of the protein and, consequently, on its function.

'Targeted top down' approach for the investigation of UTI: A 10-year follow-up study in a cohort of 1000 children.

INTRODUCTION: Investigations following urinary tract infection (UTI) aim to identify children who are prone to renal scarring, which may be preventable. In 2002, in an attempt to reduce unnecessary intervention, the present institution standardised the investigation of children with a confirmed UTI. OBJECTIVE: This study aimed to identify the significance of urological abnormalities on investigations following a UTI in children, prior to the introduction of the National Institute for Health and Care Excellence (NICE) guidelines. METHODS: Clinical information on the first 1000 patients was retrieved from a prospective UTI hospital database. The follow-up period was 10 years. RESULTS: There were 180 males and 820 females (M:F = 1:4.5). The median age of presentation was 5 years (range 11 days-16 years). A renal ultrasound (US) was performed on all patients, and was normal in 93% of cases (n = 889) (see Figure). Of the 7% who had an abnormal US (n = 71), 54 were female and 17 male (M:F = 1:3). A total of 372 DMSA scans were requested and 350 attended their appointment. Of these, 278 cases (79%) were reported as normal, while 72 had an abnormality documented. Of these 72 patients with abnormalities on DMSA scan, 49 had a repeat DMSA scan: 30 demonstrated permanent scarring, while the DMSA scan became normal in 19. Sixteen of the 278 patients whose DMSA scan was initially normal had a repeat DMSA scan due to symptoms, and all scans were normal. Twelve (1.2%) patients required surgical intervention: three underwent circumcision for recurrent UTIs; three underwent endoscopic treatment of VUR; one had a PUV resection; one underwent a cystoscopy; three had a pyeloplasty for pelvi-ureteric junction obstruction; and one had a ureteric reimplantation for vesico-ureteric junction obstruction. After initial investigations and management, 936 patients were discharged from the UTI clinic: 47 of them re-presented - 40 with recurrent UTIs and seven with dysuria. Thirty-five of the 47 children who re-presented with urological symptoms underwent a DMSA scan, which showed scarring in three (6%). DISCUSSION: Only 12% of children have a significant radiological abnormality picked up on investigation following a UTI. The present investigation approach differed from the NICE guidelines, where imaging is based on patient age and characteristics of the UTI. All children had a renal US, while DMSA scans were reserved for those children <1 year of age or those with upper tract symptoms. The present protocol recommended a renal US in all children presenting with a UTI. This promptly identified those with pelvi-ureteric junction obstruction and those with PUV, who all presented >6 months of age with a single UTI and, therefore, based on the NICE guidelines would not have undergone a renal US. Of the children who re-presented with further UTIs, a significant number were found to have dysfunctional voiding. As this link is well reported, it may be appropriate to screen for this in older children at initial presentation. Only three patients, who had a US at presentation, were subsequently found to have scarring on DMSA. After 10 years of follow-up, this could represent a false negative rate of 0.3% for the screening programme. None of the girls were found to have VUR or needed any surgical intervention, which suggested that early identification of the scarring might not have altered management. Few patients required surgical intervention, all of whom were identified early. No patient who re-presented required intervention. This would suggest that the present protocol is effective at picking up abnormalities that require surgical management. CONCLUSION: This study suggested that after a childhood UTI, the liberal use of renal ultrasound and a focused 'top down' approach to investigation is likely to identify the vast majority of children who require intervention.

Mechanism of leptin expression in breast cancer cells: role of hypoxia-inducible factor-1alpha.

We reported previously that the obesity hormone leptin is overexpressed in breast cancer biopsies. Here, we investigated molecular mechanisms involved in this process, focusing on conditions that are associated with obesity, that is, hyperinsulinemia and induction of hypoxia. By using quantitative real-time PCR, immunofluorescent detection of proteins and enzyme-linked immunosorbent assays, we found that treatment of MCF-7 breast cancer cells with high doses of insulin or the hypoxia-mimetic agent CoCl2, or culturing the cells under hypoxic conditions significantly increased the expression of leptin mRNA and protein. Notably, the greatest leptin mRNA and protein expression were observed under combined hyperinsulinemia and hypoxia or hypoxia-mimetic treatments. Luciferase reporter assays suggested that increased leptin synthesis could be related to the activation of the leptin gene promoter. DNA affinity precipitation and chromatin immunoprecipitation experiments revealed that insulin, CoCl2 and/or hypoxia treatments augmented nuclear accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) and increased its interaction with several upstream leptin regulatory sequences, especially with the proximal promoter containing four hypoxia-response elements and three GC-rich regions. By using reverse chromatin precipitation, we determined that loading of HIF-1alpha on the proximal leptin promoter concurred with the recruitment of p300, the major HIF coactivator, suggesting that the HIF/p300 complex is involved in leptin transcription. The importance of HIF-1alpha in insulin- and CoCl2-activated leptin mRNA and protein expression was confirmed using RNA interference.

Laparoscopic dismembered pyeloplasty in children younger than 2 years.

PURPOSE: Since the first laparoscopic pyeloplasty was described in a child in 1995, there have been several reports of pyeloplasty in older children. However, to date there have been few reports of laparoscopic pyeloplasty in infants and toddlers. The aim of this study was to evaluate the results of laparoscopic pyeloplasty in children younger than 2 years. MATERIALS AND METHODS: All laparoscopic Anderson-Hynes pyeloplasties performed in children younger than 2 years were retrospectively reviewed. The diagnosis of ureteropelvic junction obstruction was confirmed on renal sonography and diuretic renogram. Laparoscopic pyeloplasties were performed via a transperitoneal route as originally described, with key modifications. All children were investigated with postoperative diuretic renogram and renal ultrasonography. RESULTS: A total of 38 children with ureteropelvic junction obstruction underwent laparoscopic Anderson-Hynes pyeloplasty between January 2001 and December 2005. Of these patients 11 (7 males and 4 females) were younger than 2 years at surgery (median 1.4, range 2 to 22 months) and 1 had bilateral ureteropelvic junction obstruction, for a total of 12 primary repairs. However, 2 patients (17%) required redo laparoscopic pyeloplasty, for a total of 14 laparoscopic dismembered pyeloplasties in this age group. Operative time ranged from 70 to 140 minutes (mean 100) and median hospital stay was 2 days. Followup studies showed normal drainage in all patients except 1, who after redo pyeloplasty exhibited significantly improved but still prolonged drainage. CONCLUSIONS: This study suggests that laparoscopic pyeloplasty can now be performed in young children with good results.

Expression of the obesity hormone leptin and its receptor correlates with hypoxia-inducible factor-1 alpha in human colorectal cancer.

BACKGROUND: The obesity hormone, leptin, has been found to play a role in development and proliferation of normal and malignant tissues. Leptin activity is mediated through the leptin receptor (ObR) that is often expressed in different human cancer cells. Previously, we found that the expression of leptin and ObR can be stimulated by hypoxia-mimetic agents. The aim of this study was to analyze the abundance of and relationships among leptin, ObR and hypoxia-inducible factor-1alpha (HIF-1alpha, transcriptional regulator) in human colorectal cancer. MATERIALS AND METHODS: We investigated the expression of leptin, ObR and HIF-1alpha in colorectal cancer specimens from 135 patients who underwent curative resection. RESULTS: Immunoreactivity for leptin, ObR and HIF-1alpha protein was observed in 69 of 135 (51.1%), 129 of 135 (95.5%) and 88 of 135 (65.2%) of colorectal cancers, respectively. Statistically significant positive correlations were noted between leptin and HIF-1alpha (P = 0.005, r = 0.243), ObR and HIF-1alpha (P < 0.001, r = 0.325) as well as leptin and ObR (P < 0.001, r = 0.426) in the group of all patients as well as in various subgroups depending on clinicopathological features. CONCLUSIONS: The results indicate that the leptin system is overexpressed in human colorectal cancer and this overexpression appears to be associated with the abundance of HIF-1alpha.

The estrogen receptor alpha:insulin receptor substrate 1 complex in breast cancer: structure-function relationships.

BACKGROUND: Insulin receptor substrate 1 (IRS-1) is a signaling molecule that exerts a key role in mediating cross talk between estrogen receptor alpha (ERalpha) and insulin-like growth factor 1 (IGF-1) in breast cancer cells. Previously, we demonstrated that a fraction of IRS-1 binds ERalpha, translocates to the nucleus, and modulates ERalpha-dependent transcription at estrogen response elements (ERE). Here, we studied structure-function relationships of the ERalpha:IRS-1 complex under IGF-1 and/or estradiol (E2) stimulation. MATERIALS AND METHODS: ERalpha and IRS-1 deletion mutants were used to analyze structural and functional ERalpha/IRS-1 interactions. IRS-1 binding to ERE and IRS-1 role in ERalpha-dependent ERE transcription was examined by chromatin immunoprecipitation and gene reporter analysis, respectively. The requirement for IRS-1 in ERalpha function was tested with RNAi technology. RESULTS: Nuclear translocation of IRS-1 was induced by E2, IGF-1, and a combination of both stimuli. ERalpha/IRS-1 binding was direct and involved the activation function-1 (AF-1)/DNA binding domain (DBD) region of ERalpha and two discrete regions of IRS-1 (the N-terminal pleckstrin homology domain and a region within the C-terminus). IRS-1 knock down abrogated IGF-1-dependent transcriptional activity of unliganded ERalpha, but induced the activity of liganded ERalpha. CONCLUSIONS: ERalpha/IRS-1 interactions are direct and involve the ERalpha AF-1/DBD domain and IRS-1 domains mapping within N- and C-terminus. IRS-1 may act as a repressor of liganded ERalpha and coactivator of unliganded ERalpha.

Founder mutations in BRCA1 and BRCA2 genes.

BRCA1 and BRCA2 germline mutations contribute to a significant number of familial and hereditary breast and/or ovarian cancers. The proportion of high-risk families with breast and/or ovarian cancer cases due to mutations in these tumor suppressor genes varies widely among populations. In some population, a wide spectrum of different mutations in both genes are present, whereas in other groups specific mutations in BRCA1 and BRCA2 have been reported with high frequency. Most of these mutations are prevalent in restricted populations as consequence of a founder effect. The comparison of haplotypes between families with the same mutation can distinguish whether high-frequency alleles derive from an older or more recent single mutational event or whether they have arisen independently more than once. Here, we review some of the most well-known and significant examples of founder mutations in BRCA genes found in European and non-European populations. In conclusion, the identification of the ethnic group of families undergoing genetic counseling enables the geneticist and oncologist to make more specific choices, leading to simplify the clinical approach to genetic testing carried out on members of high-risk families. Futhermore, the high frequency of founder mutations, allowing to analyze a large number of cases, might provide accurate information regarding their penetrance.

Gastrointestinal stromal tumors (GISTs): focus on histopathological diagnosis and biomolecular features.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract that are believed to originate from a neoplastic transformation of the intestinal pacemaker cells (interstitial cells of Cajal) normally found in the bowel wall or their precursors. Although the microscopic features have been known for a long time, the defining characteristic of GIST is the presence of the cell-surface antigen CD117 (KIT), which is demonstrated by immunohistochemistry. KIT, which is a growth factor transmembrane receptor, is the product of the proto-oncogene c-kit (chromosome 4). Surgical removal remains the only curative treatment for patients with GISTs. Tumor size, mitotic index, anatomic location, tumor rupture and disease-free interval are the classic characteristics used to predict the clinical course of patients who undergo complete gross resection. Most GISTs express constitutively activated mutant isoforms of KIT or kinase platelet-derived growth factor receptor alpha (PDGFRA) that are potential therapeutic targets for imatinib mesylate. Imatinib mesylate is a rationally designed, molecularly specific oral anticancer agent that selectively inhibits several protein tyrosine kinases central to the pathogenesis of human cancer and which has demonstrated remarkable clinical efficacy in patients with chronic myeloid leukemia and malignant GISTs. More recently Sunitinib, a new KIT/PDGFRA kinase inhibitor, has been tested in patients with GIST resistant to imatinib, with promising results.

4843delC of the BRCA1 gene is a possible founder mutation in Southern Italy (Sicily).

BACKGROUND: The frequency and the type of BRCA1 mutations vary widely and might have different geographic and ethnic distribution. Most of these alterations are generally found in isolated populations as a consequence of the founder effect. The object of this study was to determine whether 4843delC, a deleterious mutation of the BRCA1 gene, might be due to a founder effect originating in the Sicilian region of Italy. This mutation was described by us for the first time and identified in two unrelated Sicilian families with hereditary breast/ovarian cancer. The two families were from the same geographical area (south-western area of Palermo, Sicily). The homogeneity of the ethnic group of the two families and the Single Nucleotide Polymorphism (SNPs) analysis of probands led us to perform a study of the allelotype of the various members. PATIENTS AND METHODS: The analysis of the haplotype of the probands and of several family members was conducted by means of a study of the highly polymorphic microsatellites within or flanking the BRCA1 gene. RESULTS: This analysis revealed the presence of a common allele associated with the mutation. CONCLUSIONS: We therefore conclude that 4843delC of the BRCA1 gene is a possible founder mutation in the Sicilian population.

Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

Is screening ultrasound of the urinary tract indicated in infants with hypertrophic pyloric stenosis?

AIM: Few retrospective studies have evaluated infants with hypertrophic pyloric stenosis (HPS) for associated urological anomalies. They have led to contradictory conclusions. The aim of this study was to evaluate the incidence of urinary tract anomalies in infants with HPS and to establish the clinical significance of this association. METHODS: One hundred and twenty-two infants (100 boys) who underwent pyloromyotomy between 1992 and 2002 were prospectively evaluated. Screening ultrasound (Us) of the urinary tract was performed in 107 infants, while 15 did not attend their ultrasound appointment. RESULTS: Renal ultrasound was abnormal in 4 (4%) of 107 screened patients with HPS. Three patients were found to have mild hydronephrosis and, in one patient, a small, normal kidney was detected. Two patients with hydronephrosis had Us follow-up and the third patient underwent Tc-99 mercaptoacetyl triglycine (MAG 3) scan. In all three patients, the hydronephrosis resolved completely on follow-up scan. CONCLUSION: The incidence of abnormal renal ultrasound in children with HPS is similar to the reported incidence of 3-6% determined with routine ultrasound screening of healthy newborns. The abnormalities detected were not clinically relevant and did not require surgical intervention. We do not recommend screening of the urinary tract in infants with HPS.

Novel strategies for immortalization of human hepatocytes.

Normal somatic cells have a finite life span due in part to their inability to maintain telomere length and chromosome stability. Immortalization strategies based on recent advances in telomere biology and aging research have led to the creation of genetically stable, nontumorigenic immortalized cell lines. Reversible immortalization, using the Cre-lox recombination and excision system, has been developed for the expansion of primary cells for cell based clinical therapies. Immortalized human hepatocyte cell lines with differentiated liver functions would find broad applications in biomedical research, especially for pharmacology and toxicology, artificial liver support, and hepatocyte transplantation. The biological basis of these new immortalization methods and their application to human hepatocytes is reviewed.

Hepatocyte differentiation initiates during endodermal-mesenchymal interactions prior to liver formation.

Previous studies with embryonic tissue explants showed that cellular interactions with mesenchyme are required for endodermal cells to differentiate into hepatocytes. However, these studies assayed hepatocyte characteristics that were evident after days of culture, leaving open the question of whether the primary inductive interactions initiated hepatocyte differentiation, or whether subsequent steps, such as may occur during cell aggregation to form the liver, were necessary. Using the technique of in situ hybridization, we find that serum albumin mRNA, a liver-specific gene product, is first detected in hepatic precursor cells of the endoderm as early as 9.5 days of mouse embryo development, a full day prior to cell aggregation and liver formation. The endodermal cells express albumin mRNA upon migration into strands of connective tissue matrix within mesenchyme. Thus, the onset of differentiation of the endoderm is coincident with its interaction with mesenchyme. Early albumin transcripts are initiated at the same site of the albumin promoter as in adult hepatocytes, suggesting that at least a subset of the transcription factors that control albumin transcription in the adult may be involved in executing the early steps of hepatic determination. We also observe a sharp increase in albumin mRNA levels shortly after the definitive formation of the liver, apparently reflecting cell interactions that enhance hepatocyte differentiation. Hepatocyte differentiation is therefore similar in several respects to pancreatic exocrine cell development, and may represent a general pattern for gut-derived tissues. For both cell types, early interactions with mesenchyme are coincident with the initial expression of differentiated gene products at a low level in proliferating endoderm, and the initial pattern of expression is amplified upon organ formation.

The initiation of new gene transcription during Xenopus gastrulation requires immediately preceding protein synthesis.

The incubation of Xenopus embryo fragments in cycloheximide at 5 or 10 micrograms ml-1 rapidly inhibits protein synthesis to 10% or less of control levels. In most batches of embryos, treatment with cycloheximide for up to 1 h causes no obvious cellular damage and protein synthesis is fully restored to normal levels 5 h later. Transcript analysis with RNA probes shows that the inhibition of protein synthesis at late blastula or early gastrula stages completely suppresses the normal initiation of actin gene transcription at the mid-late gastrula stage. This applies to muscle-specific actin genes, whose transcription is initiated by induction, as well as to cytoskeletal actin genes not activated by induction. Two-dimensional gel protein analysis shows that cycloheximide irreversibly inhibits only 10% of all genes normally expressed at a postneurula stage and that all of these are genes whose expression is normally initiated during or soon after gastrulation. Cycloheximide treatment causes a limited reduction of DNA synthesis, and no reduction of overall RNA synthesis. We conclude that the initiation of new gene transcription during gastrulation in Xenopus is dependent on the immediately preceding synthesis of certain proteins.

Associated urological anomalies in children with unilateral renal agenesis.

PURPOSE: Unilateral renal agenesis has been noted in 1:1,000 autopsies. Recently an increased incidence of vesicoureteral reflux was reported in patients with a solitary kidney. We determined the incidence of associated renal abnormalities in children with unilateral renal agenesis. MATERIALS AND METHODS: We retrospectively reviewed 46 consecutive cases of unilateral renal agenesis diagnosed at our hospital between January 1985 and February 1998. Patient age at diagnosis ranged from newborn to 12.5 years (mean 2.8 years). There were 24 boys and 22 girls. The left kidney was absent in 27 patients and the right kidney was absent in the remaining 19. A total of 24 patients were evaluated for urinary tract infection and in the other 22 a solitary kidney was found during examination for congenital malformations, enuresis or abdominal pain. The diagnosis was made in all patients by abdominal ultrasound and confirmed by excretory urography, or diethylenetriaminepentaacetic acid or dimercapto-succinic acid scan. A voiding cystourethrogram was performed in 40 patients (87%). RESULTS: Associated urological anomalies were present in 22 of the 46 patients (48%) with unilateral renal agenesis, including primary vesicoureteral reflux in 13 (28%), ureterovesical junction obstruction in 5 (11%), ureteropelvic junction obstruction in 3 (7%), and ureterovesical and ureteropelvic junction obstruction in 1 (2%). Of the 22 patients 14 (64%) underwent surgical intervention. CONCLUSIONS: Nearly half of the patients with unilateral renal agenesis had associated urological anomalies. Vesicoureteral reflux was the most common associated anomaly and it was usually of high grade. Early recognition and treatment of urological anomalies in a patient with a solitary kidney are imperative to decrease the long-term risk of renal damage.