Understanding the complex events leading to formation of an epithelial-based organ such as the breast requires a detailed insight into the crosstalk between epithelial and stromal compartments. These interactions occur both through heterotypic cellular interactions and between cells and matrix components. While in vivo models may partially capture these complex interactions, there is a need for in- vitro models to study these events. In this review we discuss cell-cell interactions in breast development focusing on the stem cell niche and branching morphogenesis. Given the recent understanding that the basic developmental events underlying branching morphogenesis are closely related to pathways important to cancer progression, i.e. epithelial plasticity and epithelial to mesenchymal transition (EMT), we will also discuss aspects relevant to cancer progression. In cancer, the adoption of mesenchymal phenotype by the malignant cells allows stromal invasion and subsequent intravasation to blood- or lymphatic vessels, a route that is a prerequisite for metastasis. A number of publications have demonstrated that tumor initiating cells, sometimes referred to as cancer stem cells adapt an EMT phenotype that renders them more resistant to apoptosis and drug therapy. The mechanism behind this phenomenon is currently unknown but this may partially explain relapse in breast cancer patients. Increased understanding of branching morphogenesis in the breast gland and the regulation of EMT and its reverse process mesenchymal to epithelial transition (MET) may hold the keys for future development of methods/drugs that neutralize the invading properties of cancer cells. Epithelial cells serve multiple functions in the human body. These include barrier functions , hormonal secretion , exocrine secretion , absorption, filtration and gas exchange . To serve its function, epithelial cells have adhesion properties that generate tight layer(s) of squamous, cuboidal or columnar epithelium dependent on location and function within the human body. Due to the immediate exposure of epithelial tissue to external environment cellular remodeling and renewal occurs relatively fast, meaning that new cells are continuously replacing older cells. Epithelial organs therefore contain stem cells that are responsible for the continuous cellular remodeling . FurtherThe branching nature of the epithelial ducts in the breast requires a level of phenotypic plasticity enabling cells to invade the underlying stroma. Cells need to transit from robust epithelial cell-cell binding to a more mobile state to facilitate migration. The cells can achieve this using distinct mechanisms including collective migration , 8 or epThe breast gland, somewhat uniquely, develops in different stages separated in time often by years or decades. In early embryonic mammary gland development, the formation of the mammary epithelial placodes in the skin is a critical event. These epithelial buds invade the underlying mesenchyme to form a rudimentary ductal systems embedded in stroma that develops along with the mammary epithelium . Most stIn humans, breast glands are similar in both sexes until the onset of puberty. In women, the second phase of development occurs during puberty. This phase is characterized by marked stromal and epithelial proliferation via hormonal stimulation. Estrogen signaling through the estrogen receptor α (ERα) is the primary force driving ductal branching and elongation, while progesterone signaling through its cognate receptor is required for proliferation and terminal differentiation in the TDLU . The epiThe final major developmental phase involves pregnancy and lactation. During pregnancy, further branching occurs and the cells within the TDLUs proliferate extensively to prepare for the final functional stage of lactation. The luminal epithelial cells finally reach their terminally differentiated state during the lactation period. At all other time periods, the luminal epithelial cells are considered to be in an immature, non-functional state. After lactation, the gland undergoes involution, characterized by massive epithelial apoptosis and return to a near pre-pregnancy non-functioning state. It is well established that the duration of the interval between the pubertal stage and first pregnancy is major risk factor for breast cancer. In that regard it has been proposed that repeated hormone stimulation of the stem cell niche in the course of repeated menstrual cycles prior to first pregnancy may be an important early event in breast carcinogenesis . Overall−CD29hiCD24+ immunophenotype, can at a single cell level regenerate the mouse mammary epithelium. The immature mammary gland stem cells have been located in the so-called peripheral cap cells of the mouse terminal end buds. Furthermore, in addition to the mammary stem cell, there is evidence for a hierarchical structure within the mammary epithelial tree, with both lobule- and duct-restricted progenitor cells [The putative mammary epithelial stem cell is responsible for growth, maintenance and late developmental generation and re-generation (at repeated pregnancies) seen through these distinct different phases of early and late development. The mouse mammary stem cell has been fairly well characterized using genetic techniques for tracking and transplantation assays in cleared mammary fat pad model , 19. Thior cells . While por cells .In comparison to the mouse, the human mammary epithelial stem cell is less well defined. There are, however, several studies that have isolated and/or characterized human breast epithelial cells with stem cell properties that have to a varying degree been able to recapitulate the human breast epithelial phenotype –25. UsinThe growth and branching morphogenesis of breast epithelium is under instructive influences of the surrounding stroma. The breast stroma is composed of collagenous extracellular matrix (ECM) and resident stromal cells including fibroblasts, immune cells, adipocytes and endothelial cells , 27. In Endothelial cells have previously been pictured as passive constituents of the conductive system transporting oxygen and nutrients towards tissues and waste products away. In addition, they have more recently been identified as critical regulators of the stem cell niche and organogenesis in many tissues, such as brain, liver and pancreas –35. In tAnalogous to the lung, we have shown that microvessels in a normal human breast are abundant and in close proximity to the epithelial cells of the TDLUs . StudiesWhen BRENCs are co-cultured in 3D rBM with D492, a breast epithelial cell line with stem cell properties, we observe increased branching morphogenesis, supporting the data that BRENCs contribute to regulation of branching epithelial morphogenesis. D492 was initially established from EpCAM positive, MUC1 negative suprabasal cells. D492 generate luminal and myoepithelial cells in culture and in 3D rBM it forms elaborate branching structures reminiscent of TDLU in situ . InteresEMT has in recent years caught the attention of the clinical research community as an intrinsic mechanism in pathogenic processes such as fibrosis and progression of many types of cancer including breast cancer –49. EMT One of the critical epithelial characteristics lost during EMT is cell-to-cell adhesion. This property is mediated by adherens junctions that are based on transmembrane proteins including cadherins, connecting adjacent cells via homophilic attachments made by their extracellular domains. Importantly, monitoring a switch from the expression of epithelial cadherin (E-cadherin) to a type of cadherin expressed by neurons and fibroblasts (N-cadherin), is the most commonly used method to assess EMT on a molecular level . AlteratExtracellular signals triggering EMT may channel through multiple pathways including the transforming growth factor beta (TGFβ), Notch, WNT, hedgehog, receptor tyrosine kinases (RTKs) and nuclear factor kappa-light chain enhancer of activated B-Cells (NFkB) , 53. TheIn view of the importance of EMT in promoting pathogenesis, identifying cells that may serve as paracrine source of TGFß or other EMT inducing ligands in cancerous or fibrotic tissue is being increasingly emphasized. Likely candidates include leukocytes, platelets, endothelial cells and cancer associated fibroblasts (CAFs) –57. ImmuThe ability of TGFß to confer EMT seems to be context specific and in some models epithelial cells are susceptible to other factors. For instance, contact dependent activation of the Notch pathway through the ligand Jagged 2 has been shown to induce EMT in colorectal cancer cells that do not adapt the phenotype in response to TGFß treatment , 59. TheThe canonical Wnt signaling pathway is directly related to the regulation of E-cadherin adhesion properties through control of its stabilizing partner ß-catenin. ß-catenin also functions as a transcription factor that can quickly transfer from cytosol to the nucleus in response to WNT signals where it activates promoters of target genes by displacing the co-repressor Groucho . Wnt sigMicroRNAs (miRNAs) have been implicated at several levels to be involved in the regulation of EMT . AlthougIn a recent study we demonstrated that Sprouty-2 a conserved negative feedback regulator of receptor tyrosine kinases affected branching morphogenesis and EMT of D492 cells in 3D culture. In this study we showed that knocking down Sprouty-2 in D492 resulted in large hyperplasia-like branching structure and in coculture with endothelial cells almost all colonies displayed mesenchymal phenotype . These dhigh/CD24low) and demonstrated by clonal inoculation into NOD/SCID mice that these cells contained cancer stem cells (CSCs) as observed by clonal growth and ability to regenerate the phenotype of the original tumor [high/CD24low expression [high/CD24low cell population with “epitheloid” phenotype rather than the CD44high/CD24negative with mesenchymal phenotype is more efficient in generating tumors in nude mice [In epithelial cancer, the EMT phenotype is associated with increased aggressiveness and metastatic behavior  Fig. 4)4). In bral tumor . Mani etpression . Also, tpression . It is, ude mice . It is, ude mice . The relTypically, tissue stem cells proliferate slowly and are therefore more resistant to chemotherapy as many cancer drugs specifically target proliferating cells by inducing DNA damage, causing growth arrest and apoptosis. Survival statistics underlining increased mortality in breast cancer patients with mesenchymal elements in the primary tumor, e.g. triple negative and claudin-low breast cancers, suggests that EMT cells are less sensitive to chemotherapeutic agents. This is further supported by the finding that cell populations displaying an EMT and CSC phenotype are likely to be present within residual breast cancer after conventional chemotherapy .At present there is not a full understanding of the inherent drug resistance of the EMT phenotype but several mechanisms could be at play. Firstly, the mesenchymal phenotype may be associated with higher expression of drug transporters such as ABCB1  and ABCG2 that actively export cytotoxic drugs. Up-regulation of these protein pumps has been suggested as an important protection mechanism of the cancer stem cells that contribute to treatment failures and relapse. Although the EMT phenotype may be a step closer to stem cell character than the epithelial state, evidence supporting this mechanism is lacking and treatment of patients with ABCB1 inhibitors such as tariquidar and zosuquidar have not improved therapeutic response . AnotherIn addition to all the above mentioned mechanisms the number of gene expression changes in cells undergoing EMT may also directly involve genes affecting the sensitivity to apoptosis signals. In fact, accumulating data implicates key EMT transcription factors directly in acquisition of chemo- and radio-resistant cancer phenotype. As an example, a study of ovarian cancer cells suggest that key EMT regulatory factors Slug and Snail directly promote both chemo- and radio-resistance by suppressing p53-mediated apoptosis . Snail hSince the TGFβ signaling pathway has been extensively linked to carcinogenesis and cancer progression, the pharmaceutical industry has been interested in developing drugs antagonizing its effect . The relSince EMT in normal and cancerous tissue is likely to be dynamically regulated by the epigenetic machinery, therapeutically targeting mechanism involved in regulating the epigenetic machinery is an attractive strategy. It has been shown that drugs (e.g. 5-azacytidine) that suppress normal promoter methylation inhibit EMT. The histone deacetylase inhibitor Panobinostat (LBH589) is being considered for the treatment of triple negative breast cancer and preliminary studies show that the drug prevents EMT in cell lines through its influence on ZEB transcription factors and E-Cadherin. Furthermore, this novel drug inhibits formation of metastasis by the breast cancer cell line MDA-MB-231 , 86. TheThe drug Eribulin mesilate that influences microtubule assembly has been shown to improve survival in late-stage breast cancer patients through an unknown mechanism. A recent study suggests that the influence of the drug is mainly through suppression of mesenchymal conversion, further stressing the importance of cytoskeleton reorganization for the completion of EMT .The anti-diabetic drug metformin, that is known to possess anti-cancer properties is reported to influence EMT. According to a recent study, this effect is probably due to activation of the AMPK pathway that is central to the regulation of energy metabolism and can be triggered by TGFβ signals . A selecThe branching epithelium of the adult female mammary gland undergoes repeated rounds of proliferation, differentiation and apoptosis during each menstruation cycle that is further exaggerated during pregnancy, lactation and involution. This continuous cellular remodeling has highlighted the importance of stem and progenitor cells within the mammary epithelium that provide the glandular epithelium with new functional cells. Furthermore, there is increasing interest in these stem cells, as they are believed to be targets for carcinogenic agents due to their longevity in tissues. It is debatable, however, whether or not it is the stem cells themselves, their progenitors or the more differentiated cells that have acquired stem cell properties and represent the true tumor initiating cells. It is of great importance that we are able learn more about the cellular hierarchy in the mammary gland and how the cross talk between the epithelium and stroma is maintained and how this goes awry during breast cancer progression. The inherent plasticity of the epithelium reflected in its ability to respond to external cues and change phenotype towards mesenchymal morphology via EMT is important, particularly since accumulating evidences implicates EMT in drug resistance and tumor relapse.


Renal Cell Carcinoma (RCC) is a heterogeneous set of diseases. Even within the major histological subtype of clear cell renal cell carcinoma (ccRCC), there is substantial variability in patient prognosis. The ultimate goal of organizing this heterogeneous disease is to elucidate prognostic elements and aid in clinical and therapeutic decision-making . In orgaCurrently, there are no clinically implemented biomarkers to predict which patients have a higher likelihood of disease recurrence. In an age of precision medicine, can a prognostic test be developed to aid clinicians in management of patients with early stage RCC? How do we decide which patients require yearly follow-up, which patients need to receive adjuvant therapy, or which patients need to enroll in a clinical trial to decrease their chance of recurrent metastatic disease? We have arrived in an era where adjuvant therapy is still reporting mixed results, but with one positive study, and many more studies to report in the near future , we willIn ccRCC, previous work has attempted to uncover such prognostic factors. The Recurrence Score (RS) and ClearCode34 are two of many efforts to develop transcript signatures with prognostic significance. The RS is a 16 gene panel validated in large patient cohorts to provide more accurate prognostic guidance than clinical and pathological variables alone . AdditioIn this month’s Oncotarget,  describeNext, in vitro analysis characterized the impact of SLINKY on phenotypes of ccRCC cell lines. Knocking down SLINKY by siRNA resulted in significantly less cell proliferation, but no change in migration. BrdU labeling experiments illustrated that SLINKY-knockdown leads to cell cycle arrest during the G1 phase. Further, transcriptomic analysis uncovered a group of 93 genes whose expression significantly changed with SLINKY knockdown. This set of 93 genes was then re-applied to the TCGA dataset and found to correlate with SLINKY expression as well as predict overall patient survival, illustrating that the genetic program dictated by SLINKY in vitro is similar to the changes initiated by SLINKY in patients.To elucidate SLINKY’s role, the Stanford group searched for the binding partner for this lincRNA. Protein microarray studies and RNA immunoprecipitation revealed that HNRNPK bound tightly to SLINKY. Furthermore, knocking down HNRNPK recapitulated the proliferation defects as well as global transcriptional changes as the siRNA knockdowns of SLINKY.Ultimately, this work suggests that SLINKY- HNRNPK complex might repress a tumor suppressor, and that low levels of intact SLINKY- HNRNPK results in appropriate transcription of the growth-arresting signal. In aggressive tumors, overexpression of the SLINKY- HNRNPK complex represses the tumor suppression, leading to increased tumor growth. Regardless of SLINKY’s physiological role, the fact that SLINKY is specifically expressed in cancerous tissues and that increased SLINKY expression predicts decreased overall survival, makes it a worthy candidate as a prognostic ccRCC biomarker.Single unit biomarkers provide the most simple and direct means of classifying disease. Further work will need to explore the heterogeneity of SLINKY expression across individual primary tumors. Interestingly, overexpression of SLINKY’s binding partner, HNRNPK, is known to be a negative predictor of survival in multiple cancer types . Now knowing a key binding partner for HNRNPK, future work may more accurately illustrate the role of this protein in tumorigenesis. Previous work argues that in myeloid malignancies HNRNPK is a haploinsufficient tumor suppressor while in solid tumors it functions an oncogene . Teasing


In Dysfunctional voiding, failure of the external sphincter-pelvic floor complex to relax during micturition results in bladder outflow obstruction with a spectrum of presentation from more benign lower urinary tract dysfunction including recurrent urinary tract infections, to significant upper tract pathology and end-stage renal failure. There is no underlying neurological or anatomical cause and the condition is postulated to be a largely learnt behavior. Diagnosis relies on non-invasive urodynamics and in particular uroflowmetry, plus or minus EMG, which is also used in biofeedback, the mainstay of treatment. The etiology, presentation, diagnosis, and treatment with particular emphasis on non-invasive urodynamics are covered. Dysfunctional voiding (DV), or micturition that is obstructed due to failure of the external urethral sphincter-pelvic floor complex to relax appropriately , has the highest rates of urinary tract infection (UTI), urinary incontinence (UI), vesicoureteric reflux (VUR), and bowel dysfunction of any functional lower urinary tract disorder and similar rates of frequency and urgency to overactive bladder (OAB) . Whilst Storage phase facilitated by hypogastric plexus stimulation (T10-L2) resulting in sympathetic relaxation of the detrusor muscle and stimulation of the urethral sphincter. Adequate compliance of the bladder wall allows storage of urine at low bladder pressures .Voiding or micturition phase where parasympathetic stimulation from the sacral micturition center (S2-S4) via the pelvic nerve facilitates detrusor contraction and internal sphincter relaxation, and somatic input via the pudendal nerve relaxes the external urethral sphincter. This allows the lowest possible detrusor pressure able to overcome outflow resistance during voiding with complete emptying of the bladder.Neurological control of the bladder governs two basic phases:Smooth coordination between the two phases of storage and micturition depends on intact neural pathways in the central nervous system in the cortical, pontine, and sacral micturition centers, which allow social planning and timing, coordination, and amplification, respectively.The bladder is an unusual autonomic visceral organ in that it interfaces with a voluntarily controlled somatic sphincter. The rectum is similar. Once toilet-training is achieved, voluntary initiation of voiding over a wide range of bladder volumes can be initiated at will in an “on-off” manner facilitated via a series of positive feedback loops using supraspinal pathways. The initial concept that babies micturate via a reflex spinal pathway is no longer held with evidence that neonates are aware of voiding with involvement of CNS centers , 3 and lBy far the commonest condition causing LUTD in children is overactive bladder (OAB) associated with uninhibited detrusor contractions during the storage phase of bladder function on invasive urodynamics. The classic symptom is that of urgency with additional holding maneuvers, urge incontinence, urinary frequency, DUI, and NE. The voiding phase is not generally implicated.DV on the other hand is dysfunction of the sphincter during voiding and refers only to the voiding phase in anatomically and neurologically intact patients. Simultaneous contraction of the sphincter and detrusor in the presence of a neurological condition is described as detrusor sphincter dyssynergia. These two terms are not interchangeable, as whilst the dyscoordination between detrusor and sphincter may be similar the underlying etiology is not .Beer first described voiding difficulty, recurrent infections, and sphincteric incoordination in neurologically normal children in 1915 . Hinman The current definition of dysfunctional voiding given by the International Children’s Continence Society (ICCS)  is:prevalence in the general population is not known because of diagnostic failure, overshadowing by storage symptoms and recurrent urinary tract infections, and confusion with terminology. Reliable surveys in children with wetting problems have reported anything from 4.2 to 32% [The true estimate of DV 2 to 32% .The causes of dysfunctional voiding are likely to be multi-factorial.A number of authors have reported congenital syndromes presenting with severe obstructed voiding in the absence of neuroanatomical abnormalities and associated upper tract deterioration. The early and severe presentation , rules out learnt maladaptation and points to congenital or genetic causes possibly associated with subclinical changes in the pons and pontine micturition center –13. DV hp < 0.001)Delayed acquisition of daytime bladder control (p < 0.001)Persistent daytime wetting (p < 0.001) following a period of previously being dryA relapse in daytime wetting  showed that the children of parents who initiated toilet training after 24 months of age (considered to be late toilet training) were more likely to experience the following than those who initiated toilet training between 15–24 months:Mothers who were older and with more educational qualifications and mothers who felt that their child was developmentally delayed and/or had a more difficult temperament started toilet training later. The authors surmise that delaying onset of toilet training until after 2 years of age prolongs the exposure time to potential stressors that can interfere with acquisition of bladder control. A limitation of the study was that there was no information available about the methods of toilet training used or the type of daytime wetting experienced subsequently .A small Swedish study longitudinally followed 59 children with non-invasive bladder monitoring from zero to 6 years and demonstrated acquisition of bladder control for day and night-time respectively at ages 3.5 and 4 years, together with later commencement in toilet training. Awareness of voiding however was noted from 18 months of age and the authors suggest that this is the opportune time to commence training. Children with a small post-void residual at 6 months of age and a large bladder capacity were more likely to become dry by day earlier [Using similar study methods to the Swedish study, 47 Vietnamese children had bladder assessments from zero to 3 years of age in association with early assisted toilet training and were compared with the Swedish cohort. Ninety-eight percent of Vietnamese children were toilet trained by 2 years of age in contrast to only 5% of Swedish children having commenced training. In addition, the study demonstrated resolution of dyscoordination between detrusor and sphincter muscles with minimal to no post-void residual urine by 9 months of age in the Vietnamese children compared to 36 months of age in Swedish children, suggesting that early toilet training confers protective bladder factors .It is thought that DV develops due to learnt and habitual dysfunction of the pelvic floor and that this is probably the commonest cause of the condition. There are many potential triggers or reasons why a child may tense their pelvic floor and engage their external urethral sphincter voluntarily during voiding; difficult toilet training in association with difficult child temperament and control issues, painful urination caused by urinary tract infection or local irritation in association with vulvitis or balanitis, dislike of urination in strange places outside of the home, fear of unclean toilets, social embarrassment and needing to eliminate only within a safe, sound-proof, personal space like home. Some reports suggest that DV is related to emotional and psychosocial problems or maturational delay and there are reports of patients developing dysfunctional voiding following childhood sexual abuse .Certain medical conditions are associated with DV. It is not uncommon for the child with urge incontinence to develop an overactive and high tone pelvic floor in response to recurrent detrusor overactive contractions in an attempt to avoid wetting, although there is no evidence that OAB necessarily evolves into DV with time . LikewisDysfunctional voiding can present at any age but most typically after toilet training and before puberty with recurrent urinary tract infections, day or night-time urinary incontinence, difficulties with voiding or urethral/suprapubic pain. The condition has a broad spectrum of presentation and outcomes from relatively harmless to the life-threatening complication of end-stage renal failure (ERF). The florid form presents similarly to the classical neuropathic or posterior urethral valve bladder with associated bilateral hydronephrosis and ERF. Bladder outflow obstruction (BOO) caused by the urethral sphincter may result in a number of physical changes in the urinary tract from distal to proximal with associated symptoms and signs; see Table BOO associated with DV results in voiding difficulty, but confusingly, children most commonly present with secondarily induced storage symptoms including urinary urgency and frequency and urge incontinence. These symptoms may be the only presenting features and typically result from detrusor overactivity, urinary infections or reduced bladder capacity consequent to large residual volumes or compensatory detrusor hypertrophy resulting in a functionally small capacity bladder. Urodynamic studies are required to distinguish between them. Others present with a history of recurrent urinary tract infection or evidence of incomplete bladder emptying on ultrasound scan. Recurrent UTI can occur due to incomplete emptying, trabeculation, or diverticulae of the bladder and VUR.Around half of children present with voiding difficulty and some present with unexplained urinary retention. They may describe difficulty with voiding in public places, delay in initiating urinary stream, infrequent voiding or requirement of visual, audible, or physical signals (such as voiding in the bath) to allow relaxation and micturition. Adults are more likely to present with actual voiding symptoms than children both due to expressive abilities and the increased prevalence of urinary retentive features in adults.Detrusor underactivity and acontractility may occur as an end point in a spectrum of storage abnormality in relation to DV commencing with initial reactive high detrusor pressures with subsequent detrusor decompensation resulting in low pressure retention. Voiding is accomplished by straining to increase abdominal pressure in order to initiate and maintain voiding. Symptoms and signs include infrequent voiding ≤3×/day , large-volume post-void residuals with overflow incontinence and UTI. Children with an over-distended bladder may be continent or have episodes of hesitancy, urge incontinence, or overflow incontinence related to increased intra-abdominal pressure. They tend to have dampness rather than a soaking of clothes and the diary will show infrequent spontaneous voiding of perhaps once or twice per day.The most severe form of DV is often called Hinman–Allen syndrome or non-neurogenic neurogenic bladder. These patients are found to have a trabeculated and irregularly shaped bladder with high voiding pressures, bilateral high-grade VUR, and a large residual urine volume. This can lead to recurrent UTI, renal scarring, hypertension, and progressive renal failure .Fowler’s syndrome is typically seen in young women aged 20 to 35 years, presenting with urinary retention with increased urethral sphincter tone with polycystic ovarian disease. Often there is a common natural history of relatively mild voiding dysfunction such as infrequent voiding or an intermittent stream. The first retention episode usually occurs following a triggering event such as an operation or childbirth. Investigations reveal abnormal sphincters, as assessed by EMG, transvaginal ultrasonographic volume and urethral pressure profile, with increased sphincter volume and concentric needle EMG demonstration of abnormal decelerating bursts and complex repetitive discharges. Whether Fowler’s syndrome represents a distinct cause of urinary retention or results from a maladaptive behavior and is similar to DV is unclear . Sacral History taking should be targeted at symptoms of both the storage and voiding phases with particular emphasis on any difficulties with initiating or maintaining urinary stream and any toileting avoidance behaviors. Features not commonly associated with OAB alone should raise suspicions of DV including features of hesitancy, obstructive urinary stream, normal-to-large capacity MVV, evidence of incomplete bladder emptying, and recurrent UTIs. It is vital not to forget a detailed bowel history and to exclude constipation based on Rome IV criteria , as wellTools of investigation including bladder and bowel diaries, questionnaires, urinalysis, and ultrasound of the urinary tract (pre-and post-void) should be routinely carried out . UltrasoThe key to diagnosis of DV is evidence of obstructed voiding during uroflowmetry preferably with accompanying pelvic floor EMG.Uroflowmetry is a simple, non-invasive and relatively inexpensive tool of investigation that makes it ideal for children. It assesses urinary voiding quantifying volume of urine passed per unit time with maximal or peak flow (q max ml/s) and average flow (q ave. ml/s), as well as voided volume, voiding time, and time to Qmax. The addition of post-void bladder ultrasound scanning using a small hand-held bladder scanner immediately after micturition allows measurement of post-void residual. The equipment is simple to use and most children feel comfortable with it as the uroflowmeter is usually used with a toilet-like commode that can be accommodated in a bathroom or private space allowing representative voiding see Fig. . In ordeStaccato flow caused by dynamic urethral obstruction during voiding is considered classical of DV. To label flow as staccato the fluctuations should be more than the square root of the maximum flow rate. However, staccato voiding is not exclusive to DV, and one group found that in only one-third of children demonstrating a staccato uroflow was pelvic floor activity the cause as demonstrated by EMG . The samWhere appropriate, further investigation may be required in the form of MRI spinal cord , cystoscopy , videourodynamics , and upper tract assessment.Initial steps in management are dependent upon whether there is evidence of damage or significant risk of damage to the kidneys. Those with high risk factors or renal impairment may require additional investigation such as invasive videourodynamics and early institution of clean intermittent catheterization and control of bladder storage pressures. If the risk of damage is low, which is probably in the majority of cases, treatment should have the primary objective to retrain the patient to relax the bladder outlet during voiding with a patient-centric focus. Behavioral therapy is based on the presumption that the disorder is a learned one and hence potentially reversible. It has been shown to be useful in children from the age of 4 years. A proposed management pyramid for children with DV is shown in Fig. The starting point for all childhood LUTD is urotherapy, and there is no exception in DV. The ICCS defines urotherapy as education of the child and parent as to normal bladder and bowel function, a drinking and voiding timetable to include timed voiding/double voiding and adequate fluid intake, correct toileting posture (see below) and aggressive management of constipation . SuccessIt is useful as a baseline to get the patient to concentrate on correct toileting posture first and certainly this does not require a specialist therapist and can be taught in a clinic outpatient appointment. This requires the child to be sitting securely on the toilet seat with buttock and foot support with comfortable hip abduction that does not activate abdominal muscle contraction and simultaneous pelvic floor activity. The theory is that the trunk or core muscles are part of an abdominal capsule made up of the pelvic floor, transversus abdominis, the diaphragm and the lumbar spine and multifidus. These muscles act as a continuum and are affected by breathing, lumbar spine positioning, and abdominal activity. In order for the pelvic floor to be maximally relaxed, the trunk must be fully stabilized by proper supported sitting with a fully relaxed abdominal wall, a neutral lumbar spine position (not extended or rounded), and the thighs at the level of the hips, slightly abducted with the feet in a neutral and supported position , 41 see. It is uBiofeedback is a form of learning or re-education in which the participant is retrained in a normally unconscious physiological process, in this case voiding, via directly tangible signals which may be visual or auditory allowing control and modification of the process and in this case direct relaxation of the pelvic floor. Success of the therapy increases with motivated and adherent patients and enthusiastic, trained therapists and may require multiple sessions. Wennergren and colleagues in 1995 first described physiotherapists working with girls using exercises designed to increase the child’s awareness of their pelvic floor musculature with the focus placed on relaxation as opposed to contraction (which tends to be the focus in adult patients) with improvement described in 12/16 . Since tJust as uroflowmetry provides the ideal investigation for diagnosis in children due to its simplicity, ease, and non-invasiveness, so it also provides the ideal platform for biofeedback training by having patients view their own voiding curve in real time to identify negative features (staccato dips) and correlate positive features with desired activity. The visual reinforcement can be magnified with auditory reinforcement either with the patient’s own urinary stream or software that links flow to sound. This requires the patient to remain in the hospital for a period of time to produce a number of voids whilst receiving immediate feedback on the uroflow curve.Some patients also benefit from biofeedback concentrating on pelvic floor muscle isolation and this can be done using perineal EMG surface electrode feedback. There are a number of commercial animated software programmes that are visually engaging for children and with which they generally have positive associations. This can help simplify muscle isolation and encourage patient interest, particularly in those children who are younger or who have attention or motivation issues. The software controller effectively becomes the pelvic floor. The other advantage is that whilst these programmes can be combined with uroflowmetry, they can be carried out independent of voiding uroflowmetry, and this can be time saving. Certainly, EMG has been shown to be as effective as uroflowmetry biofeedback with both animated and non-animated systems. Anecdotally, we find that non-animated systems are easier for older, more cognitively able children, and animated systems are more suited towards younger children or children with developmental difficulties such as autism, learning impairment or, attention deficit hyperactivity disorder. One study has found that animated perineal EMG biofeedback was as efficacious as non-animated, but yielded results more quickly (in 3.6 versus 7.6 sessions) .Real-time ultrasound, either transabdominal at the level of the bladder base or transperineal showing anterior and cranial movement of the bladder neck on contraction of the pelvic floor, with equal attention to relaxation of the pelvic floor, has also been shown to be useful .It is important to measure treatment success and failure using the same tools used to non-invasively evaluate the patient initially including bladder and bowel diaries, uroflow and EMG parameters, post void residual urine measurement, and the recurrence of UTI. Risk factors for failure of biofeedback training include bladder capacity less than 60% of predicted volume and patient noncompliance .Observation during biofeedback often reveals children with pelvic floor dysfunction together with evidence of overactive bladder symptoms who may benefit from anticholinergic medication in conjunction with biofeedback treatment providing they can empty their bladders completely.Alpha adrenergic receptors have been demonstrated in the bladder neck and throughout the human urethra and alpha blockers result in decreased bladder outlet resistance with several reports of success in treating children with incomplete bladder emptying . One stuBotulinum toxin inhibits acetylcholine release at the presynaptic neuromuscular junction and results in flaccid muscular paralysis and there are several small reports of successful intrasphincteric botulinum toxin-A in the treatment of biofeedback-resistant dysfunctional voiding , 51. FolSacral neuromodulation has been used for intractable lower urinary tract dysfunction and in two series in children with DV. The response to neuromodulation (Interstim device) was modest  but coulUrethral dilatation has fallen from grace having been found to result in no sustained voiding improvement .CIC allows regular complete emptying of the bladder and is the mainstay of treatment for those high-risk bladders threatening the upper tracts or atonic retentive bladders.Dysfunctional voiding is a voiding disorder associated with failure of the external urethral sphincter to relax during micturition in the absence of neurological or anatomical abnormality, with potential consequences for the entire urinary tract as well as quality of life.Presentation may be masked by storage symptoms, failure to elicit difficulties with voiding on history, emphasis on urinary tract infections, or vaginal reflux. Diagnosis is made by establishing functional obstructed voiding using non-invasive urodynamics classically (but not exclusively) with a staccato-flow pattern. Urotherapy and non-invasive uroflowmetry, plus or minus surface pelvic floor EMG, provides the mainstay of treatment.Neurological impairmentCongenital anatomical obstruction of the urethraFailure of pelvic floor and external urethral sphincter relaxation during voidingSmall-capacity bladderWhat is the cause of dysfunctional voiding?Urinary urgencyRecurrent urinary tract infectionsDifficulty initiating urinary streamWeakness in lower limbsHow might dysfunctional voiding present?Physical examinationRenal tract ultrasound with post void residual measurement.48-h bladder input-output diaryUroflowmetry +/- EMGHow is a suspected case of dysfunctional voiding confirmed?Commonly leads to kidney failureCan present with secondary urinary incontinenceOnly occurs in children who have suffered abuseUncommon condition in childhoodWhich statement is correct regarding dysfunctional voiding?usually occurs with a staccato-shaped flowshould only be carried out with two channel EMGmay present with any of the known uroflowmetry curvesrequires the placement of a urethral catheterUroflowmetry in dysfunctional voiding:Urotherapy with correct toileting postureAnticholinergic medicationNeuromodulationAlpha-blocker medicationWhat is the first-line treatment for dysfunctional voiding?Abdominal contraction with inhalation helps relax the pelvic floorFoot support with knees in line with the hips helps relax pelvic girdle musclesA neutral lumbar spine allows relaxation of the pelvic floorLegs should be wide apart with hips tilted forwardWith regards to correct toileting position


The indirect comparison of two interventions can be valuable in many situations. However, the quality of an indirect comparison will depend on several factors including the chosen methodology and validity of underlying assumptions. Published indirect comparisons are increasingly more common in the medical literature, but as yet, there are no published recommendations of how they should be reported. Our aim is to systematically review the quality of published indirect comparisons to add to existing empirical data suggesting that improvements can be made when reporting and applying indirect comparisons.Reviews applying statistical methods to indirectly compare the clinical effectiveness of two interventions using randomised controlled trials were eligible. We searched (1966–2008) Database of Abstracts and Reviews of Effects, The Cochrane library, and Medline. Full review publications were assessed for eligibility. Specific criteria to assess quality were developed and applied. Forty-three reviews were included. Adequate methodology was used to calculate the indirect comparison in 41 reviews. Nineteen reviews assessed the similarity assumption using sensitivity analysis, subgroup analysis, or meta-regression. Eleven reviews compared trial-level characteristics. Twenty-four reviews assessed statistical homogeneity. Twelve reviews investigated causes of heterogeneity. Seventeen reviews included direct and indirect evidence for the same comparison; six reviews assessed consistency. One review combined both evidence types. Twenty-five reviews urged caution in interpretation of results, and 24 reviews indicated when results were from indirect evidence by stating this term with the result.This review shows that the underlying assumptions are not routinely explored or reported when undertaking indirect comparisons. We recommend, therefore, that the quality of indirect comparisons should be improved, in particular, by assessing assumptions and reporting the assessment methods applied. We propose that the quality criteria applied in this article may provide a basis to help review authors carry out indirect comparisons and to aid appropriate interpretation. A and B, is of interest. If both interventions (A and B) have at some point been compared with a third common intervention (denoted C) in separate randomised controlled trials, then an indirect comparison is possible. If trials exist that compare A and B directly, then direct evidence also exists in addition to the indirect evidence.Systematic reviews of randomised controlled trials are the highest quality evidence to support healthcare decisions. When the relative effectiveness of interventions is of interest, evidence from trials that compare the interventions directly  and evidence from indirect comparisons may be sought within a review. A systematic review of randomised controlled trials that directly (head-to-head) compare two interventions would generally be regarded as the highest quality evidence to support healthcare decisions on the comparative effectiveness of two interventions. In many clinical areas this high quality evidence may not exist or may be inconclusive and utilising alternative sources of evidence such as an indirect comparison could be appropriate. For example, pharmaceutical companies may be reluctant to compare a new drug against the effective standard drug in a head-to-head trial in case results do not favour the new drug. Furthermore, indirect evidence can be more reliable than direct evidence in some cases, for instance, when direct evidence is biased due to the methodological inadequacies of trials that compare the treatments directly et al, who recommend that the indirect comparison methodology should preserve the within-trial randomisation Numerous approaches exist to undertake an indirect comparison, a review of which has been undertaken by Glenny et althe ‘adjusted’ method by Bucher meta-regression A relative to C and B relative to Chypothesis tests, that test for a difference between treatments effects of A relative to C and B relative to Cexamination of the overlap of confidence intervals for treatments effects of A against treatment B ignoring treatment C and therefore break within trial randomisation. Naive indirect comparison methods are therefore not recommended and are considered to be equivalent to observational data and subject to similar biases In contrast, the ‘naive’ method would compare treatment A relative to C is not actually observed in the B vs. C trials , the similarity assumption is difficult to assess. No well-established methods exist to determine when the similarity assumption holds; however, comparing patient or trial characteristics across the trials involved in the indirect comparison, and investigating the effect of patient or trial characteristics on the indirect comparison result using subgroup analysis, sensitivity analysis, or meta-regression, may indicate whether similarity is reasonable The core assumption underlying indirect comparison methodology is similarity of treatment effects A vs. C trials, and the similarity within the head-to-head B vs. C trials. Standard methods to assess homogeneity exist Other key assumptions that underlie indirect comparison methodology are homogeneity and consistency. Homogeneity concerns the similarity within the head-to-head The assumptions of similarity, homogeneity and consistency can be thought of as an extension of the usual homogeneity assumption in standard meta-analysis. Assessment of the assumptions is vital to ensure the results of indirect comparisons are valid and interpreted appropriately. Since no guidelines concerning the reporting of indirect comparisons and assessment methods exist, the importance of a review of the reporting and methodological quality of the indirect comparison methods applied in published reviews is clear.et al searched for reviews that applied indirect comparison methodology and summarised the methods and results of the reviews Existing research articles have summarised the indirect comparison methodology applied in published reviews and relevant methodological problems. Recently, Song et al published a summary of methodological problems identified by surveying published reviews of mixed treatment comparison meta-analysis. The methodological problems reviewed were: the mixed treatment comparison method used; whether the similarity assumption and consistency assumption was mentioned; whether efforts were made to investigate or improve the similarity for mixed treatment comparisons; and whether direct and indirect evidence was combined or compared The primary aim of this article is to report a systematic review of the reporting and methodological quality of published indirect comparisons using specifically devised quality assessment criteria. These criteria may provide a basis for the future development of a quality assessment tool for the evaluation and critical appraisal of indirect comparisons to aid appropriate interpretation. The review also adds empirical data to the existing evidence and highlights further the importance of improving reporting quality with some preliminary recommendations made.Inclusion Criteria:A and B) based on randomised controlled trials.Reviews that applied statistical methods to indirectly compare the clinical effectiveness of two interventions .Indirect comparisons based on non-randomised trials.Reviews that indirectly compared interventions qualitatively .Reviews that indirectly compare more than two interventions simultaneously .The following databases were searched using specific search terms : The DatThe full publication was obtained for each review located by the search and independently assessed against the eligibility criteria by two reviewers using an eligibility form. After assessment, differences in the assessment results were discussed. Reports were scrutinised to ensure that only the latest version of updated reviews was included.Information was extracted using a data extraction form regarding: general characteristics of the reviews, such as, the inclusion criteria in terms of patients, interventions, trial design, and primary outcomes; the indirect and direct comparisons made; the number of trials and patients in the indirect comparison; the type of data and measure of effect for the primary outcome; and whether the review was based on individual patient data or aggregate data.We also extracted information regarding the indirect comparison method; the consideration and assessment of the similarity, homogeneity, and consistency assumptions; reporting of results; and interpretation of the evidence. More specifically, we extracted the indirect comparison method reported or applied and the type of results presented . Regarding the similarity assumption, we extracted information such as: the assumption's phrasing; any reported assessment methods; whether sensitivity analysis, subgroup analysis, or meta-regression was applied to investigate if the indirect comparison result varied; any remarks regarding the results of such methods; and whether patient or trial characteristics across all trials included in the indirect comparison were reported, compared, or comparable. For the homogeneity assumption, we extracted details such as: the assumption's phrasing; the assessment method reported or applied; whether the homogeneity assumption was satisfied based on quantitative results or concluding statements; whether a fixed effects or random effects model was applied; whether sensitivity analysis, subgroup analysis, or meta-regression was applied across trials in each trial set involved in the indirect comparison; and any remarks regarding the results of these methods. Regarding the consistency assumption, we extracted information such as: the assumption's phrasing; the assessment method reported or applied; whether the assessment method was satisfied based on quantitative results or concluding statements; whether direct and indirect evidence was combined and the type of results presented ; whether patient or trial characteristics across all trials were reported, compared, or comparable; whether three-arm trials were included using direct evidence rather than indirect evidence from the trial; reasons given for using indirect and direct evidence; and whether results from each head-to-head trial were reported. For reporting of results we extracted details, such as, whether the meta-analytic result for each of the two trial sets involved in the indirect comparison was presented and the type of results given ; whether results from all the individual trials' were reported and the type of results given ; and whether the review indicated which results were based on indirect evidence. Regarding interpretation we extracted information, such as, whether the review indicated that direct and indirect evidence are not equivalent; and whether the review indicated that more head-to-head trials were needed.The data extracted related to the review's primary outcome where stated, or the outcome for which results were reported first in the absence of a specific primary outcome. When reviews did not specifically report the number of trials (or patients) in the indirect comparisons the number of trials (or patients) were calculated based on the data from direct comparisons. The review author was not contacted in the case of unclear or missing data as it was considered that the quality assessment should be based solely on the reported information.The general characteristics of reviews were summarised. Categorical data were summarised using frequencies.We independently assessed the quality of the reporting and application of indirect comparison methods in each review using specific quality criteria. Differences in the assessment results were discussed. Initially, the criteria were compiled from recommendations given in published literature We considered higher quality reviews to be reviews that applied indirect comparison methods that preserved randomisation and that presented a measure of treatment effect and measure of precision. Regarding similarity, reviews were classed as higher quality when they stated the similarity assumption and a method to assess the assumption; applied a suitable assessment method, such as, sensitivity analysis, subgroup analysis or meta-regression including all the trials in the indirect comparison; and presented and compared patient or trial characteristics for all trials in the indirect comparisons. Regarding homogeneity, we considered higher quality reviews to be reviews that applied a suitable method to assess homogeneity  and if heterogeneity was evident that it was accounted for using a random effects model and explored using sensitivity analysis, subgroup analysis or meta-regression. Regarding consistency, reviews were classed as higher quality when they assessed consistency; did not combine indirect and direct evidence in the presence of inconsistency; and compared patient or trial characteristics for all trials contributing direct and indirect evidence. We classed reviews that included three arm trials as lower quality when the review author ignored the direct evidence in the trial. We classed reviews as higher quality when justification for including direct and indirect evidence was given; and when the results from trials contributing direct evidence were presented. Regarding interpretation, we considered reviews to be of higher quality when the review author explained that direct and indirect comparisons are not equivalent to avoid misinterpretation of the results; and when the review author considered the strength of direct evidence. For reporting, reviews were judged to be of higher quality when the review author presented the two meta-analytic results used in the indirect comparison and the individual trials' results; and when the review author indicated when results of indirect comparisons were reported.See Most indirect comparisons (30 reviews) were based on fewer than 15 trials. The indirect comparisons made are reported in Reviews were focussed in a variety of clinical areas: circulatory (11 reviews); musculo-skeletal (nine reviews); reproductive (four reviews); HIV (three reviews); psychological (three reviews); cancer (two reviews); gastrointestinal (two reviews); post-operative (two reviews); psychiatric (two reviews); diabetes (one review); ocular (one review) and other clinical areas (three reviews). A range of outcomes were studied in the reviews as described in Dichotomous outcome data predominated (32 reviews) with treatment effects summarised using the risk ratio (16 reviews), the odds ratio (13 reviews), or the risk difference (three reviews); continuous outcomes were presented using the mean difference (six reviews) and the standardised mean difference (three reviews); count data were reported using the rate ratio (one review); and time to event data were summarised with the hazard ratio (one review). One review stated that individual patient data were analysed but the remainder were based on aggregate data.A variety of interventions were compared indirectly. Thirty-four reviews indirectly compared pharmacological treatments: drugs (20 reviews), doses or regimens (seven reviews), and drug combinations (seven reviews). Nine reviews compared non-pharmacological interventions: vitamin/mineral supplements (two reviews), testing methods (two reviews), and treatment delivery (five reviews).Adequate statistical methods, that is, methods that preserved randomisation within trials, were applied in 41 reviews (95%): 23 reviews applied the adjusted method, six reviews used meta-regression, five reviews compared the overlap of confidence intervals, and seven reviews used significance tests. Two reviews (5%) applied inadequate methods (naïve method).Of the 41 reviews that used adequate methods, only 25 (61%) presented a measure of treatment effect and its precision for the indirect comparison .The similarity assumption was stated in 11 reviews (26%) using various terminology and described in different sections of the review manuscript; the assumption was described in the introduction (one review), methods (two reviews), results (two reviews), discussion (five reviews), and appendix (one review) .None of the reviews explicitly described a method to examine the assumption of similarity within the grouping the trials according to a particular characteristic, indirectly comparing interventions for each grouping  (seven reviews);conducting meta-regression including trial-level summaries that may modify the treatment effect (four reviews);selecting a trial group based on a particular characteristic and indirectly comparing interventions using the selected trial subset  (eight reviews).Analyses varied greatly in terms of the number of variables studied.A summary of patient and trial characteristics were presented in 38 reviews (88%), although the number of characteristics varied substantially across reviews. Only eleven reviews (26%) compared characteristics between the two trials sets contributing to the indirect comparison: four reviews reported that characteristics were comparable; five reviews stated characteristics were dissimilar  but continued to estimate the indirect comparison; and two reviews did not state whether or not characteristics were comparable, thus were unclear regarding comparability, but did discuss the similarities and differences of characteristics among the trials.Three reviews included one trial per treatment comparison therefore homogeneity assessment was not applicable. To determine the presence of heterogeneity, 24 reviews (60%) implemented adequate methods, namely the Chi-squared test, I-squared statistic, or estimation of between-trial variability. Twelve reviews (30%) did not report an adequate method or results of a homogeneity assessment for the relevant group of trials. The assessment method was unclear or it was unclear whether the assessment had included the group of trials of interest in four reviews (10%).Based on the I-squared statistic, Chi-square test, or statements reported, the homogeneity assumption seemed reasonable in eight reviews. There was evidence of heterogeneity in 15 reviews, 11 of which applied a random effects model. In seventeen reviews homogeneity was not tested or reported, hence the presence of homogeneity was unclear.For the 32 reviews for which statistical heterogeneity may exist, twelve reviews (38%) implemented adequate methods: subgroup analysis (seven reviews), sensitivity analysis (two reviews), or meta-regression (three reviews) to explore clinical and/or methodological factors as a potential explanation of statistical heterogeneity within the trial sets. Nineteen reviews (59%) did not explore potential causes of heterogeneity for relevant trial groups. One review (3%), classified as unclear, did not indicate the trial set on which the assessment was applied.Seventeen reviews (40%) included direct and indirect evidence in the review for the same comparison. Six of these reviews (35%) assessed consistency of the treatment effects: one review used a hypothesis test to compare the direct and indirect estimates of treatment effect; and five reviews discussed the consistency of direct and indirect treatment effects. Eleven of the reviews (65%) did not assess consistency of the treatment effects.Of the six reviews that did evaluate consistency, four reported consistent evidence and two reported inconsistency. One review that reported consistency combined direct and indirect effect measures using meta-analysis to produce a pooled effect estimate. Both of the reviews that reported inconsistency investigated differences and did not combine evidence types.Patient and trial characteristics were compared across direct and indirect evidence trials in five reviews (29%) of which two reported comparability, one reported non-comparability, and two did not report results.Twelve reviews included information from three-arm trials, but only three of these reviews (25%) correctly analysed these data as direct evidence rather than indirect evidence.Justification for including indirect evidence and direct evidence was provided in eight reviews (47%), reasons were: limited number of trials providing direct evidence (five reviews), aimed to compare direct and indirect evidence (two reviews), and both reasons (one review).Six reviews (35%) did not present the results from each trial contributing direct evidence.Twenty-five reviews (58%) made a distinction between indirect comparisons and direct comparisons. Twenty-four reviews (56%) stated that more direct evidence trials were needed.Thirty-seven reviews (86%) presented meta-analysis results from each of the two trial sets involved in the indirect comparison. Twenty-four reviews (56%) highlighted when the result was an indirect comparison by stating this term with the result. The treatment effect estimated from each trial was reported in 23 reviews (53%).Guidelines for reporting conventional pair-wise meta-analyses and for producing high quality systematic reviews are already available Firstly, the method of analysis, the assumptions made and methods for assessing the plausibility of assumptions, particularly that of similarity should be clearly stated within the A vs. C, B vs. C, and A vs. B) would be similar across all trials irrespective of which interventions where included in the trial . If the true treatment effect comparing any two interventions is modified by a particular trial or patient characteristic and all the trials involved in the indirect comparison are not alike with respect to the characteristic, then the assumption will be violated. One approach to assess the similarity assumption is to compare patient characteristics and trial features descriptively across all trials contributing to the indirect comparison. This can help identify variability in any important characteristic that could modify the treatment effects and hence violate the similarity assumption. If characteristics are similar, the similarity assumption is more likely to hold than if characteristics are dissimilar. However, if characteristics vary but are not expected to modify treatment effects then the assumption may still be satisfied. This of course assumes that there are no unknown characteristics that would modify the result. The characteristics studied should be chosen using expert, evidence-based information, as should be the case in any standard meta-analysis. In our review, only 11 reviews (26%) undertook some kind of comparison of trial or patient characteristics. Bucher et al compared characteristics across the two trial sets (A vs. C and B vs. C) by calculating a summary measure for each of the trial sets and then comparing the summary measures et al. Secondly, the potential for modification of the result of the indirect comparison can be explored using appropriate characteristics by sensitivity analyses, subgroup analysis, or meta-regression, although the usual limitations of these methods should be kept in mind Although a formal statistical test for similarity is not available, there are approaches that can be used to assess how reasonable is the assumption. The similarity assumption holds when the true treatment effects comparing any two interventions  that did not make this distinction. When interpreting indirect evidence, consideration should be given to the generalisability of the patients included in the trials involved in the indirect comparison, just as the generalisability of patients included in trials in a direct comparison should be considered when interpreting direct evidence. Moreover, the results of the assessment of the assumptions can help determine the reliability of the indirect evidence; if the assumptions appear reasonable, the indirect evidence should be valid. In the same way, the assessment of the homogeneity assumption can help determine the reliability of the direct evidence.The results of the indirect comparison, direct comparison, individual trial results, and the meta-analytic treatment effects from each of the two trial sets involved in the indirect comparison, should be reported. Also, review authors should clearly indicate which results are based on indirect evidence; our findings showed that 19 reviews did not make this indication.One important aspect not examined in this review is that indirect comparisons should be based on meta-analysis results which are a component of a systematic review as for any other meta-analysis. The usual rigorous methodology and assessment of risk of bias should be undertaken as part of the systematic review Song et al included 88 reviews, substantially more than the 43 reviews included in this overview Song et al. Similarly, 58 reviews were included by Song et al which are not included in this assessment. The reason for this disparity is partly due to differences in eligibility criteria, search strategies and search terms. Even so, the results of this review mostly support the findings of Song et al but consider the quality of aspects in more depth than previous research. Song et al found that trial similarity was discussed or explicitly mentioned in 45% of reviews, where as we found that 26% of reviews explicitly stated the assumption. Song et al reported that 26% reviews carried out subgroup or meta-regression to identify or adjust for possible treatment effect modifiers; we found that 44% of reviews undertook similar methods. We found that 26% of reviews compared trial and patient characteristics across all the trials used in the indirect comparison; Song et al stated that 30% of reviews compared characteristics. Consistency of direct and indirect evidence was assessed in 71% of reviews that applied the naive approach or adjusted indirect comparison method as described by Song et al, where as we established that 35% assessed the consistency of evidence. Song et al found that 12% of these reviews combined direct and indirect evidence; we found that 6% of reviews combined evidence.The recently published article by et al highlighted the methodological flaws in published indirect comparisons and made recommendations regarding suitable methodology. Our review identifies the importance of improving reporting quality and adds empirical data to the existing evidence regarding methodological quality. The specifically devised quality assessment criteria applied in this review provides a grounding to help review authors carry out indirect comparisons and to aid appropriate interpretation.Song et al reported that 63% of reviews applied the adjusted method or naive approaches, where as only 20% of reviews compared multiple treatments simultaneously using meta-analysis. These results show that this article is applicable to the main body of published reviews.The main limitation of this review is that generalisability is restricted because reviews that compared more than two interventions simultaneously, for example, using mixed treatment comparison meta-analysis models were excluded because additional quality criteria and search terms would apply to these reviews. Detailed quality assessment criteria would include modelling details such as allowance for multi-arm trials, specification of variance structures, and assessment of consistency of indirect evidence using different common interventions . For this reason, reviews that compared more than two interventions simultaneously will be considered separately following a search using adapted search terms. In total, 21 reviews of randomised trials using mixed treatment comparison methodology were excluded from this overview. However, it is worth noting that the methodology for undertaking a simple indirect comparison is much more accessible than for complex mixed treatment comparisons and therefore widely applicable. Interestingly, Song et al although slightly different sets of reviews were included in each article.A further limitation of this review is that we may not have retrieved all published reviews including an indirect comparison because some reviews may not have been indexed using the search terms specified. However there is no reason to believe that the reviews we identified would differ to those we did not identify and hence our sample should be a representative sample of published indirect comparisons in the medical literature. In fact the conclusions reached in this article are comparable to the article by Song Lastly, a thorough assessment of quality would require clinical knowledge of the individual review topic areas. Clinical knowledge would allow assessment of the similarity assumption as potential patient characteristics that could influence the result of the indirect comparison may be known to those working within the individual review areas.In conclusion, indirect comparisons can be extremely valuable and their use is increasing in the literature. However, the validity of the indirect comparison relies on underlying assumptions similar to standard meta-analysis. This review shows that these assumptions are not routinely explored when undertaking and reporting indirect comparisons. We recommend therefore, that the methodological and reporting quality of indirect comparisons should be improved and propose that the quality criteria applied in this article may provide a basis to help review authors carry out indirect comparisons and to aid appropriate interpretation.Table S1Search terms for databases.(0.06 MB DOC)Click here for additional data file.Table S2Characteristics of included reviews. *Outcome name (primary outcome or main outcome); data type; measure of effect. **NT: number of trials; NP: number of patients.(0.21 MB DOC)Click here for additional data file.Table S3Quality assessment results for individual reviews. Abbreviations: c: comparable/consistent, f: fixed effects model, h: homogeneity reported or determined from results, he: heterogeneity reported or determined from results, n: no, na: not applicable, nc: not comparable/consistent, nr: not reported, nt: no three-arm trials, r: random effects model, t: using a statistical test, u: unclear, y: yes.(0.18 MB DOC)Click here for additional data file.


Escherichia coli (UPEC) represent the predominant cause of urinary tract infections (UTIs). A key UPEC molecular virulence mechanism is type 1 fimbriae, whose expression is controlled by the orientation of an invertible chromosomal DNA element—the fim switch. Temperature has been shown to act as a major regulator of fim switching behavior and is overall an important indicator as well as functional feature of many urologic diseases, including UPEC host-pathogen interaction dynamics. Given this panoptic physiological role of temperature during UTI progression and notable empirical challenges to its direct in vivo studies, in silico modeling of corresponding biochemical and biophysical mechanisms essential to UPEC pathogenicity may significantly aid our understanding of the underlying disease processes. However, rigorous computational analysis of biological systems, such as fim switch temperature control circuit, has hereto presented a notoriously demanding problem due to both the substantial complexity of the gene regulatory networks involved as well as their often characteristically discrete and stochastic dynamics. To address these issues, we have developed an approach that enables automated multiscale abstraction of biological system descriptions based on reaction kinetics. Implemented as a computational tool, this method has allowed us to efficiently analyze the modular organization and behavior of the E. coli fimbriation switch circuit at different temperature settings, thus facilitating new insights into this mode of UPEC molecular virulence regulation. In particular, our results suggest that, with respect to its role in shutting down fimbriae expression, the primary function of FimB recombinase may be to effect a controlled down-regulation (rather than increase) of the ON-to-OFF fim switching rate via temperature-dependent suppression of competing dynamics mediated by recombinase FimE. Our computational analysis further implies that this down-regulation mechanism could be particularly significant inside the host environment, thus potentially contributing further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs.Uropathogenic  Escherichia coli (UPEC) bacteria, whose wide-spread and increasing antibiotic-resistance has made the development of alternative anti-UPEC treatments progressively more important and urgent. UPEC's foremost virulence factor is hair-like surface structures called type 1 fimbriae. Thus, one such potentially promising therapeutic approach may be to manipulate bacteria's own cellular circuitry toward inducing UPEC to turn off their fimbriae expression—rendering individual microbes benign. This task requires detailed understanding of molecular mechanisms involved, which may be significantly aided by in silico modeling. However, for UPEC fimbriation control circuit and many other systems, low-level all-inclusive quantitative models inevitably become too computationally demanding to remain practical, while high-level qualitative representations frequently prove inadequate owing to the substantial organizational and behavioral complexity of biological processes involved.Urinary tract infections (UTIs) represent a major growing threat to global public health. With over 15 million cases a year in the United States alone, UTIs are characterized by very high recurrence/reinfection rates, particularly among women and minority groups We have developed an automated multiscale model abstraction methodology that helps address these problems by systematically generating intermediate-level representations that rigorously balance computational efficiency and modeling accuracy. Here, we use such an approach to examine how different temperature settings quantitatively affect UPEC transitions between fimbriate and afimbriate phases, to gain new understanding of the underlying modular circuit switch control logic, and to suggest further insights into ways this knowledge could potentially be used in therapeutic applications. Escherichia coli (UPEC)—the main causative agent that accounts for 80–90 percent of all community-acquired UTIs in the United States in vivo during UTIs E. coli strains as well E. coli to establish quiescent pathogen reservoirs shielded from native host defenses and antibiotic treatments as well as serve to seed subsequent UTIs in a type 1 fimbriae-dependent manner Type 1 fimbriae (pili) represent the foremost virulence factor in lower urinary tract infections (UTIs) by uropathogenic E. coli is randomly phase variable, whereby individual cells stochastically switch between fimbriate (ON) and afimbriate (OFF) states with rates regulated by various internal as well as environmental conditions E. coli type 1 fimbriae, this phase variation is controlled by the fim circuit switch that functions based on the inversion of a 314bp chromosomal region, fimS, bounded by two 9bp inverted repeats left and right (IRL and IRR) fimS element contains the promoter for fimA and other genes encoding structural subunits of type 1 fimbriae. As a result, an individual E. coli cell expresses type 1 fimbriae when the fim switch is in the ON position and rapidly becomes afimbriate when the switch flips into the OFF position fimS requires either fim switch is strongly controlled by temperature in a complex manner E. coli to rapidly undergo afimbriation under appropriate conditions As illustrated in This work investigates the logic and behavior of the gene regulatory circuit, which controls the ON/OFF switching of type 1 fimbriae expression, by starting with the reaction-level description of its underlying biochemical and biophysical molecular interaction mechanisms. We are particularly interested in the role of environmental cues in this process and, specifically, of temperature as it is known to control many gene regulatory circuits in bacteria—often those responsible for virulence functions in silico analysis can greatly benefit from applications of appropriate intermediate-level system model abstractions—whereby multiple individual biological interactions are aggregated into significantly few(er), but quantitatively analogous functional processes. An optimized model abstraction scheme then looks to accurately capture the target characteristics of biological system behavior, while trading off some tightly controlled degree of precision for significant computational gains. Additionally, the resulting abstracted model of the system may also be useful in helping to uncover any general high-level logical patterns embedded within the biological networks involved, which can otherwise be obscured by the low-level molecular interaction mechanics.Although these computational limitations may often render any direct implementations of the all-inclusive low-level quantitative models impractical, the use of entirely high-level qualitative representations frequently becomes inadequate as well, owing to the substantial multiscale dynamical and functional complexity that biological systems can manifest. In such cases, Our method initiates the abstraction procedure with a detailed reaction-level representation of biological processes in question. This enables it to utilize basic biochemical and biophysical principles to rigorously implement many of the existing as well as potentially allow for the development and incorporation of novel abstraction techniques, However, such an approach to model complexity reduction could also lead to a further problem: while most abstractions used in the analysis of biomolecular networks have traditionally been implemented manually and on the mechanism-by-mechanism basis, doing so accurately in a general biological systems setting becomes tedious and time-consuming. The resulting model translation and transformation errors also tend to increase when progressively more intricate organism-scale physiological processes—from cell differentiation and tissue development to cancer, infection, host-pathogen interaction dynamics, etc.—are considered.automating the abstraction process via a set of algorithms developed for and implemented in the reb2sac computational tool E. coli phage The strategy used here is able to substantially overcome these issues by E. coli, fim mode through the corresponding ON-to-OFF inversion of fimS. Notably, while the behavior of most molecular processes depends on temperature, in this system global regulatory proteins fim circuit is subjected to in the physiological range—including those of a host. For instance, fimB / fimE promoters and represses their expression lrpfim switching via its ability to introduce sharp bends into the target DNA fimS DNA element and associated regulatory sites are what largely serves to kinetically effect the ON-to-OFF fim switch circuit dynamics. As the latter physiologically initiates the transition of an individual bacterium from the virulent fimbriate to the largely benign afimbriate phase and given the wide-spread emergence of antibiotic-resistant UPEC, a better understanding of such processes could benefit the development of novel clinical UPEC UTI therapies by, among other things, providing deeper insights into mechanisms potentially able to medically abrogate UPEC virulence by exploiting its internal molecular circuitry responsible for regulating the state of fimS in order to inhibit type 1 fimbriae expression.Here, we use such computational analysis aided by automated model abstraction to examine the behavior of the basic genetic regulatory network responsible for the ON/OFF switching of type 1 fimbriae expression in uropathogenic fim switch. As discussed earlier, we then abstract this detailed representation by utilizing reb2sac, which enables us to successfully circumvent the otherwise significant computational issues involved. The accuracy of our abstracted model analysis with respect to the target system property—i.e., the temperature dependence of the fim switch turn-off rate—is further validated by comparing its results with those computed via the unabstracted detailed model as well as with those derived from empirical observations (where available). This, in turn, serves to explicitly demonstrate how automated model abstractions can be used to help substantially improve the speed and efficiency of biological molecular systems analysis, while also maintaining precision and improving interpretability of results. For instance, the abstracted representation has allowed us to better understand the general circuit-level organization of the regulatory logic behind the UPEC fimbriation switch and to identify the two key subnetworks—fim switch configuration—involved in its engineering design. Our conclusions also confirm that temperature has a major and non-trivial role in determining ON/OFF switching of fimbriae expression as well as suggest new insights into the role of Towards this end, the paper begins by considering a detailed reaction-level discrete and stochastic description of the biological molecular network controlling the E. coli fimbriation switch system, which has resulted in a detailed model of the fim circuit that comprises 52 reactions and 31 species  can be effectively and efficiently estimated by using the described model abstraction methods. Here, fim switch model at – respectively – 7 and 15 additional temperature points, where experimental data are not available  molecular dynamics in gene regulatory, signal transduction, and other biological systems In recent years, rapid advances of experimental biology made it practical to study both molecular- and network-scale organization of many biological and physiological processes in much greater detail than was previously feasible. This, in turn, has made computational analysis not only possible, but also essential to any efforts aimed at understanding the increasingly intricate structures and functions of multiscale biological systems that are being uncovered through empirical means. However, this growing wealth of knowledge about fim switch circuit). The latter helps contribute additional insights into the underlying system organization and physiology as well as make their often intricate logic easier to understand.Going forward, these considerations appear to suggest that “model abstractions”—whereby, for instance, multiple biological network interactions comprising individual biomolecular mechanisms are rigorously and systematically aggregated into a few easily tractable, but functionally analogous components—will continue to become an increasingly useful tool in the general context of computational and systems biology. Importantly, model abstractions can serve not only to substantially reduce the computational requirements associated with the analysis of specific multiscale biological processes, but may also lead to identification of functional units that correspond to biologically meaningful modules or motifs  has been able to suggest a possible explanation for this ostensible contradiction by identifying a potentially key regulatory role of E. coli ON-to-OFF switching rates in the host environment. This process can help to further prolong or abridge the persistence of the fimbriate phase in individual bacteria, which may be crucial for UPEC survival when colonizing bladder and invading urothelium, while trying to escape immune system responses and effects of antibiotic treatments, fim switch inversion control, which could be of importance in a highly variable and often rapidly fluctuating environment of the urinary tract.As a result, we have been able to gain a number of insights into the internal dynamics of this clinically relevant system, including into the strong temperature dependence of putative UPEC afimbriation switching rates e.g., , which cin situ could be affected by the various aspects of complex host-pathogen interactions noted previously, it may be worth considering that to date much of the discussion has been framed in the context of such immune response processes as cytokine production, resulting inflammation, and potential subsequent exfoliation of infected bladder epithelial cells that generally lead to the increase in local tissue temperature E. coli's ability to control this process through variations in fim switch shutdown as discussed here, could offer promising potential for contributing further understanding towards the development of novel remedial approaches.While the extent to which these innate mechanisms are able to curtail or enhance virulence of UPEC ephedrine and artemisinin—with more on the way increase in temperature, our analysis indicates that the adaptive feedback strategy evolved by UPEC tends to bring about PPL conditions, whereby ON-to-OFF type 1 fimbriation circuit switch may become maximally sensitized to in vivo. On the other hand, our conclusions also support the notion that decreasing the temperature of UPEC environment may increase shutdown rates of type 1 fimbriation circuit switch  that leads to its own mode of thermoregulation in sutu, causing them to either become innately less virulent—for example, as has been discussed here in the context of UPEC fimbriation circuit switching—or making them more susceptible to the immune response as well as antibiotic and other treatments, thus potentially contributing to the ongoing enhancement of existing and the development of novel therapeutic applications.It is important to note, however, that this merely suggests the possibility and does not engender any further assessment of potential efficacy such therapies may have in clinical UTI settings. The latter requires a more extensive follow on investigation—particularly in view of additional host-pathogen interaction dynamics, the multicellular nature of the system and commensurably greater complexity of intra-/inter-cellular networks it comprises, the epidemiology of autoinfection processes involved in promoting UTIs from and diversity of the endogenous bacterial flora, etc. as well as any associated difficulties in developing detailed models of the intra-host pathogen environment. Such challenges are often due to our understanding of biomolecular functions involved being insufficiently detailed and/or tissue-specific processes adding further layers of complexity to the overall infection dynamics. For instance, while this work has been able to use modeling and computational analysis in order to explore certain aspects of type 1 fimbriae switch control, the latter are primarily relevant to lower urinary tract infections. In contrast, upper UTIs are predominantly promulgated by P fimbriae—a distinct UPEC adhesive factor, which is regulated by significantly different biomolecular circuitry  events that are randomly promoted by cis-regulatory genomic elements, which are present in low integer counts. Under these conditions, biomolecular systems can manifest emergent and unintuitive behaviors that may greatly deviate from the predictions of macroscopic continuous and deterministic classical chemical kinetics  fim switch circuit requires the use of a mesoscopic discrete and stochastic process description based on the chemical master equation (CME) Previous works by Wolf & Arkin, Blomfeld et al., and others have helped elucidate and ascertain the importance of discrete and stochastic mechanisms in the i) the system is in the same state at time ii) the probability that it is in a different state at time propensity function and it is further assumed that This approach considers the behavior of biomolecular systems at the individual reaction level by exactly tracking the time-evolution of the discrete number probability distribution for all molecular species present in the system and by correspondingly treating each reaction as a separate random event. An intuitive basis for the (forward) CME can be described as follows: given Taking the limit Unfortunately, solving the CME exactly for most biologically, physiologically, or clinically meaningful systems is typically not feasible either analytically or numerically due to the intrinsic complexity of its differential-difference form. To address this problem, a number of alternative methods—focusing on approximate analytical solutions, general computational techniques, and a range of specific applications—have been developed i) the waiting time to the next reaction, ii) the index of the next reaction, Our numerical simulations approach is based on the SSA and, specifically, is implemented as a streamlined version of Gillespie's Direct Method reb2sac, each simulation starts with the switch in the ON position and is run for up to one cell generation of 20 minutes as in Using this implementation of the SSA in fim circuit  the production-degradation processes of ii) the processes regulating the configuration of the fim switch itself.The reaction network graph examination carried out as part of the motif recognition, data flow, system organization, and abstraction analysis has led us to identify two major modules responsible for dynamically controlling the fimS inversion process. What is less immediately obvious, however, is the key role they play in receiving environmental signals, including temperature, and feeding this information into the fim configuration subnetwork for integration into the switch inversion decision. The temperature regulation facet of this process is effected by the substantial thermal sensitivity of the fimE and fimB promoter repression. Notably, such temperature control is relatively stronger across much of the physiological regime relevant to the fim switch circuit operation than the effect of As discussed earlier, FimB and FimE site-specific recombinases are essential to fimbriation circuit switching as enablers of the also see .reb2sac—a (bipartite) graph representation of biochemical networks may be more desirable i) the efficient traversal of the reaction network, which is crucial for pattern matching and subsequent model abstraction; and (ii) an optimized implementation of the stochastic simulation algorithm without the need for constructing additional data structures— such as dependency graphs—which minimizes the number of updates.The reaction-based description of FimB and FimE regulation subnetwork used here is given in fim switch regulatory proteins, leading to ON-to-OFF phase inversions and thus involving the fimS invertible DNA element itself. This subnetwork is derived from the 18 configurations that the switch DNA region can be in based on the occupation of various binding sites by regulatory proteins. The reaction-level description of this module is given in The second major subnetwork centers around binding and unbinding reactions of fimS DNA and the fimE / fimB described earlier, another main mode of temperature control in the E. coli fimbriation switch circuit is through its effect on the abundance of the lrp expression is up-regulated as the former increases owing to the reduction in Besides the regulatory factor binding/unbinding to/from detailed reaction-level representations of biomolecular networks allow for very comprehensive descriptions of biological mechanisms, such low-level models can lead to substantial computational costs as well as may, potentially, obscure the overall system structure and dynamics. The problem could be further exacerbated by the particular choices of initial and environmental conditions that biological systems are embedded in. For example, while this paper discussed the behavior of the fim circuit in E. coli growing on minimal liquid medium, the in situ observed switching characteristics may be altered on rich liquid or solid medium While SSA offers a powerful method for numerically analyzing the behavior of discrete-stochastic biomolecular interaction networks, relying on just one or several simulation runs in order to gain a general understanding of a biological system subject to stochastic decision-making, such as UPEC fimbriation ON/OFF switching, could often be misleading because—similarly to the use of CCK—randomly-simulated individual sample trajectories of the underlying stochastic process are frequently insufficient to characterize its overall probabilistic dynamics abstracted representations—which could be efficiently accomplished through systematic and, given the complexity of most biological processes, automatic application of various model approximations and simplifications—becomes essential One approach toward addressing such challenges is the ongoing development of advanced analytical and numerical approximation methods—whether with respect to time  pre-processing; (ii) main abstraction loop; and (iii) post-processing. Pre-processing is used to modify the structure of the input model so that the appropriate abstraction methods in the main loop can be applied more effectively. For example, if a model initially contains irrelevant reactions with respect to a particular species or dynamical property that the user is interested in analyzing—these reactions are removed at the pre-processing step to help speed up the abstraction process. The main loop contains abstraction methods that are applied repeatedly until the structure of the model no longer changes. In the case of gene regulatory networks, abstraction methods such as operator site reduction are typically placed in the main loop. Post-processing is used to transform the model into a form suitable for subsequent application of follow-up analysis methods—e.g., stochastic simulation, Markov chain analysis, etc.Specifically, as shown in reb2sac automated abstraction tool to simplify the detailed model by systematically going through the fim switch network and applying various qualifying simplifications and/or approximations as appropriate. The resulting abstracted model is indeed significantly simpler computationally and more understandable logically than the detailed one. For example, the production-degradation reaction scheme of fimB and fimE promoter sites as “rapid” and the corresponding number of the operator sites (one) as “low”—and by then applying the rapid-equilibrium and quasi-steady-state approximations to these processes. The tool then continues to examine the dynamics of other species and finds that the concentrations of As discussed earlier, transforming a detailed biological system model into an abstracted one can substantially increase the efficiency of its computational analysis as well as potentially improve our understanding of its overall structure and function. In this work, we have used the of  and   is reducfim switching probability, these abstractions reduce the detailed subnetwork of fim element configuration subnetwork. For instance, the reaction process corresponding to the fim switch inversion through state 6 Click here for additional data file.