Proximal hypospadias repair remains challenging. Our approach to the first stage of two-stage proximal hypospadias repairs has evolved from using Byars' flaps to preputial inlay grafts in ...anatomically suitable cases and pedicled preputial flaps in more complex repairs. We reviewed our outcomes, hypothesizing that inlay grafts and pedicled preputial flaps were associated with lower complication risks than Byars’ flaps.
A single institution, retrospective, cohort study of consecutive two-stage, primary, proximal hypospadias repairs performed from 2007 to 2017 was conducted. Patients with <6 months follow-up and incomplete operative reports were excluded. Risk of complications (fistula, dehiscence, diverticulum, meatal stenosis, stricture) were evaluated following urethroplasty and stratified by first-stage repair technique. As technique refinements have been made since 2012, comparisons between two temporal subgroups (those who underwent repair in 2007–2012 and in 2013–2017) were made.
78 of 127 patients met inclusion criteria. Overall complication rate was 47% (Summary Table). Median follow-up was 25.4 months (range 6.4–128.5 months) after urethroplasty. Pedicled preputial flaps (hazards ratio HR 0.30; 95% Confidence Interval CI 0.14–0.65) and inlay grafts (HR 0.32; 95% CI 0.11–0.95) were associated with lower complication risks compared to Byars' flaps (Summary Table). Median time to complication was significantly shorter for Byars’ flaps (5.7 months) than for inlay grafts (40.6 months) and pedicled preputial flaps (79.2 months) by Kaplan Meier analysis. Temporal subgroup comparisons showed that overall complication rates decreased from 70% to 31% (p = 0.001), but differences in complication rates by first-stage technique were not statistically significant.
In our cohort, repairs with Byars' flaps had the highest complication rate, which is consistent with our observations that urethras tubularized from Byars’ flaps lack appropriate backing and are hypermobile and irregular. To overcome these shortcomings, modifications were made to our approach to two-stage proximal hypospadias repairs with the use of inlay grafts and pedicled preputial flaps quilted to the underlying corporal bodies to optimize the stability of the urethral plate. Our preliminary results are promising.
Approach to the first stage of two-stage repairs affects outcomes. Pedicled preputial flaps and inlay grafts were associated with lower complication risks than Byars’ flaps. Refinement of technique and patient selection may have resulted in fewer complications in the short term. However, long-term follow-up is needed.Summary Table. Overall Complications and Unadjusted Cox Proportional Hazards Model Comparing Complications by First-stage Repair TechniqueOverall ComplicationsAll (N = 78)Inlay Graft (N = 13)Pedicled Preputial Flap (N = 33)Byars' Flaps (N = 21)Combined (N = 11)Complicationsa37 (47%)4 (31%)11 (33%)17 (81%)4 (36%)Median Number of Complications Per Patient0 (0–2)0 (0–2)0 (0–2)1 (0–2)0 (0–1)Type of Complications (n, % of cohort)bFistula18 (23%)2 (15%)6 (18%)8 (38%)2 (18%)Glans/Partial dehiscence10 (13%)1 (8%)06 (29%)3 (30%)Stricture6 (8%)2 (15%)1 (3%)3 (14%)0Diverticulum3 (4%)01 (3%)2 (10%)0Meatal Stenosis3 (4%)03 (9%)00Dehiscence2 (2.5%)01 (3%)1 (5%)0Unadjusted Cox Proportional Hazards Model Comparing Complications by First-stage Repair TechniqueRepairHazard Ratio95% Confidence IntervalsInlay Graft versus Byars' Flaps0.320.11–0.95Pedicled Preputial Flap versus Byars' Flaps0.300.14–0.65Combined versus Byars' Flaps0.480.18–1.31aThis notes the number of patients who developed complications. If a patient developed two complications, they were only counted once for this variable.bThis notes the number of patients with each type of complication.
There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used ...the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments.
We analyzed the methylation status of the 5' regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients.
The 5' regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P < or = 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P < or = 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P < or = 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P < or = 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals.
Our results indicate that methylation of the 5' region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.
A major challenge in value-based health care is the lack of standardized health outcomes measurements, hindering optimal monitoring and comparison of the quality of health care across different ...settings globally. The International Consortium for Health Outcomes Measurement (ICHOM) assembled a multidisciplinary international working group, comprised of 26 health care providers and patient advocates, to develop a standard set of value-based patient-centered outcomes for breast cancer (BC). The working group convened via 8 teleconferences and completed a follow-up survey after each meeting. A modified 2-round Delphi method was used to achieve consensus on the outcomes and case-mix variables to be included. Patient focus group meetings (8 early or metastatic BC patients) and online anonymized surveys of 1225 multinational BC patients and survivors were also conducted to obtain patients' input. The standard set encompasses survival and cancer control, and disutility of care (eg, acute treatment complications) outcomes, to be collected through administrative data and/or clinical records. A combination of multiple patient-reported outcomes measurement (PROM) tools is recommended to capture long-term degree of health outcomes. Selected case-mix factors were recommended to be collected at baseline. The ICHOM will endeavor to achieve wide buy-in of this set and facilitate its implementation in routine clinical practice in various settings and institutions worldwide.
The Glans-Meatus-Shaft (GMS) Score is a pre-operative phenotypic scoring system used to assess hypospadias severity and risk for post-operative complications. The ‘M’ component is based on ...pre-operative meatal location, but meatal location sometimes changes after penile degloving, resulting in ‘meatal mismatch.‘
To identify: 1) the incidence and clinical predictors of meatal mismatch, and 2) the association of meatal mismatch with post-operative urethrocutaneous fistula development.
We performed a retrospective cohort study on patients who underwent primary hypospadias repair at a single center from 2011 to 2018. Meatal mismatch was defined as: upstaging (meatus moving more proximally after degloving), downstaging (moving more distally after degloving), or none. Covariates included: pre-degloving meatal location, chordee severity, penoscrotal anatomy, pre-operative testosterone, and number of stages for repair. To test the association between meatal mismatch and fistula development, we constructed two, nested, multivariable Cox proportional hazards regression models with and without meatal mismatch and compared them with the likelihood ratio test. A sensitivity analysis excluded patients with <6 months of follow-up.
Of 485 patients, 99 (20%) exhibited meatal mismatch, including 75 (15%) with upstaging and 24 (5%) patients with downstaging (Figure). Meatal mismatch was significantly associated with penoscrotal webbing, number of stages for repair, and pre-degloving meatal location, with downstaging being associated with more proximal meatal location. Over a median follow-up of 7.3 months (interquartile range 2.0–20.9), fistulae developed in 56 (12%) patients. On multivariable analysis, meatal upstaging was associated with a 3-fold increased risk of fistula development (Hazards Ratio HR: 3.04, 95% Confidence Interval CI: 1.44–6.45) compared to no mismatch. Meatal downstaging had similar risk of fistula development compared to no mismatch (HR: 0.99, 95% CI: 0.29–3.35). Multi-stage compared to single-stage repair was associated with reduced risk of fistula development (HR: 0.24, 95% CI: 0.09–0.66). The likelihood ratio test favored the model that included meatal mismatch. The sensitivity analysis showed similar findings.
Our short-term results suggest that meatal mismatch may be an important additional consideration to the GMS score as a tool to assess hypospadias severity, counsel families, and predict outcomes. Longer-term studies are needed to enhance the precision of risk stratification in hypospadias.
Meatal mismatch occurred in 20% of patients undergoing hypospadias repair. Among this cohort, meatal upstaging was associated with a 3-fold increased risk of post-operative urethrocutaneous fistula development. Display omitted Summary Table 1Sensitivity Analysis (restricted to patients with ≥6 months of follow-up, N=275) multivariable Cox regression models with and without meatal mismatch. Likelihood ratio test showed better fit with full model (p=0.04).Summary Table 1VariableBase Model HR (95% CI), p-valueFull Model HR (95% CI), p-valuePre-degloving Meatal LocationGlanularReferentReferentCoronal0.54 (0.09–3.24), p = 0.500.58 (0.10-3.53), p = 0.56Distal/Midshaft1.61 (0.38-6.91), p = 0.522.24 (0.50-10.03), p = 0.29Proximal3.33 (0.69-16.18), p = 0.147.18 (1.21-42.52), p = 0.001Chordee Severity (n = 457)None (0°)ReferentReferentMild (1–29°)3.41 (0.43-26.80), p = 0.243.90 (0.50-30.50), p = 0.20Moderate (30–60°)3.91 (0.48-31.66), p = 0.203.61 (0.45-29.01), p = 0.23Severe (≥61°)4.11 (0.50-34.02), p = 0.193.95 (0.48-32.51), p = 0.20# of Stages of RepairSingle-stageReferentReferentMulti-stage1.97 (1.01-3.84), p = 0.0471.76 (0.89-3.47), p = 0.11Pre-operative TestosteroneNoReferentReferentYes1.65 (0.92–2.98), p = 0.101.43 (0.78–2.62), p = 0.25Meatal MismatchNo mismatch–ReferentMeatal downstaging–0.88 (0.25-3.01), p = 0.84Meatal upstaging–3.04 (1.34-6.88), p = 0.007
Objectives
Variant cytochrome P450 (CYP) 3A4 alleles cannot explain human variation in CYP3A4 expression. This study investigated whether common single‐nucleotide polymorphisms (SNPs) in multidrug ...resistance 1 (MDR1), encoding P‐glycoprotein, or the pregnane X receptor (PXR) were associated with basal or inducible CYP3A4 expression.
Methods
MDR1 G2677T and C3435T SNPs and a PXR 6–base pair (bp) deletion were genotyped in the deoxyribonucleic acid from 144 human livers in 3 cohorts each phenotyped for basal or rifampin (INN, rifampicin)–inducible hepatic CYP3A4 expression (or both) and in 57 human small bowel biopsy specimens from 3 cohorts each phenotyped for either basal or rifampin‐induced CYP3A4 expression (or both).
Results
Hepatic CYP3A4 expression/function was significantly higher in persons homozygous for the MDR1 2677T (Ser893) allele compared with persons homozygous for 2677G (Ala893) in all 3 hepatic cohorts. For example, homozygous MDR1 2677 TT livers had higher midazolam hydroxylase activity than homozygous 2677 GG livers (1831 ± 1336 pmol · min−1 · mg protein−1 versus 1060 ± 552 pmol · min−1 · mg protein−1, P = .03). In 2 of the 3 groups the association was observed in men but not in women. For example, homozygous MDR1 2677 TT male hepatocytes had significantly higher testosterone 6β‐hydroxylase activity compared with homozygous 2677 GG livers (0.120 ± 0.06 pmol · min−1 · mg protein−1 versus 0.069 ± 0.04 pmol · min−1 · mg protein−1, P = .0002). Conversely, rifampin induction of testosterone 6β‐hydroxylation in primary human hepatocytes was significantly higher in persons homozygous for 2677G (12.0 ± 5.7–fold) compared with MDR1 homozygous TT carriers (7.3 ± 4.6–fold) (P = .01). Suggestive evidence for higher CYP3A4 expression in MDR1 2677T carriers was also observed in human intestines. CYP3A4 expression was also related to a 6‐bp deletion in PXR in 2 of the liver cohorts. Two‐factor ANOVA analysis revealed a significant interaction between the MDR1 2677 SNP and the PXR 6‐bp deletion influencing CYP3A4 expression (P = .007).
Conclusions
Individuals homozygous for the MDR1 2677T allele show enhanced constitutive CYP3A4 expression in the liver and intestine, as compared with those homozygous for the MDR1 2677G allele, particularly in men. Conversely, the magnitude of CYP3A4 induction by rifampin is greater in persons with the MDR1 2677G allele and is inversely related to baseline CYP3A4 expression. MDR1 likely influences basal CYP3A4 expression by limiting the intracellular concentration of an endogenous regulator. MDR1 genotype may be a useful predictor of basal CYP3A4 and the extent of some CYP3A4‐mediated drug interactions in humans. Moreover, the influence of MDR1 genotype on CYP3A4 expression adds additional complexity to determining the relative contribution of the MDR1 alleles to the disposition of substrates shared by CYP3A4 and MDR1/P‐glycoprotein.
Clinical Pharmacology & Therapeutics (2006) 79, 325–338; doi: 10.1016/j.clpt.2005.11.013
Besides their use in mRNA expression profiling, oligonucleotide microarrays have also been applied to single‐nucleotide polymorphism (SNP) and loss of heterozygosity (LOH) or allelic imbalance ...studies. In this report, we evaluate the reliability of using whole genome amplified DNA for analysis with an oligonucleotide microarray containing 11 560 SNPs to detect allelic imbalance and chromosomal copy number abnormalities. Whole genome SNP analyses were performed with DNA extracted from osteosarcoma tissues and patient‐matched blood. SNP calls were then generated by Affymetrix® GeneChip® DNA Analysis Software. In two osteosarcoma cases, using unamplified DNA, we identified 793 and 1070 SNP loci with allelic imbalance, respectively. In a parallel experiment with amplified DNA, 78% and 83% of these SNP loci with allelic imbalance was detected. The average false‐positive rate is 13.8%. Furthermore, using the Affymetrix® GeneChip® Chromosome Copy Number Tool to analyze the SNP array data, we were able to detect identical chromosomal regions with gain or loss in both amplified and unamplified DNA at cytoband resolution.
To date, there are no efficacious translational solutions for end-stage urinary bladder dysfunction. Current surgical strategies, including urinary diversion and bladder augmentation ...enterocystoplasty (BAE), utilize autologous intestinal segments (e.g. ileum) to increase bladder capacity to protect renal function. Considered the standard of care, BAE is fraught with numerous short- and long-term clinical complications. Previous clinical trials employing tissue engineering approaches for bladder tissue regeneration have also been unable to translate bench-top findings into clinical practice. Major obstacles still persist that need to be overcome in order to advance tissue-engineered products into the clinical arena. These include scaffold/bladder incongruencies, the acquisition and utility of appropriate cells for anatomic and physiologic tissue recapitulation, and the choice of an appropriate animal model for testing. In this study, we demonstrate that the elastomeric, bladder biomechanocompatible poly(1,8-octamethylene-citrate-co-octanol) (PRS; synthetic) scaffold coseeded with autologous bone marrow-derived mesenchymal stem cells and CD34
hematopoietic stem/progenitor cells support robust long-term, functional bladder tissue regeneration within the context of a clinically relevant baboon bladder augmentation model simulating bladder trauma. Partially cystectomized baboons were independently augmented with either autologous ileum or stem-cell-seeded small-intestinal submucosa (SIS; a commercially available biological scaffold) or PRS grafts. Stem-cell synergism promoted functional trilayer bladder tissue regeneration, including whole-graft neurovascularization, in both cell-seeded grafts. However, PRS-augmented animals demonstrated fewer clinical complications and more advantageous tissue characterization metrics compared to ileum and SIS-augmented animals. Two-year study data demonstrate that PRS/stem-cell-seeded grafts drive bladder tissue regeneration and are a suitable alternative to BAE.
We investigated the role of hydrogen sulphide (H
2S) in intracellular pH (pH
i) regulation in vascular smooth muscle cells and its contribution on vasodilation. NaHS, a H
2S donor, decreased pH
i in ...a concentration-dependent manner ranging from 10
μM to 1
mM. Neither inhibition of the Na
+/H
+ exchanger with 5-(
N-ethyl-
N-isopropyl) amiloride, (EIPA, 10
μM), nor plasmalemmal Ca
2+-ATPase with CdCl
2 (20
nM) alters the effect of NaHS on pH
i. Blockade of the
Cl
-
/
HCO
3
-
exchanger with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) significantly attenuated the pH
i lowering effect of NaHS. Moreover, NaHS significantly increased the activity of
Cl
-
/
HCO
3
-
exchanger when measured with NH
4Cl prepulse method. DIDS attenuated the vasorelaxation induced by NaHS whereas EIPA and CdCl
2 did not cause any change. In conclusion, H
2S induced intracellular acidification via activation of
Cl
-
/
HCO
3
-
exchanger, which is, at least partially, responsible for H
2S-mediated vasorelaxation.
Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family ...with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling. The TXNRD1 mutation p.Pro190Leu affecting a highly conserved amino acid residue was identified by whole-exome sequencing of blood DNA from the index patient. The detected mutation and its segregation within the family – all siblings of the index patient were homozygous and the parents heterozygous – were confirmed by Sanger sequencing. TXNRD1 activity was determined in subcellular fractions from a skeletal muscle biopsy and skin fibroblasts of the index patient and the expression levels of the mutated protein were assessed by 75Se labeling and Western blot analysis. As result of the mutation, the activity of TXNRD1 was reduced in the patient's fibroblasts and skeletal muscle (to 34±3% and 16±8% of controls, respectively). In fibroblasts, we detected reduced 75Se-labeling of the enzyme (41±3% of controls). An in-depth in vitro kinetic analysis of the recombinant mutated TXNRD1 indicated 30–40% lowered kcat/Se values. Therefore, a reduced activity of the enzyme in the patient's tissue samples is explained by (i) lower enzyme turnover and (ii) reduced abundance of the mutated enzyme as confirmed by Western blotting and 75Se labeling. The mutant fibroblasts were also found to be less resistant to a hydrogen peroxide challenge. Our data agree with a potential role of insufficient ROS detoxification for disease manifestation in genetic generalized epilepsy.
•Mutation of TXNRD1 was identified in a family with genetic generalized epilepsy.•Patient's cells show reduced TXNRD1 activity and increased sensitivity to H2O2.•The homozygous p. Pro190Leu mutation decreases turnover and reduces steady-state cellular levels of TXNRD1.
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