Evaluation of robotic-assisted laparoscopic and open pyeloplasty in children: single-surgeon experience.

INTRODUCTION: Robotic-assisted laparoscopic pyeloplasty (RALP), the most commonly undertaken paediatric robotic urologic surgery, has not been compared against open pyeloplasty (OPN) by a single surgeon. Here, we describe our experience and outcomes. METHODS: Children undergoing RALP or OPN from 2007 to 2013 were reviewed. Clinical success was defined as resolution of presenting symptoms and improved/stable hydronephrosis on ultrasound. RESULTS: RALP and OPN cohorts comprised 52 and 40 patients, respectively. RALP patients were significantly older (6.8 vs 1.2 years, p<0.01) and heavier (28.4 vs 8.4 kg, p<0.01). Operative times for RALP were longer (203.3 vs 135.0 min, p<0.01), but decreased significantly with increasing experience (r(2)=0.42, p<0.01). Seven type-IIIb Clavien-Dindo complications occurred in RALP patients compared with two in OPN cases. There were no differences in postoperative narcotic administration (p=0.92) or duration of stay in hospital (DOSH) (p=0.93). A total of 11/40 (28%) OPN patients required epidural analgesia but none were placed in the RALP cohort. A total of 49/52 (94%) RALP patients and 40/40 OPN cases had successful outcomes. Three RALP patients required revision RALP. CONCLUSIONS: These data show that outcomes for RALP and OPN were comparable. An initial learning curve with RALP is to be expected, but operative times for RALP approached those for OPN. Previously reported benefits of RALP (reduced analgesic requirements, DOSH) were not observed. This difference may have been due to comparison of a heterogeneous cohort. Close evaluation of complications allowed for improved placement of stents in RALP.

The effects of donor and recipient practices on transplant center finances.

Over the past several years we have noted a marked decrease in this profitability of our kidney transplant program. Our hypothesis is that this reduction in kidney transplant institutional profitability is related to aggressive donor and recipient practices. The study population included all adults with Medicare insurance who received a kidney transplant at our center between 1999 and 2005. Adopting the hospital perspective, multi-variate linear regression models to determine the independent effects of donor and recipient characteristics and era effects on total reimbursements and total hospital margin. We note statistically significant decreased medical center incremental margins in cases with ECDs (-$5887) and in cases of DGF (-4937). We also note an annual change in the medical center margin is independently associated with year and changes at a rate of -$5278 per year, related to both increasing costs and decreasing Medicare reimbursements. The financial loss associated with patient DGF and the use of ECD kidneys may resonate with other centers, and could hinder efforts to expand kidney transplantation within the United States. The Centers for Medicare and Medicaid Services (CMS) should consider risk-adjusted reimbursement for kidney transplantation.

Financial implications of pancreas transplant complications: a business case for quality improvement.

We quantified the financial implications of surgical complications following pancreas transplantation. We reviewed medical and financial records of 49 pancreas transplant recipients at the University of Michigan Health System (UMHS) between 1/6/2002 and 11/22/2004. The association of donor, transplant recipient and financial variables was assessed. The median costs to UMHS of procedures and follow-up were $92,917 for recipients without surgical complications versus $108,431 when a surgical complication occurred, a difference of $15,514 (p = 0.03). Median reimbursement by the payer was $17,363 higher in patients with a surgical complication (p = 0.001). Similar trends (higher insurer costs) were noted when stratifying by payer (public and private) and specific procedure (SPK and PAK). All parties (patient, physician, payer and medical center) should benefit from quality improvement, with payers having a financial interest in pancreas transplant surgical quality initiatives.

Decision Making in Fuzzy Discrete Event Systems1.

The primary goal of the study presented in this paper is to develop a novel and comprehensive approach to decision making using fuzzy discrete event systems (FDES) and to apply such an approach to real-world problems. At the theoretical front, we develop a new control architecture of FDES as a way of decision making, which includes a FDES decision model, a fuzzy objective generator for generating optimal control objectives, and a control scheme using both disablement and enforcement. We develop an online approach to dealing with the optimal control problem efficiently. As an application, we apply the approach to HIV/AIDS treatment planning, a technical challenge since AIDS is one of the most complex diseases to treat. We build a FDES decision model for HIV/AIDS treatment based on expert's knowledge, treatment guidelines, clinic trials, patient database statistics, and other available information. Our preliminary retrospective evaluation shows that the approach is capable of generating optimal control objectives for real patients in our AIDS clinic database and is able to apply our online approach to deciding an optimal treatment regimen for each patient. In the process, we have developed methods to resolve the following two new theoretical issues that have not been addressed in the literature: (1) the optimal control problem has state dependent performance index and hence it is not monotonic, (2) the state space of a FDES is infinite.

Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations.

BACKGROUND & AIMS: Nonalcoholic chronic pancreatitis is usually idiopathic and often associated with cystic fibrosis gene (CFTR) mutations. It is unknown whether pancreatitis risk correlates with having 1 or 2 CFTR mutations, abnormal epithelial ion transport, or mutations of other genes. METHODS: We tested 39 patients with idiopathic chronic pancreatitis (mean age at diagnosis, 33 years) for common mutations of CFTR and of genes encoding a trypsin inhibitor (PSTI) and trypsinogen (PRSS1). To exclude hereditary pancreatitis, we initially relied on family history and subsequently tested for PRSS1 mutations. Twenty subjects were tested for rare CFTR mutations (DNA sequencing) and 11 were tested for extrapancreatic CFTR function (clinical and physiologic evaluation). RESULTS: Mutations were identified in 24 of 39 subjects. Nine patients had cystic fibrosis-causing mutations, 8 of whom also had mild-variable mutations. Eight others had only mild-variable mutations. Nine subjects had the N34S PSTI mutation and 1 had hereditary pancreatitis (R122H, PRSS1). Pancreatitis risk was increased approximately 40-fold by having 2 CFTR mutations (P < 0.0001), 20-fold by having N34S (P < 0.0001), and 900-fold by having both (P < 0.0001). Subjects with 2 CFTR mutations had abnormal nasal epithelial ion transport and clinical findings suggesting residual CFTR function between that in cystic fibrosis and in carriers. By contrast, subjects with only PSTI mutations had normal CFTR function. CONCLUSIONS: CFTR-related pancreatitis risk correlates with having 2 CFTR mutations and reduced extrapancreatic CFTR function. The N34S PSTI mutation increased risk separately. Testing for pancreatitis-associated CFTR and PSTI genotypes may be useful in nonalcoholic pancreatitis.

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation.

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC(50) = 85 microM) and activated CFTR in Calu-3 cells (EC(50) = 47 microM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.

The role of human papillomavirus deoxyribonucleic acid assay and repeated cervical cytologic examination in the detection of cervical intraepithelial neoplasia among human immunodeficiency virus-infected women. Cervical Disease Study Group of the American Foundation for AIDS Research Community Based Clinical Trials Network.

OBJECTIVES: We sought to measure the characteristics of a quantitative human papillomavirus deoxyribonucleic acid assay and repeated cervical cytologic examination in screening for cervical intraepithelial neoplasia among human immunodeficiency virus-infected women. STUDY DESIGN: Human immunodeficiency virus-infected women with screening CD4+ lymphocyte counts of < or = 500 cells/mm3 (n = 103) were examined by quantitative human papillomavirus deoxyribonucleic acid assay and serial cervical cytologic examination and by colposcopy with biopsy and endocervical curettage during the course of 1 year. RESULTS: Quantitative measures of total human papillomavirus deoxyribonucleic acid and high-risk human papillomavirus deoxyribonucleic acid were strongly associated with any cervical intraepithelial neoplasia (P = .005) and high-grade cervical intraepithelial neoplasia (P = .0006), but they improved the sensitivity and negative predictive value of baseline screening only slightly when combined with cervical cytologic examination. Incident cervical intraepithelial neoplasia occurred frequently (20%) during 1 year of follow-up and was more common among human papillomavirus-infected women. Repeated cytologic examination identified 60% of women with new cervical intraepithelial neoplasia. CONCLUSION: Human immunodeficiency virus-infected women with at least mild immunosuppression have a high incidence of cervical intraepithelial neoplasia, which warrants close follow-up. Those with high baseline human papillomavirus deoxyribonucleic acid levels may be at the highest risk for incident cervical intraepithelial neoplasia.

Cystic fibrosis mutations and genetic predisposition to idiopathic chronic pancreatitis.

Idiopathic chronic pancreatitis is a leading cause of chronic pancreatitis. Work from this and other groups has shown that idiopathic chronic pancreatitis is associated with mutations of the cystic fibrosis gene (CFTR). Many idiopathic pancreatitis patients have compound heterozygote genotypes in which both copies of the CFTR gene are abnormal. In these patients, the pancreatic disease can be viewed as a mild variant of cystic fibrosis, in which there is sufficient residual CFTR function to prevent lung disease. This article summarizes the evidence associating these abnormal CFTR genotypes with idiopathic chronic pancreatitis and reviews the implications of this association for the pathogenesis, classification, and prevention of pancreatitis.

ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation.

Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.

Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its pre-mRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing mutant (deltaF508) pre-mRNA targets and tested this against a number of PTMs capable of binding to the CFTR target intron 9 and trans-splicing in the normal coding sequences for exons 10-24 (containing F508). When 293T cells were cotransfected with both constructs, they produced a trans-spliced mRNA in which normal exon 10-24 replaced mutant exon 10. To test whether SMaRT produced mature CFTR protein, proteins were immunoprecipitated from lysates of cotransfected cells and detected by Western blotting and PKA-phosphorylation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This proof-of-concept study demonstrates that exon replacement by SMaRT can repair an abnormal pre-mRNA associated with a genetic disease.

Correction of aberrant splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by antisense oligonucleotides.

The CFTR splicing mutation 3849 + 10 kb C --> T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 + 10 kb C --> T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2'-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose- and sequence-dependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders.

Are mutations in the cystic fibrosis gene important in chronic pancreatitis?

The leading causes of chronic pancreatitis are alcohol and idiopathic pancreatitis. The importance of genetic factors in chronic pancreatitis has been uncertain. Recently, however, it was learned that many patients with idiopathic chronic pancreatitis have mutations of the cystic fibrosis gene. This article reviews the evidence that links mutations of this gene to unexplained pancreatitis, and discusses the implications of this for the evaluation, pathogenesis, classification, and possible prevention of pancreatitis.

Cystic fibrosis in the pancreas: recent advances provide new insights.

Idiopathic chronic pancreatitis accounts for up to one third of chronic pancreatitis cases. The most common inherited disease of the exocrine pancreas is cystic fibrosis, which is caused by mutations of a gene encoding an ion transport protein. It was discovered during the past year that many patients with idiopathic chronic pancreatitis have mutations of the gene that causes cystic fibrosis. This article reviews the evidence associating mutations of this gene with chronic pancreatitis and discusses the implications of this association for the evaluation, pathogenesis, classification, and possible prevention of pancreatitis.

Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis.

BACKGROUND: It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS: We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS: Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION: In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.

Early bactericidal activity of isoniazid in pulmonary tuberculosis. Optimization of methodology. The DATRI 008 Study Group.

Early bactericidal activity (EBA) of antituberculosis drugs is the rate of decrease in the concentration of tubercle bacilli sputum during the initial days of therapy. The study reported here was designed to optimize the methodology for obtaining precise EBA measurements. The study compared the results with two versus five treatment days; overnight sputum collections with early morning collections; and quantitative smears for acid-fast bacilli (AFB) with quantitative cultures. Isoniazid (INH) was used as a model drug. Among 28 smear-positive patients enrolled in the study in five cities in the United States, 16 were evaluable (INH-susceptible tuberculosis [TB] and adequate sputum collections). The mean baseline bacterial load was 6.69 log10 cfu/ml (SE = 0.24). Quantitative culture of 10- or 12-h sputum collections obtained on two baseline days and treatment Day 5 was the optimal method for EBA measurement. The mean 5-d EBA was 0.21 log10 cfu/ml/d (SE = 0.03; p < 0.001) and the EBA appeared to be constant during the first five treatment days. On the basis of these data, multiarm studies of investigational drugs will require 25 evaluable subjects per arm to detect (80% power and two-tailed alpha of 0.05) an EBA at least 50% as large as the EBA of INH. In countries with a low incidence of TB, the usefulness of this methodology for rapidly assessing new antituberculosis agents may be limited by the relatively large number of subjects required to compare EBA values across treatment arms.

Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells.

Airway goblet cells secrete mucin in response to ATP and uridine 5'-triphosphate (UTP), but the underlying signal transduction pathways are poorly understood. Cultures of SPOC1 cells (L. H. Abdullah, S. W. Davis, L. Burch, M. Yamauchi, S. H. Randell, P. Nettesheim, and C. W. Davis. Biochem. J. 316: 943-951, 1996) secreted mucin on exposure to phorbol 12-myristate 13-acetate (PMA) [apparent affinity (K0.5) approximately 100 nM] and ionomycin (K0.5 approximately 5 microM) almost fivefold over baseline. Thapsigargin also elicited secretion (K0.5 approximately 20 nM). Ionomycin and PMA together elicited approximately twice the secretion of either agent alone. Overnight exposure to half-maximal PMA abolished the response to maximal doses of UTP and PMA, whereas ionomycin was fully effective. Protein kinase C (PKC) activity in the membrane fraction was increased by maximal doses of PMA and UTP, whereas ionomycin had no effect. PKC inhibitors were relatively ineffective against PMA- and UTP-induced mucin secretion. Human and canine goblet cells in epithelial explants, by video microscopy, underwent exocytosis with ionomycin (1 microM) and PMA (0.1 or 1 microM). SPOC1 cell mucin secretion was not stimulated by forskolin, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate. Cystic fibrosis transmembrane conductance regulator was not detected in SPOC1 cells by Western blotting, and its mRNA was detected by reverse transcriptase polymerase chain reaction (PCR) only as a very weak band and after 55 PCR cycles. Multidrug resistance (MDR1), however, was readily detected by Western blotting, and its mRNA was detected as a major band after 35 PCR cycles. Thus airway goblet cell mucin secretion, distal to receptor activation, may be regulated independently by Ca(2+)- and PKC-dependent pathways. Cystic fibrosis transmembrane conductance regulator and cyclic nucleotides, however, may not play a major role in this secretion.

Gene targeting of a CFTR allele in HT29 human epithelial cells.

HT29 cells endogenously express the cystic fibrosis transmembrane conductance regulator (CFTR) and have been used previously as a model to examine cellular regulation of CFTR expression and chloride secretory function. Homologous recombination has been used to specifically disrupt CFTR transcription in the HT29-18-C1 subclone. Experiments demonstrate successful disruption of a CFTR allele by DNA constructs, which target insertion of the neomycin phosphotransferase gene into CFTR exon 1 via homologous recombination. The mutation of one allele is a partial knockout because this cell line has multiple CFTR alleles. The mutation is confirmed by polymerase chain reaction (PCR) and genomic Southern blot analysis. A 52-68% reduction in CFTR mRNA levels is observed in the mutant cell line by both Northern and PCR analysis. However, Western blots show no decrease in total CFTR protein levels. Consistent with the lack of reduction in CFTR protein, the partial knockout mutant does not demonstrate alterations in cyclic AMP or calcium stimulation of chloride efflux or net osmolyte loss. Results suggest that posttranscriptional regulation of CFTR levels may contribute to maintenance of cellular chloride transport function.

Polarized expression of cAMP-activated chloride channels in isolated epithelial cells.

We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.