Autoreactive antibodies control blood glucose by regulating insulin homeostasis.

Homeostasis of metabolism by hormone production is crucial for maintaining physiological integrity, as disbalance can cause severe metabolic disorders such as diabetes mellitus. Here, we show that antibody-deficient mice and immunodeficiency patients have subphysiological blood glucose concentrations. Restoring blood glucose physiology required total IgG injections and insulin-specific IgG antibodies detected in total IgG preparations and in the serum of healthy individuals. In addition to the insulin-neutralizing anti-insulin IgG, we identified two fractions of anti-insulin IgM in the serum of healthy individuals. These autoreactive IgM fractions differ in their affinity to insulin. Interestingly, the low-affinity IgM fraction (anti-insulin IgMlow) neutralizes insulin and leads to increased blood glucose, whereas the high-affinity IgM fraction (anti-insulin IgMhigh) protects insulin from neutralization by anti-insulin IgG, thereby preventing blood glucose dysregulation. To demonstrate that anti-insulin IgMhigh acts as a protector of insulin and counteracts insulin neutralization by anti-insulin IgG, we expressed the variable regions of a high-affinity anti-insulin antibody as IgG and IgM. Remarkably, the recombinant anti-insulin IgMhigh normalized insulin function and prevented IgG-mediated insulin neutralization. These results suggest that autoreactive antibodies recognizing insulin are key regulators of blood glucose and metabolism, as they control the concentration of insulin in the blood. Moreover, our data suggest that preventing autoimmune damage and maintaining physiological homeostasis requires adaptive tolerance mechanisms generating high-affinity autoreactive IgM antibodies during memory responses.

Interleukin-6: An Under-Appreciated Inducer of Thermogenic Adipocyte Differentiation.

Adipose tissue inflammation is a key factor leading to obesity-associated immune disorders, such as insulin resistance, beta cell loss in the pancreatic islets, meta-inflammation, and autoimmunity. Inhibiting adipose tissue inflammation is considered a straightforward approach to abrogate these diseases. However, recent findings show that certain pro-inflammatory cytokines are essential for the proper differentiation and functioning of adipocytes. Lipolysis is stimulated, and the thermogenic competence of adipocytes is unlocked by interleukin-6 (IL-6), a cytokine that was initially recognized as a key trigger of adipose tissue inflammation. Coherently, signal transducer and activator of transcription 3 (STAT3), which is a signal transducer for IL-6, is necessary for thermogenic adipocyte development. Given the impact of thermogenic adipocytes in increasing energy expenditure and reducing body adiposity, functions of IL-6 in the adipose tissue have gained attention recently. In this review, we show that IL-6 signaling may protect from excess fat accumulation by stimulating thermogenesis in adipocytes.

Loss of Uncoupling Protein 1 Expression in the Subcutaneous Adipose Tissue Predicts Childhood Obesity.

Stimulation of thermogenesis by inducing uncoupling protein 1 (UCP1) expression in adipocytes is thought to promote weight loss by increasing energy expenditure, and it is postulated that the human newborn has thermogenic subcutaneous fat depots. However, it remains unclear whether a relevant number of UCP1-expressing (UCP1+) adipocytes exist in the early postnatal life. Here we studied the distribution of UCP1 and the expression of thermogenic genes in the subcutaneous adipose tissues of the human fetus, infant and child. We show that the deep layer of human fetal and neonatal subcutaneous fat, particularly the abdominal wall, is rich in UCP1+ adipocytes. These adipocytes develop in the late third trimester and persist throughout childhood, expressing a panel of genes linked to mitochondrial biogenesis and thermogenesis. During the early childhood adiposity rebound-a critical phase that determines obesity risk later in life-the absence of adipose tissue UCP1 expression in children with normal body mass index (BMI) correlates with an obesity-associated gene expression signature. Finally, UCP1 expression is negatively correlated with BMI z-score and adipocyte size in infants and children. Overall, our results show that the absence of UCP1 expression in adipose tissue is an early indicator of adipose tissue expansion in children.

The environmental obesogen bisphenol A increases macrophage self-renewal.

Self-renewal of macrophages is important for the healthy development and replenishment of tissue-resident macrophage pools. How this mechanism is controlled by endocrine signals is still largely unexplored. Here, we show that the endocrine disruptor bisphenol A (BPA) increases macrophage self-renewal. This effect was associated with phosphorylation of extracellular signal-regulated kinase (ERK) and a slight increase in the expression of liver X receptor alpha (LXRα). We found that LXRα inhibition induced, while LXRα activation impeded, macrophage self-renewal. LXRα signaling hence may protect from excessive macrophage expansion. Self-renewing macrophages, however, had negligible LXRα expression when compared with quiescent macrophages. Accordingly, tissue-resident macrophage pools, which are dominated by quiescent macrophages, were rich in LXRα-expressing macrophages. Overall, we show that BPA increases macrophage self-renewal and that this effect, at least in part, can be inhibited by increasing LXRα expression. Since BPA is accumulated in the adipose tissue, it has the potential to increase self-renewal of adipose tissue macrophages, leading to a condition that might negatively impact adipose tissue health.

STAT6 mediates the effect of ethanol on neuroinflammatory response in TBI.

Traumatic brain injury (TBI) and ethanol intoxication (EI) frequently coincide, particularly in young subjects. However, the mechanisms of their interaction remain poorly understood. Among other pathogenic pathways, TBI induces glial activation and neuroinflammation in the hippocampus, resulting in acute and chronic hippocampal dysfunction. In this regard, we investigated the role of EI affecting these responses unfolding after TBI. We used a blunt, weight-drop approach to model TBI in mice. Male mice were pre-administered with ethanol or vehicle to simulate EI. The neuroinflammatory response in the hippocampus was assessed by monitoring the expression levels of >20 cytokines, the phosphorylation status of transcription factors and the phenotype of microglia and astrocytes. We used AS1517499, a brain-permeable STAT6 inhibitor, to elucidate the role of this pathway in the EI/TBI interaction. We showed that TBI causes the elevation of IL-33, IL-1ß, IL-38, TNF-α, IFN-α, IL-19 in the hippocampus at 3 h time point and concomitant EI results in the dose-dependent downregulation of IL-33, IL-1ß, IL-38, TNF-α and IL-19 (but not of IFN-α) and in the selective upregulation of IL-13 and IL-12. EI is associated with the phosphorylation of STAT6 and the transcription of STAT6-controlled genes. Moreover, ethanol-induced STAT6 phosphorylation and transcriptional activation can be recapitulated in vitro by concomitant exposure of neurons to ethanol, depolarization and inflammatory stimuli (simulating the acute trauma). Acute STAT6 inhibition prevents the effects of EI on IL-33 and TNF-α, but not on IL-13 and negates acute EI beneficial effects on TBI-associated neurological impairment. Additionally, EI is associated with reduced microglial activation and astrogliosis as well as preserved synaptic density and baseline neuronal activity 7 days after TBI and all these effects are prevented by acute administration of the STAT6 inhibitor concomitant to EI. EI concomitant to TBI exerts significant immunomodulatory effects on cytokine induction and microglial activation, largely through the activation of STAT6 pathway, ultimately with beneficial outcomes.

Transcriptional control of apoptotic cell clearance by macrophage nuclear receptors.

Apoptotic cell clearance by macrophages is key for normal tissue development and homeostasis. Nuclear receptors, such as peroxisome proliferator activated receptors (PPARs), liver X receptor (LXR), retinoic acid receptor (RAR), retinoid X receptor (RXR) and glucocorticoid receptor (GR) orchestrate this vital process. The underlying mechanism involves the transcriptional control of key genes of apoptotic cell recognition and internalization, such as Cd36, Mertk, Axl, C1qa, Tgm2, Abca1. In addition, apoptotic cell uptake leads to M2 activation of macrophages, and this process is also controlled at the gene transcription level by nuclear receptors. Apoptotic cells provide signals for nuclear receptors, which in turn accelerate the safe disposal of apoptotic debris, which eventually allows renewal of the tissues, and impedes the development of inflammation and autoimmunity against dying cells. Nuclear receptor signaling is vulnerable to endocrine disruptors, which may interfere with the ability of macrophages to phagocytose and acquire M2 activation. This review summarizes the mechanisms, which allow nuclear receptors to control apoptotic cell clearance by macrophages.

Adipose tissue macrophages in non-rodent mammals: a comparative study.

The stromal vascular fraction (SVF) of adipose tissue in rodents and primates contains mesenchymal stem cells and immune cells. SVF cells have complex metabolic, immune and endocrine functions with biomedical impact. However, in other mammals, the amount of data on SVF stem cells is negligible and whether the SVF hosts immune cells is unknown. In this study, we show that the SVF is rich in immune cells, with a dominance of adipose tissue macrophages (ATMs) in cattle (Bos primigenius taurus), domestic goat (Capra aegagrus hircus), domestic sheep (Ovis aries), domestic cat (Felis catus) and domestic dog (Canis familiaris). ATMs of these species are granulated lysosome-rich cells with lamellipodial protrusions and express the lysosome markers acid phosphatase 5 (ACP-5) and Mac-3/Lamp-2. Using ACP-5 and Mac-3/Lamp-2 as markers, we additionally detected ATMs in other species, such as the domestic horse (Equus ferus caballus), wild boar (Sus scrofa) and red fox (Vulpes vulpes). Feline and canine ATMs also express the murine macrophage marker F4/80 antigen. In the lean condition, the alternative macrophage activation marker CD206 is expressed by feline and canine ATMs and arginase-1 by feline ATMs. Obesity is associated with interleukin-6 and interferon gamma expression and with overt tyrosine nitration in both feline and canine ATMs. This resembles the obesity-induced phenotype switch of murine and human ATMs. Thus, we show, for the first time, that the presence of ATMs is a general trait of mammals. The interaction between the adipose cells and SVF immune cells might be evolutionarily conserved among mammals.

Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms.

The alternatively activated or M2 macrophages are immune cells with high phenotypic heterogeneity and are governing functions at the interface of immunity, tissue homeostasis, metabolism, and endocrine signaling. Today the M2 macrophages are identified based on the expression pattern of a set of M2 markers. These markers are transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors with diverse and often yet unexplored functions. This review discusses whether these M2 markers can be reliably used to identify M2 macrophages and define their functional subdivisions. Also, it provides an update on the novel signals of the tissue environment and the neuroendocrine system which shape the M2 activation. The possible evolutionary roots of the M2 macrophage functions are also discussed.

The invertebrate midintestinal gland ("hepatopancreas") is an evolutionary forerunner in the integration of immunity and metabolism.

The immune system has an impact on the metabolic performance in vertebrates, thus the metabolic effects of immune cells are receiving intense attention today in the biomedical field. However, the evolutionary origin of the immunity-metabolism interaction is still uncertain. In this review, I show that mollusks and crustaceans integrate immune functions to a metabolic organ, the midintestinal gland ("hepatopancreas"). In these animals, the epithelial cells of the midintestinal gland are major sources of immune molecules, such as lectins, hemocyanin, ferritin, antibacterial and antiviral proteins, proteolytic enzymes and nitric oxide. There is crosstalk between midintestinal gland cells and phagocytes, which aids the initiation of the immune response and the clearance of pathogens. The midintestinal gland is thereby an integrated organ of immunity and metabolism. It is likely that immunity was the primary function of the midintestinal gland cells and that their role in the intermediate metabolism has evolved during the course of their further specialization.

FMRF-amide is a glucose-lowering hormone in the snail Helix aspersa.

Although glucose is metabolically the most important carbohydrate in almost all living organisms, still little is known about the evolution of the hormonal control of cellular glucose uptake. In this study, we identify Phe-Met-Arg-Phe-amide (FMRFa), also known as molluscan cardioexcitatory tetrapeptide, as a glucose-lowering hormone in the snail Helix aspersa. FMRFa belongs to an evolutionarily conserved neuropeptide family and is involved in the neuron-to-muscle signal transmission in the snail digestive system. This study shows that, beyond this function, FMRFa also has glucose-lowering activity. We found neuronal transcription of genes encoding FMRFa and its receptor and moreover the hemolymph FMRFa levels were peaking at metabolically active periods of the snails. In turn, hypometabolism of the dormant periods was associated with abolished FMRFa production. In the absence of FMRFa, the midintestinal gland ("hepatopancreas") cells were deficient in their glucose uptake, contributing to the development of glucose intolerance. Exogenous FMRFa restored the absorption of hemolymph glucose by the midintestinal gland cells and improved glucose tolerance in dormant snails. We show that FMRFa was released to the hemolymph in response to glucose challenge. FMRFa-containing nerve terminals reach the interstitial sinusoids between the chondroid cells in the artery walls. We propose that, in addition to the known sites of possible FMRFa secretion, these perivascular sinusoids serve as neurohemal organs and allow FMRFa release. This study suggests that in evolution, not only the insulin-like peptides have adopted the ability to increase cellular glucose uptake and can act as hypoglycemic hormones.

Leptin receptor deficient diabetic (db/db) mice are compromised in postnatal bone regeneration.

Increased fragility fracture risk with improper healing is a frequent and severe complication of insulin resistance (IR). The mechanisms impairing bone health in IR are still not fully appreciated, which gives importance to studies on bone pathologies in animal models of diabetes. Mice deficient in leptin signaling are widely used models of IR and its comorbidities. Leptin was first recognized as a hormone, regulating appetite and energy balance; however, recent studies have expanded its role showing that leptin is a link between insulin-dependent metabolism and bone homeostasis. In the light of these findings, it is intriguing to consider the role of leptin resistance in bone regeneration. In this study, we show that obese diabetic mice lacking leptin receptor (db/db) are deficient in postnatal regenerative osteogenesis. We apply an ectopic osteogenesis and a fracture healing model, both showing that db/db mice display compromised bone acquisition and regeneration capacity. The underlying mechanisms include delayed periosteal mesenchymatic osteogenesis, premature apoptosis of the cartilage callus and impaired microvascular invasion of the healing tissue. Our study supports the use of the db/db mouse as a model of IR associated bone-healing deficits and can aid further studies of mesenchymatic cell homing and differentiation, microvascular invasion, cartilage to bone transition and callus remodeling in diabetic fracture healing.

Autoimmune kidney disease and impaired engulfment of apoptotic cells in mice with macrophage peroxisome proliferator-activated receptor gamma or retinoid X receptor alpha deficiency.

Autoimmune glomerulonephritis is a common manifestation of systemic lupus erythematosus (SLE). In this study, we show that mice lacking macrophage expression of the heterodimeric nuclear receptors PPARγ or RXRα develop glomerulonephritis and autoantibodies to nuclear Ags, resembling the nephritis seen in SLE. These mice show deficiencies in phagocytosis and clearance of apoptotic cells, and they are unable to acquire an anti-inflammatory phenotype upon feeding of apoptotic cells, which is critical for the maintenance of self-tolerance. These results demonstrate that stimulation of PPARγ and RXRα in macrophages facilitates apoptotic cell engulfment, and they provide a potential strategy to avoid autoimmunity against dying cells and to attenuate SLE.

Prevention of Chronic Morbidities in Extremely Premature Newborns with LISA-nCPAP Respiratory Therapy and Adjuvant Perinatal Strategies.

Less invasive surfactant administration techniques, together with nasal continuous airway pressure (LISA-nCPAP) ventilation, an emerging noninvasive ventilation (NIV) technique in neonatology, are gaining more significance, even in extremely premature newborns (ELBW), under 27 weeks of gestational age. In this review, studies on LISA-nCPAP are compiled with an emphasis on short- and long-term morbidities associated with prematurity. Several perinatal preventative and therapeutic investigations are also discussed in order to start integrated therapies as numerous organ-saving techniques in addition to lung-protective ventilations. Two thirds of immature newborns can start their lives on NIV, and one third of them never need mechanical ventilation. With adjuvant intervention, these ratios are expected to be increased, resulting in better outcomes. Optimized cardiopulmonary transition, especially physiologic cord clamping, could have an additively beneficial effect on patient outcomes gained from NIV. Organ development and angiogenesis are strictly linked not only in the immature lung and retina, but also possibly in the kidney, and optimized interventions using angiogenic growth factors could lead to better morbidity-free survival. Corticosteroids, caffeine, insulin, thyroid hormones, antioxidants, N-acetylcysteine, and, moreover, the immunomodulatory components of mother's milk are also discussed as adjuvant treatments, since immature newborns deserve more complex neonatal interventions.

Stimulator of Interferon Genes (STING) Triggers Adipocyte Autophagy.

Innate immune signaling in adipocytes affects systemic metabolism. Cytosolic nucleic acid sensing has been recently shown to stimulate thermogenic adipocyte differentiation and protect from obesity; however, DNA efflux from adipocyte mitochondria is a potential proinflammatory signal that causes adipose tissue dysfunction and insulin resistance. Cytosolic DNA activates the stimulator of interferon response genes (STING), a key signal transducer which triggers type I interferon (IFN-I) expression; hence, STING activation is expected to induce IFN-I response and adipocyte dysfunction. However, we show herein that mouse adipocytes had a diminished IFN-I response to STING stimulation by 2'3'-cyclic-GMP-AMP (cGAMP). We also show that cGAMP triggered autophagy in murine and human adipocytes. In turn, STING inhibition reduced autophagosome number, compromised the mitochondrial network and caused inflammation and fat accumulation in adipocytes. STING hence stimulates a process that removes damaged mitochondria, thereby protecting adipocytes from an excessive IFN-I response to mitochondrial DNA efflux. In summary, STING appears to limit inflammation in adipocytes by promoting mitophagy under non-obesogenic conditions.

Metabolic impact of adipose tissue macrophages in the early postnatal life.

Adipose tissue macrophages (ATMs) play key roles in metabolic inflammation, insulin resistance, adipose tissue fibrosis, and immune disorders associated with obesity. Research on ATM biology has mostly been conducted in the setting of adult obesity, since adipocyte hypertrophy is associated with a significant increase in ATM number. Signals that control ATM activation toward a proinflammatory or a proresolving phenotype also determine the developmental program and lipid metabolism of adipocytes after birth. ATMs are present at birth and actively participate in the synthesis of mediators, which induce lipolysis, mitobiogenesis, and mitochondrial uncoupling in adipocytes. ATMs in the newborn and the infant promote a lipolytic and fatty acid oxidizing adipocyte phenotype, which is essential to support the lipid-fueled metabolism, to maintain nonshivering thermogenesis and counteract an excessive adipose tissue expansion. Since adipose tissue metabolism in the early postnatal life determines obesity status in adulthood, early-life ATM functions may have a life-long impact.

Mitochondrial RNA stimulates beige adipocyte development in young mice.

Childhood obesity is a serious public health crisis and a critical factor that determines future obesity prevalence. Signals affecting adipocyte development in early postnatal life have a strong potential to trigger childhood obesity; however, these signals are still poorly understood. We show here that mitochondrial (mt)RNA efflux stimulates transcription of nuclear-encoded genes for mitobiogenesis and thermogenesis in adipocytes of young mice and human infants. While cytosolic mtRNA is a potential trigger of the interferon (IFN) response, young adipocytes lack such a response to cytosolic mtRNA due to the suppression of IFN regulatory factor (IRF)7 expression by vitamin D receptor signalling. Adult and obese adipocytes, however, strongly express IRF7 and mount an IFN response to cytosolic mtRNA. In turn, suppressing IRF7 expression in adult adipocytes restores mtRNA-induced mitobiogenesis and thermogenesis and eventually mitigates obesity. Retrograde mitochondrion-to-nucleus signalling by mtRNA is thus a mechanism to evoke thermogenic potential during early adipocyte development and to protect against obesity.

Histamine and H1 -histamine receptors faster venous circulation.

The study has analysed the action of histamine in the rabbit venous system and evaluated its potential role in contraction during increased venous pressure. We have found that a great variety exists in histamine sensitivity and H(1) -histamine receptor expression in various types of rabbit veins. Veins of the extremities (saphenous vein, femoral vein, axillary vein) and abdomen (common iliac vein, inferior vena cava) responded to histamine by a prominent, concentration-dependent force generation, whereas great thoracic veins (subclavian vein, superior vena cavas, intrathoracic part of inferior vena cava) and a pelvic vein (external iliac vein) exhibited slight sensitivity to exogenous histamine. The lack of reactivity to histamine was not due to increased activity of nitric oxide synthase (NOS) or heme oxygenase-1. H(1) -histamine receptor expression of veins correlated well with the histamine-induced contractions. Voltage-dependent calcium channels mediated mainly the histamine-induced force generation of saphenous vein, whereas it did not act in the inferior vena cava. In contrast, the receptor-operated channels were not involved in this response in either vein. Tyrosine phosphorylation occurred markedly in response to histamine in the saphenous vein, but not in the inferior vena cava. Histamine induced a prominent ρ kinase activation in both vessels. Protein kinase C and mitogen-activated protein kinase (MAPK) were not implicated in the histamine-induced intracellular calcium sensitization. Importantly, transient clamping of the femoral vein in animals caused a short-term constriction, which was inhibited by H(1) -histamine receptor antagonist in vivo. Furthermore, a significantly greater histamine immunopositivity was detected in veins after stretching compared to the resting state. We conclude that histamine receptor density adapts to the actual requirements of the circulation, and histamine liberated by the venous wall during increased venous pressure contributes to the contraction of vessels, providing a force for the venous return.

Lipotoxicity in macrophages: evidence from diseases associated with the metabolic syndrome.

Accumulation of lipid metabolites within non-adipose tissues can induce chronic inflammation by promoting macrophage infiltration and activation. Oxidized and glycated lipoproteins, free fatty acids, free cholesterol, triacylglycerols, diacylglycerols and ceramides have long been known to induce cellular dysfunction through their pro-inflammatory and pro-apoptotic properties. Emerging evidence suggests that macrophage activation by lipid metabolites and further modulation by lipid signaling represents a common pathogenic mechanism underlying lipotoxicity in atherosclerosis, obesity-associated insulin resistance and inflammatory diseases related to metabolic syndrome such as liver steatosis and chronic kidney disease. In this review, we discuss the latest discoveries that support the role of lipids in modulating the macrophage phenotype in different metabolic diseases. We describe the common mechanisms by which lipid derivatives, through modulation of macrophage function, promote plaque instability in the arterial wall, impair insulin responsiveness and contribute to inflammatory liver, muscle and kidney disease. We discuss the molecular mechanism of lipid activation of pro-inflammatory pathways (JNK, NFkappaB) and the key roles played by the PPAR and LXR nuclear receptors-lipid sensors that link lipid metabolism and inflammation.

Inflammation as death or life signal in diabetic fracture healing.

Increased apoptosis of chondrocytes and osteoblasts and prolonged survival of osteoclasts lead to early destruction of callus tissue and impair bone remodeling in fracture healing of diabetic patients. Diabetes is accompanied by an increased inflammatory state, reactive oxygen species (ROS) generation and accumulation of advanced glycation end products (AGEs), a heterogenous group of toxic metabolites that can induce inflammation. Prolonged hyperglycemia and insulin resistance correlate with increased apoptosis rate and, accordingly, the proapoptotic role of several inflammatory mediators, ROS and AGEs has been also documented. In this review we summarize the most recent reports supporting the idea that inflammatory signaling increases chondrocyte and osteoblast death and prolongs osteoclast survival, resulting in impaired bone regeneration in diabetes. Antagonising inflammatory signal pathways and solution of inflammation may deserve greater attention in the management of diabetic fracture healing.

Double unfurled dartos flap technique in the surgical treatment of recurrent urethrocutaneous fistulas.

BACKGROUND: The management of recurrent urethrocutaneous fistula (RUCF) is a challenging problem that poses a serious difficulty for the hypospadias surgeon. We report here a novel technique in which a double unfurled dartos subcutaneous flap is utilized in the treatment of patients with RUCF. METHODS: We retrospectively reviewed the records of all our patients who underwent surgical treatment of urethrocutaneous fistula with this new operative method after previously failed fistula repair. The main novelty of this technique is the use of two opposite medium thickness flaps, unfurled from the inner surface of the dartos fascia and spread over the fistula and each other so as to cover the urethral suture line completely, and fixed to the surrounding corporal tissue. RESULTS: Eight patients with 11 RUCFs (with localizations varying from subcoronal to penoscrotal fistula opening) underwent surgical correction with the new method. There had previously been at least 3 recurrences in 6 of these patients, and different closure techniques had been used. RUCF diameter was <4 mm in all patients. Surgery was performed at the earliest following a 6-month healing period since the last fistula repair attempt. All of the RUCFs were repaired successfully with the technique. After a follow-up of at least 6 months, none of the 8 patients had developed recurrence of the fistula, and there were no postoperative complications. CONCLUSION: The double unfurled dartos subcutaneous flap method appears to be a simple and appropriate procedure with which to repair recurrent mid-shaft and proximal urethral fistulas after failed hypospadias repair.