Instructional Approaches: Anatomy Education of Physical Therapists.

The profession of physical therapy has undergone an evolution since its inception. Since the early 1900s it has grown from a technical training program to a doctorate level degree. Human anatomy courses remain a requirement for physical therapist educational curricula. However, changes in anatomy pedagogy have been trending within health profession educational models, leading to questions regarding which method is best for student learning. The objective of this study was to determine if anatomy instructional method used within physical therapist educational curricula impacted current anatomy knowledge. Licensed physical therapists were recruited to complete a demographic survey and a questionnaire to demonstrate their knowledge of anatomy topics. Anatomy topics included six regional components: (1) upper limb; (2) lower limb; (3) thorax and abdomen; (4) pelvis; (5) spine; and (6) head. Each regional component contained five questions regarding systemic subsets related to joints and osteology, muscles, nervous system, vasculature, and special areas (e.g., spatial orientations, structures within spaces, pathways of nerves). Within the thorax and abdominal region, data analysis indicated that the dissection instruction method, when compared to no laboratory instruction, led to statistically significant greater anatomical knowledge (P = 0.02). Dissection also showed greater means when compared to the no laboratory method (P = 0.02) and the prosection method in the head region (P = 0.01). However, the variance explained by instructional method was small. This study adds empirical evidence regarding current anatomy knowledge exhibited by physical therapists as the level of anatomical knowledge exhibited small differences based on instructional methods.

Roles for urothelium in normal and aberrant urinary tract development.

Congenital anomalies of the kidney and urinary tract (CAKUTs) represent the leading cause of chronic kidney disease and end-stage kidney disease in children. Increasing evidence points to critical roles for the urothelium in the developing urinary tract and in the genesis of CAKUTs. The involvement of the urothelium in patterning the urinary tract is supported by evidence that CAKUTs can arise as a result of abnormal urothelial development. Emerging evidence indicates that congenital urinary tract obstruction triggers urothelial remodelling that stabilizes the obstructed kidney and limits renal injury. Finally, the diagnostic potential of radiological findings and urinary biomarkers derived from the urothelium of patients with CAKUTs might aid their contribution to clinical care.

Isolated Diffuse Choroidal Hemangioma: A Variant of Sturge-Weber Syndrome?

The authors report a case of a diffuse choroidal hemangioma in the left eye of a 17-year-old girl. No evidence of leptomeningeal angiomatosis was found on magnetic resonance imaging and neither the medical history nor the clinical examination revealed a port wine birthmark on the left side of the face. Fine telangiectatic vessels were found on the bulbar conjunctiva of the ipsilateral eye. Because of the shared destination of the vessel-trigeminal-ectoderm complex that migrates toward the optic placode, forming choriocapillaris, upper facial skin (upper eyelid), and conjunctival vessels, an embryological basis can be offered for ophthalmic angiomatosis of Sturge-Weber syndrome. The authors propose that vascular angiomatosis of upper eyelid skin and conjunctiva are phenotypically equivalent and may be present or absent, independent of each other. Their proposed expansion of Sturge-Weber syndrome diagnostic criteria needs to be validated by a comprehensive review of ophthalmic features of Sturge-Weber syndrome. [J Pediatr Ophthalmol Strabismus. 2020;57:e43-e47.].

Impact of successful pediatric ureteropelvic junction obstruction surgery on urinary HIP/PAP and BD-1 levels.

INTRODUCTION: In the pediatric patient whose ureteropelvic junction obstruction (UPJO) is not always symptomatic, imaging is the most common means of detecting surgical success. There is interest, however, in other means of post-operative monitoring. A panel of antimicrobial peptides (AMPs) has been previously found to be elevated in UPJO, but the impact of surgical correction on these AMPs is unknown. OBJECTIVE: To determine if elevated levels of candidate urinary AMP biomarkers of urinary tract obstruction decrease following UPJO repair. STUDY DESIGN: Pediatric patients undergoing surgical correction of an UPJO were recruited for participation. Bladder urine from uninfected consenting/assenting patients was collected immediately prior to surgery and then at least 6 months afterward. Based on prior studies demonstrating significant elevation of beta defensin 1 (BD-1), hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), cathelicidin (LL-37), and neutrophil gelatinase-associated lipocalin (NGAL) in patients with UPJO versus control patients, we performed enzyme-linked immunosorbent assays on these four AMPs to compare their expression before and after surgical intervention. If found to significantly decrease, AMP levels were compared to healthy controls. AMP levels were normalized to urine creatinine. Results were analyzed with paired t test or Wilcoxon test using Graphpad software. Correlation was calculated using Pearson or Spearman correlation. A p-value of <0.05 was considered significant. RESULTS: 13 UPJO patients were included in this study; 9 were male (69%). Age at surgery was a median of 4.3 years (average 6.1, range 0.4-18.4 years). Follow-up urine samples were collected a median of 27.4 months after surgery (average 27.4; range 7.8-45.3 months). All 13 patients had clinical improvement and/or signs of improved hydronephrosis on post-operative imaging. HIP/PAP and BD-1 significantly decreased in post-surgical samples compared to pre-surgical samples (p = 0.02 and 0.01, respectively); NGAL and LL-37 did not significantly change. Overall, HIP/PAP decreased in 12 patients (92%) and BD-1 decreased in 11 patients (85%). BD-1 levels after successful repair were not different from healthy controls (p = 0.06). DISCUSSION: Urinary biomarkers of obstruction should detect significant obstructive pathology as well as reflect its resolution. This would enable their use in post-operative monitoring and augment current methods of determining successful surgical outcome through imaging. CONCLUSIONS: The AMPs HIP/PAP and BD-1 are significantly elevated in UPJO but then significantly decrease after pyeloplasty, with BD-1 returning to healthy control levels. As a result, these AMPs could serve as markers of successful surgical intervention.

Morphological ciliary muscle changes associated with form deprivation-induced myopia.

Myopic children have larger ciliary muscles than non-myopic children, suggesting that the ciliary muscle may have an impact on or be affected by refractive error development. The guinea pig represents an attractive model organism for myopia development research. The purpose of the study was to investigate whether form deprivation-induced myopia in one or more strains of guinea pig causes thickening of the ciliary muscle as seen in human myopia. Thirty-nine guinea pigs were bred from in-house progenitors obtained from Cincinnati Children's Hospital (Cincinnati) and the United States Army (Strain 13). At 2-4 days of age the right eyes of animals were exposed to form deprivation for 7 days while the fellow eyes served as controls. Refractive error was determined with retinoscopy while vitreous chamber depth (VCD) and axial length (AL) were determined with A-scan ultrasound. Ciliary muscle characteristics (ciliary muscle length, cross-sectional area, volume, cell number, cell size, and smooth muscle actin concentration) were determined histologically with antibody labeling and analyzed according to whether the animal developed axial myopia (anisometropia > -2.00 D with VCD and/or AL differences > 0.1 mm) or was unresponsive. This analysis method yielded four groups with Group 1 having no induced myopia but with axial elongation (n = 11), Group 2 having myopia without vitreous or axial elongation (n = 8), Group 3 having myopia with either vitreous or axial elongation (n = 11), and Group 4 having myopia with both vitreous and axial elongation (n = 8). There were no post-treatment inter-ocular differences between strains or for the overall group of animals for any ciliary muscle variable; however, a higher response group number in multivariate ordinal regression was related to having a treated compared to fellow eye that had a lower smooth muscle actin concentration (p = 0.006), with a shorter ciliary muscle length (p = 0.042), and a less oblate eye shape (p = 0.010). Guinea pig ciliary muscle length and smooth muscle actin concentration were significantly less in the treated eyes of axially myopic animals suggesting that 7 days of form deprivation induced ciliary muscle cellular atrophy or inhibited ciliary muscle growth. Form deprivation myopia in the guinea pig does not result in the increase in ciliary muscle thickness associated with human juvenile and adult myopia.

A Novel Approach to Gross Dissection of the Human Pelvis and Perineum.

Progressive curricular changes in medical education over the past two decades have resulted in the diaspora of gross anatomy content into integrated curricula while significantly reducing total contact hours. Despite the development of a wide range of alternative teaching modalities, gross dissection remains a critical component of medical education. The challenge posed to modern anatomists is how to maximize and integrate the time spent dissecting under the current curricular changes. In this study, an alternative approach to the dissection of the pelvis and perineum is presented in an effort to improve content delivery and student satisfaction. The approach involves removal of the perineum en bloc from the cadaver followed by excision of the pubic symphysis, removal and examination of the bladder and associated structures, examination and bisection of the midline pelvic organs in situ, and midsagittal hemisection of the pelvis for identification of the neurovasculature. Results indicate that this novel dissecting approach increases the number of structures identified by 46% ± 14% over current dissecting methods. Survey results indicate that students were better able to integrate lecture and laboratory concepts, understand the concepts, and successfully identify more structures using the new approach (P < 0.05). The concept of anatomic efficiency is introduced and proposed as a standard quantitative measure of gross dissection proficiency across programs and institutions. These findings provide evidence that innovative solutions to anatomy education can be found that help to maintain critical content and student satisfaction in a modern medical curriculum.

Loss-of-function variants in myocardin cause congenital megabladder in humans and mice.

Myocardin (MYOCD) is the founding member of a class of transcriptional coactivators that bind the serum-response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies, and to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by cosegregation of MYOCD variants with the phenotype in 4 unrelated families by in vitro transactivation studies in which pathogenic variants resulted in abrogated SM gene expression and by the finding of megabladder in 2 distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.

DNA copy number variations in children with vesicoureteral reflux and urinary tract infections.

Vesicoureteral reflux (VUR) is a complex, heritable disorder. Genome-wide linkage analyses of families affected by VUR have revealed multiple genomic loci linked to VUR. These loci normally harbor a number of genes whose biologically functional variant is yet to be identified. DNA copy number variations (CNVs) have not been extensively studied at high resolution in VUR patients. In this study, we performed array comparative genomic hybridization (aCGH) on a cohort of patients with a history of both VUR and urinary tract infection (UTI) with the objective of identifying genetic variations responsible for VUR and/or UTI susceptibility. UTI/VUR-associated CNVs were identified by aCGH results from the 192 Randomized Intervention for Children With Vesicoureteral Reflux (RIVUR) patients compared to 683 controls. Rare, large CNVs that are likely pathogenic and lead to VUR development were identified using stringent analysis criteria. Because UTI is a common affliction with multiple risk factors, we utilized standard analysis to identify potential disease-modifying CNVs that can contribute to UTI risk. Gene ontology analysis identified that CNVs in innate immunity and development genes were enriched in RIVUR patients. CNVs affecting innate immune genes may contribute to UTI susceptibility in VUR patients and may provide the first step in assisting clinical medicine in determining adverse outcome risk in children with VUR.

Krt5+ urothelial cells are developmental and tissue repair progenitors in the kidney.

Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5+) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk)+ RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk+ RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk+ RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk+ RUCs derive from embryonic and neonatal Krt5+ RUCs, whereas Krt5+ RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14. However, in response to UTO, postnatal day 14-labeled adult Krt5+ RUCs break their lineage restriction and robustly differentiate into Upk+ RUCs. Thus, Krt5+ RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk+ RUCs in a temporally restricted manner.

Common clinical markers predict end-stage renal disease in children with obstructive uropathy.

BACKGROUND: Obstructive uropathy (OU) is a common cause of end-stage renal disease (ESRD) in children. Children who escape the newborn period with mild-to-moderate chronic kidney disease (CKD) continue to be at increased risk. The predictive ability of clinically available markers throughout childhood is poorly defined. METHODS: Patients with OU were identified in the Chronic Kidney Disease in Children Study. The primary outcome of interest was renal replacement therapy (RRT) (cases). Controls were age matched and defined as patients within the OU cohort who did not require RRT during study follow-up. RESULTS: In total, 27 cases and 41 age-matched controls were identified. Median age at baseline and age at outcome measurement were 10 vs. 16 years, respectively. First available glomerular filtration rate (GFR) (36.9 vs. 53.5 mL/min per 1.73 m2), urine protein/creatinine (Cr) (0.40 vs. 0.22 mg/mg) and microalbumin/Cr (0.58 vs. 0.03 mg/mg), and serum CO2 (20 vs. 22 mmol/L) and hemoglobin (12.4 vs. 13.2 g/dL) differed significantly between cases and controls, respectively. GFR declined 3.07 mL/min per 1.73 m2/year faster in cases compared to that in controls (p < 0.0001). Urine protein/Cr and microalbumin/Cr increased by 0.16 and 0.11 per year more in cases compared to those in controls, respectively (p ≤ 0.001 for both). Serum phosphate increased by 0.11 mg/dL and serum albumin and hemoglobin decreased by 0.04 (g/dL) and 0.14 (g/dL) per year more for cases compared to those for controls, respectively (p < 0.05 for all). CONCLUSIONS: Age-specific baseline and longitudinal measures of readily available clinical measures predict progression to ESRD in children with mild-to-moderate CKD from OU.

The uroplakin plaque promotes renal structural integrity during congenital and acquired urinary tract obstruction.

Urinary tract obstruction represents a common cause of kidney injury across the human life span, resulting in chronic kidney disease and end-stage renal disease. Yet, the extent of obstructive renal damage can be heterogeneous between individuals, implying the existence of unknown mechanisms that protect against or accelerate kidney injury. In this study, we investigated the role of urothelial remodeling in renal adaptation during congenital and acquired obstruction. In the Megabladder ( Mgb-/-) model of congenital obstruction and unilateral ureteral ligation model of acute obstruction, progressive hydronephrosis is strongly associated with dynamic reorganization of the renal urothelium, which elaborates a continuous uroplakin (Upk) plaque. This led us to postulate that the Upk plaque prevents parenchymal injury during urinary tract obstruction. To test this hypothesis, we interbred Mgb-/- and Upk1b-/- mice, which lack the critical Upk1b subunit for Upk plaque formation. Upk1b-/-; Mgb-/- mice experienced an accelerated onset of bilateral hydronephrosis with severe (>67%) parenchymal loss, leading to renal failure and mortality in adolescence. To investigate the function of the renal Upk plaque during acute obstruction, we destabilized the Upk plaque by Upk1b deletion or genetically depleted Upk+ cells following unilateral ureteral obstruction. Both of these strategies accelerated renal parenchymal loss following ureteral ligation, attesting to a conserved, stabilizing role for Upk plaque deposition in the acutely obstructed kidney. In aggregate, these complementary experiments provide the first evidence that the Upk plaque confers an essential, protective adaptation to preserve renal parenchymal integrity during congenital and acquired urinary tract obstruction.

Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection.

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells in vitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression in vivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.

Role of renal urothelium in the development and progression of kidney disease.

The clinical and financial impact of chronic kidney disease (CKD) is significant, while its progression and prognosis is variable and often poor. Studies using the megabladder (mgb -/- ) model of CKD show that renal urothelium plays a key role in modulating early injury responses following the development of congenital obstruction. The aim of this review is to examine the role that urothelium has in normal urinary tract development and pathogenesis. We discuss normal morphology of renal urothelium and then examine the role that uroplakins (Upks) play in its development. Histologic, biochemical, and molecular characterization of Upk1b RFP/RFP mice indicated Upk1b expression is essential for normal urinary tract development, apical plaque/asymmetric membrane unit (AUM) formation, and differentiation and functional integrity of the renal urothelium. Our studies provide the first evidence that Upk1b is directly associated with the development of congenital anomalies of the urinary tract (CAKUT), spontaneous age-dependent hydronephrosis, and dysplastic urothelia. These observations demonstrate the importance of proper urothelial differentiation in normal development and pathogenesis of the urinary tract and provide a unique working model to test the hypothesis that the complex etiology associated with CKD is dependent upon predetermined genetic susceptibilities that establish pathogenic thresholds for disease initiation and progression.

Inflammation drives renal scarring in experimental pyelonephritis.

Acquired renal scarring occurs in a subset of patients following febrile urinary tract infections and is associated with hypertension, proteinuria, and chronic kidney disease. Limited knowledge of histopathology, immune cell recruitment, and gene expression changes during pyelonephritis restricts the development of therapies to limit renal scarring. Here, we address this knowledge gap using immunocompetent mice with vesicoureteral reflux. Transurethral inoculation of uropathogenic Escherichia coli in C3H/HeOuJ mice leads to renal mucosal injury, tubulointerstitial nephritis, and cortical fibrosis. The extent of fibrosis correlates most significantly with inflammation at 7 and 28 days postinfection. The recruitment of neutrophils and inflammatory macrophages to infected kidneys is proportional to renal bacterial burden. Transcriptome analysis reveals molecular signatures associated with renal ischemia-reperfusion injury, immune cell chemotaxis, and leukocyte activation. This murine model recapitulates the cardinal histopathological features observed in humans with acquired renal scarring following pyelonephritis. The integration of histopathology, quantification of cellular immune influx, and unbiased transcriptional profiling begins to define potential mechanisms of tissue injury during pyelonephritis in the context of an intact immune response. The clear relationship between inflammatory cell recruitment and fibrosis supports the hypothesis that acquired renal scarring arises as a consequence of excessive host inflammation and suggests that immunomodulatory therapies should be investigated to reduce renal scarring in patients with pyelonephritis.

Renal epithelial miR-205 expression correlates with disease severity in a mouse model of congenital obstructive nephropathy.

BACKGROUND: Congenital obstructive nephropathy (CON) is a leading cause of pediatric chronic kidney disease (CKD). Despite optimal surgical and medical care, there is a high rate of CKD progression. Better understanding of molecular and cellular changes is needed to facilitate development of improved biomarkers and novel therapeutic approaches in CON. METHODS: The megabladder (mgb) mouse is an animal model of CKD with impaired bladder emptying, hydronephrosis, and progressive renal injury. In this study, we characterize a particular microRNA, miR-205, whose expression changes with the degree of hydronephrosis in the mgb(-/-) kidney. RESULTS: Expression of miR-205 is progressively increased in the adult mgb(-/-) mouse with worsening severity of hydronephrosis. miR-205 expression is correlated with altered expression of cytokeratins and uroplakins, which are markers of cellular differentiation in urothelium. We describe the spatial pattern of miR-205 expression, including increased expression in renal urothelium and novel miR-205 expression in medullary collecting duct epithelium in the congenitally obstructed kidney. CONCLUSION: miR-205 is increased with severity of CON and CKD in the mgb(-/-) mouse and may regulate urothelial differentiation.

Polymorphisms in α-Defensin-Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux.

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real-time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk.

Uroplakin 1b is critical in urinary tract development and urothelial differentiation and homeostasis.

Proper development and maintenance of urothelium is critical to its function. Uroplakins are expressed in developing and mature urothelium where they establish plaques associated with the permeability barrier. Their precise functional role in development and disease is unknown. Here, we disrupted Upk1b in vivo where its loss resulted in urothelial plaque disruption in the bladder and kidney. Upk1b(RFP/RFP) bladder urothelium appeared dysplastic with expansion of the progenitor cell markers, Krt14 and Krt5, increased Shh expression, and loss of terminal differentiation markers Krt20 and uroplakins. Upk1b(RFP/RFP) renal urothelium became stratified with altered cellular composition. Upk1b(RFP/RFP) mice developed age-dependent progressive hydronephrosis. Interestingly, 16% of Upk1b(RFP/RFP) mice possessed unilateral duplex kidneys. Our study expands the role of uroplakins, mechanistically links plaque formation to urinary tract development and function, and provides a tantalizing connection between congenital anomalies of the kidney and urinary tract along with functional deficits observed in a variety of urinary tract diseases. Thus, kidney and bladder urothelium are regionally distinct and remain highly plastic, capable of expansion through tissue-specific progenitor populations. Furthermore, Upk1b plays a previously unknown role in early kidney development representing a novel genetic target for congenital anomalies of the kidney and urinary tract.

Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection.

Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.

Ciliary Muscle Cell Changes During Guinea Pig Development.

PURPOSE: Guinea pig ciliary muscle (CM) increases robustly in volume, length, and thickness with age. We wanted to characterize CM cells during development to determine the contributions of hypertrophy (cell size increase) and hyperplasia (cell number increase) during development. METHODS: Six pigmented guinea pig eyes were collected at each of five ages: 1, 10, 20, 30, and 90 days. Refractive errors and axial lengths were determined. Eyes were temporally marked, enucleated, hemisected, and fixed. Nasal and temporal eye segments were embedded and 30-µm serial sections were collected; the two most central slides from each hemisection were analyzed with an epifluorescence microscope and Stereo Investigator software to determine normal morphologic parameters. RESULTS: Refractive errors became less hyperopic (P = 0.0001) while axial lengths and CM lengths, cross-sectional areas, volumes, and cell sizes all increased linearly with log age (all P < 0.00001). Ciliary muscle cell numbers increased only during the first 20 days of life (P = 0.02). Nasal and temporal CM lengths (P = 0.07), cross-sectional areas (P = 0.18), and cell numbers (P = 0.70) were not different, but CM cell sizes were initially larger temporally and became larger nasally after age 30 days. CONCLUSIONS: The mechanism of guinea pig CM cell growth during the first 90 days of life was characterized by early hyperplasia combined with hypertrophic cell growth throughout development that results in larger CM lengths, cross-sectional areas, and volumes. Nasal-temporal CM development was generally symmetric, but there was more CM hypertrophy nasally at older ages.

Loss of Notch2 and Notch3 in vascular smooth muscle causes patent ductus arteriosus.

The overlapping roles of the predominant Notch receptors in vascular smooth muscle cells, Notch2 and Notch3, have not been clearly defined in vivo. In this study, we use a smooth muscle-specific deletion of Notch2 together with a global Notch3 deletion to produce mice with combinations of mutant and wild-type Notch2/3 alleles in vascular smooth muscle cells. Mice with complete loss of Notch3 and smooth muscle-expressed Notch2 display late embryonic lethality and subcutaneous hemorrhage. Mice without smooth muscle-Notch2 and only one wild-type copy of Notch3 die within one day of birth and present with vascular defects, most notably patent ductus arteriosus (DA) and aortic dilation. These defects were associated with decreased expression of contractile markers in both the DA and aorta. These results demonstrate that Notch2 and Notch3 have overlapping roles in promoting development of vascular smooth muscle cells, and together contribute to functional closure of the DA.