A Novel Mouse Model of Schistosoma haematobium Egg-Induced Immunopathology

Schistosoma haematobium is the etiologic agent for urogenital schistosomiasis, a major source of morbidity and mortality for more than 112 million people worldwide. Infection with S. haematobium results in a variety of immunopathologic sequelae caused by parasite oviposition within the urinary tract, which drives inflammation, hematuria, fibrosis, bladder dysfunction, and increased susceptibility to urothelial carcinoma. While humans readily develop urogenital schistosomiasis, the lack of an experimentally-tractable model has greatly impaired our understanding of the mechanisms that underlie this important disease. We have developed an improved mouse model of S. haematobium urinary tract infection that recapitulates several aspects of human urogenital schistosomiasis. Following microinjection of purified S. haematobium eggs into the bladder wall, mice consistently develop macrophage-rich granulomata that persist for at least 3 months and pass eggs in their urine. Importantly, egg-injected mice also develop urinary tract fibrosis, bladder dysfunction, and various urothelial changes morphologically reminiscent of human urogenital schistosomiasis. As expected, S. haematobium egg-induced immune responses in the immediate microenvironment, draining lymph nodes, and systemic circulation are associated with a Type 2-dominant inflammatory response, characterized by high levels of interleukin-4, eosinophils, and IgE. Taken together, our novel mouse model may help facilitate a better understanding of the unique pathophysiological mechanisms of epithelial dysfunction, tissue fibrosis, and oncogenesis associated with urogenital schistosomiasis.     

Ultrasonography and abdominal radiography versus intravenous urography in investigation of urinary tract infection in men: prospective incident cohort study

ObjectivesTo compare ultrasonography and abdominal radiography with intravenous urography in the investigation of urinary tract infection in men.DesignProspective study in two hospital departments. Radiological procedures and urological assessments performed on different days by different cliniciansSettingDistrict general hospital.ParticipantsConsecutive series of men (n=114) referred to the department of urology for investigation of proved urinary tract infection.InterventionsUltrasonography and intravenous urography of renal tract and assessment of urinary flow rate. Clinical assessment, cystoscopy, urodynamic studies, and transrectal ultrasonography with biopsy.ResultsImportant abnormalities were seen in 53 of 100 fully evaluated patients, the most common being a poorly emptying bladder (34). The combination of plain radiographs of kidneys, ureter, and bladder and ultrasonography detected more abnormalities than intravenous urography alone. No important abnormality was missed by this combination (sensitivity 100% and specificity 93%).ConclusionsUltrasonography with abdominal radiography is as accurate as intravenous urography in detecting important urological abnormalities in men presenting with urinary tract infection. This combination is safer than intravenous urography and should be the initial investigation for such patients. Additional determination of urinary flow rate is useful for the assessment of an incompletely emptying bladder.

What is already known on this topic

Ultrasonography alone is the primary investigation of choice for urinary tract infection in children and womenUltrasonography has limited sensitivity for renal stones and poor sensitivity for ureteric stonesUrinary infection is less common in men than women and the risk factors are different

What this study adds

Ultrasonography is as effective as intravenous urography in men with urinary tract infection only when it is combined with plain radiographyIn men aged over 50 an incompletely emptying bladder is the most common abnormalityIn such patients determination of urinary flow rate is particularly helpful     

Candiduria: problems of diagnosis and therapy]

Candiduria is rather common. Yeasts could be detected in urine that was contaminated during collection of the specimens in patients with urinary bladder colonization or the upper urinary tract infection due either to retrograde spread of the pathogen from the urinary bladder or hematogenous dissemination from a distant infection focus. Most patients with candiduria are asymptomatic. The rate of complications is not known but appears to be low since candidemia rarely results from asymptomatic candiduria unless obstruction is present or instrumental examination of the urinary tract was performed. Unfortunately, there are no reliable diagnostic tests distinguishing fungal infection and colonization. Guidelines for antifungal therapy of candiduria, based almost entirely on fantastic reports and expert opinions, rather than on controlled clinical trials, were proposed by the Infectious Diseases Society of America. Until reliable methods for distinguishing infection from colonization are developed, further use of antifungal therapy is unlike to provide information for clinicians on the pathogenesis and effective treatment of candiduria.     

Grey-Scale Ultrasound in the Imaging of Urinary Tract Disease

Grey-scale ultrasound defines smaller renal lesions that had previously been appreciated and is able to define associated lesions of the liver such as metastases and cysts. The appropriate technique to delineate the normal anatomy of the kidney is described. Ultrasound plays a central role in the identification and characterization of renal mass lesions thus leading to appropriate further work up. In renal transplant evaluation ultrasound is useful as a complementary modality to other imaging studies permitting the recognition of pelvic fluid collections, rejection, and hydronephrosis. Specific findings are present in renal abscess, perirenal abscess, and in several of the renal cystic diseases. Adrenal lesions can be identified and clarified. In the lower urinary tract, ultrasound can identify bladder and prostatic tumors.Ultrasound provides a rapid, safe and non-invasive modality which is complementary to other imaging techniques in the diagnosis of urinary tract disease.     

Controlling Schistosomiasis: Significant Decrease of Anaemia Prevalence One Year after a Single Dose of Praziquantel in Nigerien Schoolchildren

Background

In the framework of the monitoring and evaluation of the Nigerien schistosomiasis and soil-transmitted helminth control programme, a follow-up of children took place in eight sentinel sites. The objective of the study was to assess the evolution of Schistosoma haematobium infection and anaemia in schoolchildren after a single administration of praziquantel (PZQ) and albendazole.

Methods/Principal Findings

Pre-treatment examination and follow-up at one year post-treatment of schoolchildren aged 7, 8, and 11 years, including interview, urine examination, ultrasound examination of the urinary tract, and measurement of haemoglobin. Before treatment, the overall prevalence of S. heamatobium infection was 75.4% of the 1,642 enrolled children, and 21.8% of children excreted more than 50 eggs/10 ml urine. Prevalence increased with age. The overall prevalence of anaemia (haemoglobin <11.5 g/dl) was 61.6%, decreasing significantly with increasing age. The mean haemoglobinemia was 11 g/dl. In bivariate analysis, anaemia was significantly more frequent in children infected with S. haematobium, although it was not correlated to the intensity of infection. Anaemia was also associated with micro-haematuria and to kidney distensions. In a sub-sample of 636 children tested for P. falciparum infection, anaemia was significantly more frequent in malaria-infected children. In multivariate analysis, significant predictors of anaemia were P. falciparum infection, kidney distension, and the village. One year after a single-dose praziquantel treatment (administered using the WHO PZQ dose pole) co-administered with albendazole (400 mg single dose) for de-worming, the prevalence of S. haematobium infection was 38%, while the prevalence of anaemia fell to 50.4%. The mean haemoglobinemia showed a statistically significant increase of 0.39 g/dl to reach 11.4 g/dl. Anaemia was no longer associated with S. haematobium or to P. falciparum infections, or to haematuria or ultrasound abnormalities of the urinary tract.

Conclusions

The high prevalence of anaemia in Nigerien children is clearly a result of many factors and not of schistosomiasis alone. Nevertheless, treatment of schistosomiasis and de-worming were followed by a partial, but significant, reduction of anaemia in schoolchildren, not explainable by any other obvious intervention.     

Genetics of Vesicoureteral Reflux

Vesicoureteral reflux (VUR) is the retrograde passage of urine from the bladder to the upper urinary tract. It is the most common congenital urological anomaly affecting 1-2% of children and 30-40% of patients with urinary tract infections. VUR is a major risk factor for pyelonephritic scarring and chronic renal failure in children. It is the result of a shortened intravesical ureter with an enlarged or malpositioned ureteric orifice. An ectopic embryonal ureteric budding development is implicated in the pathogenesis of VUR, which is a complex genetic developmental disorder. Many genes are involved in the ureteric budding formation and subsequently in the urinary tract and kidney development. Previous studies demonstrate an heterogeneous genetic pattern of VUR. In fact no single major locus or gene for primary VUR has been identified. It is likely that different forms of VUR with different genetic determinantes are present. Moreover genetic studies of syndromes with associated VUR have revealed several possible candidate genes involved in the pathogenesis of VUR and related urinary tract malformations. Mutations in genes essential for urinary tract morphogenesis are linked to numerous congenital syndromes, and in most of those VUR is a feature. The Authors provide an overview of the developmental processes leading to the VUR. The different genes and signaling pathways controlling the embryonal urinary tract development are analyzed. A better understanding of VUR genetic bases could improve the management of this condition in children.     

Mitochondrial involvement in bladder function and dysfunction

Benign bladder pathology resulting from prostatic hypertrophy or other causes is a significant problem associated with ageing in humans. This condition is characterized by increased bladder mass, decreased urinary flow rate, decreased compliance, and these and other changes in bladder function often subject patients to increased risk of urinary tract infection. While the physiologic attributes of benign bladder pathology have been extensively described in humans and in various animal model systems, the biochemical and molecular genetic bases for that pathology have only recently been investigated in detail. Studies demonstrate that mitochondrial energy production and utilization are severely impaired in bladder smooth muscle during benign bladder disease, and to a large extent this realization has provided a rational basis for understanding the characteristic alterations in urinary flow and compliance in bladder tissue. Recent investigations targeting the detailed molecular basis for impaired mitochondrial function in the disease have shown that performance of the organellar genetic system, and to a large extent that of relevant portions of the nuclear genetic system as well, is severely aberrant in bladder tissue. In this article, we discuss the physiologic aspects of benign bladder disease, summarize biochemical evidence for the altered mitochondrial energy metabolism that appears to underlie bladder pathology, review the structure and function of the mitochondrial genetic system, and discuss molecular genetic studies of that system which have begun to provide a mechanistic explanation for the biochemical and physiological abnormalities that characterize the disease. We also discuss areas for further research which will be critically important in increasing our understanding of the detailed causes of benign bladder pathology.     

Computed tomographic excretory urography for diagnosing ectopic ureter in a female rhesus macaque (Macaca mulatta)

Ectopic ureter is a congenital abnormality where the ureter terminates at a site other than the urinary bladder. A five-year-old female rhesus monkey presented with a urine odor, a wet perineum, and persistent dribbling of urine. An ultrasound examination revealed a cyst-like structure (1 × 0.75 cm) on the left side of the bladder. Computed tomographic excretory urography (CTEU) imaging revealed a left unilateral extramural ectopic ureter, which was connected to the vagina. The perineum and wet hair were dried and disinfected with 0.4% chlorhexidine for perineal hygiene and skin care. The animal was closely monitored for potential moist dermatitis near the perineum and for urinary tract infection. The complete blood count (CBC) and blood chemistry results showed no signs of inflammation during the observation period. This is the first report of detailed diagnosis of ectopic ureter by ultrasound and CTEU in a female rhesus monkey.     

Clinical importance of significant asimptomatic bacteriuria in newborns and infants during early postnatal period

The aim of the study was to detect newborns at risk for developing renal impairment, and to point out the importance of significant asimptomatic bacteriuria in perinatal period and early infancy. Severe urinary tract anomalies are very often accompanied only by asimptomatic bacteriuria in perinatal period. Three urinalysis ware done after delivery. 212 newborns with significant asimptomatic bacteriuria underwent ultrasound examination, and were followed up to three months. Those with normal findings and with passing bacteriuria in the first 2 months were excluded. Group of 52 newborns underwent radioisotope examination. Frequency of urinary tract anomalies in newborns was 34.6%. Increased risk for renal impairment had children with urinary tract anomalies in close family, urinary tract infection or bacteriuria, EPH gestosis and prepartal symptoms of febrile infection in mother, children with IUGR, strangulated umbilical cord, prolonged jaundice and attacks of peripheral cyanosis in perinatal period.     

Neonatal outcome of fetuses with urinary tract abnormalities diagnosed by prenatal ultrasonography.

Between 1979 and 1986 an abnormality of the urinary tract was diagnosed by prenatal ultrasound examination in 93 fetuses. Postnatal investigation at a large teaching hospital showed a definite abnormality in 85 infants, 66 of whom were boys. An obstructed urinary tract, usually requiring surgery, was present in 46 infants. Other abnormalities included a multicystic kidney (in 15 infants), vesicoureteric reflux (in 9), prune-belly syndrome (in 5) and polycystic kidneys (in 5). Early recognition and treatment of urinary tract disorders in infants should be accompanied by informed prenatal counselling to minimize parents'' anxiety.     

The controversies regarding the role of estrogens in urogynecology

Estrogens are crucial for the proper functioning of genitourinary tract. Hypoestrogenism related to menopause could be linked to numerous disturbances of lower urinary tract. However, the results of most well designed clinical studies do not support use of estrogen or hormone replacement therapy for the treatment of genitourinary symptoms. According to evidence base medicine stress urinary incontinence, overactive bladder syndrome or pelvic organ prolapse are best treated by the surgery or non-hormonal drug therapy.     

Preventive and therapeutic administration of an indigenous <Emphasis Type="Italic">Lactobacillus</Emphasis> sp. strain against <Emphasis Type="Italic">Proteus mirabilis</Emphasis> ascending urinary tract infection in a mouse model

Probiotics are increasingly being considered as non-pharmaceutical and safe potential alternatives for the treatment and prevention of a variety of pathologies including urinary tract infections. These are the most common infections in medical practice and are frequently treated with antibiotics, which have generated an intense selective pressure over bacterial populations. Proteus mirabilis is a common cause of urinary tract infections in catheterised patients and people with abnormalities of the urinary tract. In this work we isolated, identified and characterised an indigenous Lactobacillus murinus strain (LbO2) from the vaginal tract of a female mouse. In vitro characterisation of LbO2 included acid and bile salts tolerance, growth in urine, adherence to uroepithelial cells and in vitro antimicrobial activity. The selected strain showed interesting properties, suitable for its use as a probiotic. The ability of LbO2 to prevent and even treat ascending P. mirabilis urinary tract infection was assessed using an experimental model in the mouse. Kidney and bladder P. mirabilis counts were significantly lower in mice preventively treated with the probiotic than in non-treated mice. When LbO2 was used for therapeutic treatment, bladder counts of treated mice were significantly lower although no significant differences were detected in P. mirabilis kidney colonisation of treated and non-treated animals. These results are encouraging and prompt further research related to probiotic strains and the basis of their effects for their use in human and animal health.     

Hand‐held,clinical dual mode ultrasound ‐ photoacoustic imaging of rat urinary bladder and its applications

Urinary bladder imaging is critical to diagnose urinary tract disorders, and bladder cancer. There is a great need for safe, non‐invasive, and sensitive imaging technique which enables bladder imaging. Photoacoustic imaging is a rapidly growing imaging technique for various biological applications. It can be combined with clinical ultrasound imaging system for hand‐held, dual modal ultrasound‐photoacoustic real‐time imaging. Structural (bladder wall) and functional (accretion of nanoparticles) bladder imaging is shown here with combined ultrasound and photoacoustic imaging in rats. Photoacoustic images of bladder wall is shown using black ink as the contrast agent. Chicken tissues were stacked on the abdomen of the animal to demonstrate the feasibility of photoacoustic imaging till a depth of 2 cm. Also, the feasibility of photoacoustic imaging for a common bladder disorder, vesicoureteral reflux is studied using urinary tract mimicking phantom. It is also shown that a clinical ultrasound system can be used for photoacoustic imaging of non‐invasive clearance study of gold nanorods from circulation by monitoring the gradual accumulation of the gold nanorods in the bladder. The time taken for accumulation of nanorods in the bladder can be used as an indicator of the clearance rate of the nanoparticle circulation from the body.      

Chronic hepatosplenomegaly in African school children: a common but neglected morbidity associated with schistosomiasis and malaria

Chronic hepatosplenomegaly, which is known to have a complex aetiology, is common amongst children who reside in rural areas of sub-Saharan Africa. Two of the more common infectious agents of hepatosplenomegaly amongst these children are malarial infections and schistosomiasis. The historical view of hepatosplenomegaly associated with schistosomiasis is that it is caused by gross periportal fibrosis and resulting portal hypertension. The introduction of ultrasound examinations into epidemiology studies, used in tandem with clinical examination, showed a dissociation within endemic communities between presentation with hepatosplenomegaly and ultrasound periportal fibrosis, while immuno-epidemiological studies indicate that rather than the pro-fibrotic Th2 response that is associated with periportal fibrosis, childhood hepatosplenomegaly without ultrasound-detectable fibrosis is associated with a pro-inflammatory response. Correlative analysis has shown that the pro-inflammatory response is also associated with chronic exposure to malarial infections and there is evidence of exacerbation of hepatosplenomegaly when co-exposure to malaria and schistosomiasis occurs. The common presentation with childhood hepatosplenomegaly in rural communities means that it is an important example of a multi-factorial disease and its association with severe and subtle morbidities underlies the need for well-designed public health strategies for tackling common infectious diseases in tandem rather than in isolation.     

Suprapubic aspiration under ultrasound guidance in children with fever of undiagnosed cause.

OBJECTIVES--To assess the ease of use of suprapubic aspiration of urine under ultrasound guidance in babies with fever of uncertain cause and to assess the importance of bacterial counts and pyuria in relation to abnormalities of the urinary tract and the importance of pyuria in the absence of bacteriuria. DESIGN--Analysis of urine samples obtained by suprapubic aspiration in babies and children from July 1991 to June 1992. The clinical records of the children with bacteriuria and sterile pyuria were examined retrospectively. SETTING--Neonatal and paediatric wards of a district general hospital. SUBJECTS--508 babies and children who had fever of uncertain cause or were seriously ill. RESULTS--No difficulties arose in the collection of 545 specimens. Bacteria were isolated from the specimens of 44 children, 24 of whom had abnormalities of the urinary tract. The bacterial count was < 10(8)/l in 18 of the children with bacteriuria, 10 of whom had abnormalities. No white cells were seen in 22 of the 46 bacteriuric specimens; nine of the children with no pyuria had vesicoureteric reflux. 439 of the 499 non-bacteriuric specimens showed no white cells. 60 children had pyuria without bacteriuria. CONCLUSIONS--The use of ultrasound guidance simplifies suprapubic aspiration of urine in babies. Low bacterial counts may be associated with abnormalities of the urinary tract. Laboratory techniques capable of detecting such counts reliably should be used. Pyuria is absent in half of babies and very young children with bacteriuria. It rarely occurs without bacteriuria, and if it does an explanation should be sought.     

Advances in stem cell therapy for the lower urinary tract

Lower urinary tract diseases are emotionally and financially burdensome to the individual and society. Current treatments are ineffective or symptomatic. Conversely, stem cells (SCs) are regenerative and may offer long-term solutions. Among the different types of SCs, bone marrow SCs (BMSCs) and skeletal muscle-derived SCs (SkMSCs) have received the most attention in pre-clinical and clinical trial studies concerning the lower urinary tract. In particular, clinical trials with SkMSCs for stress urinary incontinence have demonstrated impressive efficacy. However, both SkMSCs and BMSCs are difficult to obtain in quantity and therefore neither is optimal for the eventual implementation of SC therapy. On the other hand, adipose tissue-derived SCs (ADSCs) can be easily and abundantly obtained from "discarded" adipose tissue. Moreover, in several head-on comparison studies, ADSCs have demonstrated equal or superior therapeutic potential compared to BMSCs. Therefore, across several different medical disciplines, including urology, ADSC research is gaining wide attention. For the regeneration of bladder tissues, possible differentiation of ADSCs into bladder smooth muscle and epithelial cells has been demonstrated. For the treatment of bladder diseases, specifically hyperlipidemia and associated overactive bladder, ADSCs have also demonstrated efficacy. For the treatment of urethral sphincter dysfunction associated with birth trauma and hormonal deficiency, ADSC therapy was also beneficial. Finally, ADSCs were able to restore erectile function in various types of erectile dysfunction (ED), including those associated with diabetes, hyperlipidemia, and nerve injuries. Thus, ADSCs have demonstrated remarkable therapeutic potentials for the lower urinary tract.     

Bactofection with Toll-Like Receptor 4 in a Murine Model of Urinary Tract Infection

The role of innate immunity in the prevention of urinary tract infection is well-documented. Toll-like receptor 4 (TLR4) is a major determinant of innate immune response. In an animal model of urinary tract infection, bactofection-mediated gene transfer of TLR4 was tested in a preventive approach. Bactofection with TLR4 reduced the colonization with uropathogenic Escherichia coli by 91% in the kidney and by 41% in the bladder. Reduced colonization was associated with lower oxidative stress and expression of monocyte chemoattractant protein-1 and myeloperoxidase in the kidney. Bactofection with TLR4 was successful in the prevention of ascending pyelonephritis. Further studies should focus on long-term effects, the dose response and the potential therapeutic use in models of chronic urinary tract infection.     

Ultrasonography compared with intravenous urography in the investigation of adults with haematuria.

OBJECTIVE--To compare ultrasonography with intravenous urography in the investigation of adults with haematuria. DESIGN--Prospective study entailing the examination of all patients with both investigations concurrently. The investigations were performed independently on routine lists by different duty radiologists. Each was aware of the details of the request form but not of the findings of the other investigation. SETTING--Radiology department of a teaching hospital. PATIENTS--155 Consecutive adult patients (aged 18-93) referred from general practitioners and hospital outpatient clinics with a history of haematuria. FOLLOW UP--When results of both examinations proved normal no clinical or radiological follow up was sought. All abnormal findings of either investigation were correlated with results of subsequent imaging studies or operative findings. RESULTS--81 Patients (52%) had normal findings on urography and ultrasonography. Overall, the findings of ultrasonography concurred with those of urography in 144 cases (93%). Among the discrepant findings of the two investigations ultrasonography missed two ureteric calculi; one was in a non-dilated ureter, and in the other case ultrasonography detected the secondary ureteric dilatation. Ultrasound examination alone detected four bladder tumours not visible on urography with sizes ranging from 5 to 21 mm, representing one fifth of the 20 cystoscopically proved bladder tumours detected in the series. Ultrasonography detected all the 22 neoplastic lesions discovered in the study (20 bladder, two renal). Ultrasonography clarified the nature of renal masses evident in three urograms (simple cysts). CONCLUSIONS--Ultrasonography is a safe and accurate method of investigating the urinary tract in adults with haematuria. When combined with a single plain abdominal radiograph it proved to be superior to urography as the primary imaging study in this series. Ultrasonography should certainly be preferred to urography if cystoscopy is not planned. No urothelial tumours of the upper urinary tract were found in the series, reflecting their rarity. For those patients in whom ultrasonography and plain radiography have shown no abnormality and in whom cystoscopic appearances are normal urography would be advisable to exclude urothelial tumours of the upper urinary tract.     

Neo-regeneration of urinary bladder: a desired metaplasia of autologous membrane from rectosigmoid colon containing stem cells of intestinal crypts

The current management of diseases of urinary bladder requiring resection is by augmentation cystoplasty or transplantation of ureters. Transplantation of ureters is associated with morbidity and mortality. Ideal management will be by regenerating urinary bladder in vivo. Neo-regeneration of tissues and organs like abdominal wall, aponeurosis etc., has been attempted and patented. After neo-regeneration of mesoderm tissues and organs, regeneration of urinary bladder (developed from endoderm) was. In vivo surgical techniques were developed in dogs. It is known that the embryonic morphogenesis of urinary bladder is from uro-genital sinus of hind gut. A membrane, containing endoderm stem cells in crypts of recto-sigmoid colon, was surgically isolated and colonized with remnant of urinary bladder wall after extensive resection. Experimental study was performed in dogs, for 60 days to one and a half year. Regeneration of all the layers of tissues of the wall of urinary bladder was observed. The neo-regeneration phenomenon has been recognized as "desired metaplasia". The regenerated neo tissue/organ on histological examination and cystometry studies was found compatible with normal urinary bladder. The hypothesis, neo-regeneration and desired metaplasia, is discussed.     

The development of the bladder trigone, the center of the anti-reflux mechanism

The urinary tract is an outflow system that conducts urine from the kidneys to the bladder via the ureters that propel urine to the bladder via peristalsis. Once in the bladder, the ureteral valve, a mechanism that is not well understood, prevents backflow of urine to the kidney that can cause severe damage and induce end-stage renal disease. The upper and lower urinary tract compartments form independently, connecting at mid-gestation when the ureters move from their primary insertion site in the Wolffian ducts to the trigone, a muscular structure comprising the bladder floor just above the urethra. Precise connections between the ureters and the trigone are crucial for proper function of the ureteral valve mechanism; however, the developmental events underlying these connections and trigone formation are not well understood. According to established models, the trigone develops independently of the bladder, from the ureters, Wolffian ducts or a combination of both; however, these models have not been tested experimentally. Using the Cre-lox recombination system in lineage studies in mice, we find, unexpectedly, that the trigone is formed mostly from bladder smooth muscle with a more minor contribution from the ureter, and that trigone formation depends at least in part on intercalation of ureteral and bladder muscle. These studies suggest that urinary tract development occurs differently than previously thought, providing new insights into the mechanisms underlying normal and abnormal development.