'Rapid transit' constipation in children: a possible genesis for irritable bowel syndrome.

Children with chronic idiopathic constipation (CIC) often end up at the surgeon when medical treatments have failed. This opinion piece discusses a recently described pattern of CIC called 'Rapid transit constipation (RTC)' first identified in 2011 as part of surgical workup. RTC was identified using a nuclear medicine gastrointestinal transit study (NMGIT or nuclear transit study) to determine the site of slowing within the bowel and to inform surgical treatment. Unexpectedly, we found that RTC occured in 29% of 1000 transit studies in a retrospective audit. Irritable bowel syndrome (IBS) occurs in 7-21% of the population, with a higher prevalence in young children and with constipation type dominating in the young. While 60% improve with time, 40% continue with symptoms. First-line therapy for IBS in adults is a diet low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols which reduces symptoms in > 70% of patients. In children with functional gastrointestinal disorders, fructose intolerance occurs in 35-55%. Reducing fructose produced significant improvement in 77-82% of intolerant patients. In children with RTC and a positive breath test upon fructose challenge, we found that exclusion of fructose significantly improved constipation, abdominal pain, stool consistency and decreased laxative use. We hypothesise that positive breath tests and improvement of pain and bowel frequency with sugar exclusion diets in RTC suggest these children have IBS-C. These observations raise the possibility that many children with CIC could be treated by reducing fructose early in their diet and this might prevent the development of IBS in later life.

The effect of food withdrawal in children with rapid-transit constipation.

BACKGROUND: Rapid proximal colonic transit with anorectal holdup is a subtype of chronic constipation linked to food intolerance. We aimed to determine the effectiveness of dietary exclusion as a treatment for constipated children with rapid-transit constipation by scintigraphy. METHODS: Questionnaires on diet and symptoms were mailed out to 125 children with chronic constipation and rapid proximal colonic transit on nuclear transit study at our institute between 1998 and 2014 years. Patients were given instructions and encouraged to undertake a six-food elimination diet targeting common protein allergens (dairy, wheat, soy, eggs, nuts, seafood). Answers were completed by circling an option or on visual analogue scale. Results were evaluated statistically using GraphPad Prism 6 by a Wilcoxon matched-pairs rank test. P < 0.05 was considered significant. RESULTS: We received 44/125 responses, 26 patients [mean age 11 years (5-21)] had attempted elimination diet and 18 had not. Dairy and wheat were the most common foods eliminated and symptomatic improvement was greater for patients who had completely eliminated foods. Constipation, abdominal pain and pain on defecation were reduced (p < 0.01). Laxative usage decreased, although this was not statistically significant. Families encountered problems with dietary exclusion, particularly expense. Assistance from a dietician or nutritionist was sought by >50 % of families. CONCLUSION: Dietary exclusion is a promising strategy to treat constipation in children with rapid proximal colonic transit. However, it was hard for many families, demonstrating the need for identifying the cause more specifically and a better set of instructions for the family and/or dietitian to follow.

Precise characterization of 6Li Feshbach resonances using trap-sideband-resolved RF spectroscopy of weakly bound molecules.

We perform radio-frequency dissociation spectroscopy of weakly bound 6Li2 Feshbach molecules using low-density samples of about 30 molecules in an optical dipole trap. Combined with a high magnetic field stability, this allows us to resolve the discrete trap levels in the radio-frequency dissociation spectra. This novel technique allows the binding energy of Feshbach molecules to be determined with unprecedented precision. We use these measurements as an input for a fit to the 6Li scattering potential using coupled-channel calculations. From this new potential, we determine the pole positions of the broad 6Li Feshbach resonances with an accuracy better than 7×10(-4) of the resonance widths. This eliminates the dominant uncertainty for current precision measurements of the equation of state of strongly interacting Fermi gases. As an important consequence, our results imply a corrected value for the Bertsch parameter ξ measured by Ku et al. [Science 335, 563 (2012)], which is ξ=0.370(5)(8).

Large effects of electric fields on atom-molecule collisions at millikelvin temperatures.

Controlling interactions between cold molecules using external fields can elucidate the role of quantum mechanics in molecular collisions. We create a new experimental platform in which ultracold rubidium atoms and cold ammonia molecules are separately trapped by magnetic and electric fields and then combined to study collisions. We observe inelastic processes that are faster than expected from earlier field-free calculations. We use quantum scattering calculations to show that electric fields can have a major effect on collision outcomes, even in the absence of dipole-dipole interactions.

Universality of the three-body parameter for Efimov states in ultracold cesium.

We report on the observation of triatomic Efimov resonances in an ultracold gas of cesium atoms. Exploiting the wide tunability of interactions resulting from three broad Feshbach resonances in the same spin channel, we measure magnetic-field dependent three-body recombination loss. The positions of the loss resonances yield corresponding values for the three-body parameter, which in universal few-body physics is required to describe three-body phenomena and, in particular, to fix the spectrum of Efimov states. Our observations show a robust universal behavior with a three-body parameter that stays essentially constant.

The Kelly technique of bladder exstrophy repair: continence, cosmesis and pelvic organ prolapse outcomes.

PURPOSE: The Kelly technique of radical soft tissue mobilization, an alternative to osteotomy and modern staged repair, has been used extensively at our tertiary referral center for bladder exstrophy in the last 2 decades. We present what is to our knowledge the first long-term followup of the Kelly technique in 31 patients treated at our institution. MATERIALS AND METHODS: Patients admitted for bladder exstrophy at our institution since 1980 were identified and the medical charts were reviewed. Continence questionnaires were completed during followup appointments or by mail. Continence was defined as complete-dry greater than 3 hours during the day and night with 2 or fewer night wets per month and partial-dry 2 hours or more during the day and 3 or greater night wets per month, and/or stress incontinence. The degree of pelvic organ prolapse was assessed in females older than 12 years. RESULTS: Data were available on 31 Kelly patients, including 14 females, 4 to 25 years old and 13 patients, including 4 females, 2 to 29 years old treated with another staged technique. Of 30 Kelly patients without urinary diversion 21 (70%) were completely or partially continent. Of the 30 patients 17 voided spontaneously without clean intermittent catheterization or augmentation, of whom 12 (71%) were continent. Lower abdominal appearance was graded as abnormal in 11 of 12 male Kelly patients vs in 2 of 7 nonKelly males with pubic approximation (p = 0.01). Of the 12 females assessed none of 9 Kelly patients had prolapse, whereas 2 of 3 nonKelly patients had prolapse (p <0.05). CONCLUSIONS: The continence rate after the Kelly operation compares favorably with that in recent series. The abnormal appearance of the lower abdomen and bony pelvis in Kelly males may result from a lack of pubic approximation. Importantly pelvic organ prolapse may be decreased in women after the Kelly technique.

Phosphorylation events during Müllerian duct regression.

Regression of the fetal rat Müllerian duct in vitro was stimulated by sodium fluoride in the absence of Müllerian inhibiting substance. The action of Müllerian inhibiting substance was inhibited by sodium vanadate, adenosine 5'-triphosphate, and several related nucleotides in the presence of manganese ions. Epidermal growth factor specifically inhibited the substance, but only with manganese ions present. Insulin, platelet-derived growth factor, and nerve growth factor had no effect. These results suggest that dephosphorylation of membrane proteins mediates the action of Müllerian inhibiting substance.

Long-term urinary symptoms in adolescent and adult women with congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive condition resulting in excess androgen production. Females are typically born with ambiguous genitalia and often undergo feminising genitoplasty in infancy or childhood. Recently, there has been considerable international debate as to whether distressing urinary symptoms in CAH patients are truly present and, if so, whether these urinary problems are a consequence of the feminising genitoplasty. OBJECTIVE: To identify and assess any urinary symptoms in an Australian cohort of adolescent and adult women with CAH who have undergone feminising genitoplasty in infancy, childhood or adolescence as a part of their management. STUDY DESIGN: Females with CAH aged 12-40 years, who had undergone feminising genitoplasty, and were identified from a hospital database (n = 72). Those aged 12-15 years were assessed using the Paediatric Incontinence Symptom Index questionnaire in conjunction with sections of the Bristol Female Lower Urinary Tract Symptoms Scored Form questionnaire. Those aged 16-40 years were assessed using the Bristol Female Lower Urinary Tract Symptoms Scored Form questionnaire. Uroflowmetry studies and post-void residual volume ultrasounds were also conducted. Previously published normative data were used for the control population. RESULTS: Responses to the questionnaire indicated that CAH patients had a higher incidence of urgency, frequency, urge incontinence, unexplained incontinence and nocturnal incontinence, when compared to previously published control data. Average and maximum urine flow rates measured by uroflowmetry were within normal range; however, the 16-40-year-old age group had significantly increased mean post-void residual volumes (P < 0.001) (Summary table). DISCUSSION: The presence of lower urinary tract symptoms in these patients has previously been interpreted as a direct outcome of feminising genitoplasty; however, these results could also be accounted for by the virilisation of pelvic floor musculature. Androgens have been shown to increase skeletal muscle mass, but their exact impact on the pelvic floor musculature requires further research. Three previous studies have measured post-void residual volumes in patients with CAH, all of which found it them be raised. CONCLUSIONS: Patients with CAH appeared to have overall normal urinary flow but increased post-void residual volumes. The data suggested that this population of patients has an increased probability of incontinence, urgency, and frequency when compared to a control population. These results confirmed findings of other small studies; however, it remains unclear if these changes reflected the underlying diagnosis or were a consequence of management.

Immunohistochemical localisation of cholinergic muscarinic receptor subtype 1 (M1r) in the guinea pig and human enteric nervous system.

Little is known regarding the location of cholinergic muscarinic receptor 1 (M1r) in the ENS, even though physiological data suggest that M1rs are central to cholinergic neurotransmission. This study localised M1rs in the ENS of the guinea pig ileum and human colon using fluorescence immunohistochemistry and RT-PCR in human colon. Double labelling using antibodies against neurochemical markers was used to identify neuron subytpes bearing M1r. M1r immunoreactivity (IR) was present on neurons in the myenteric and submucosal ganglia. The two antibodies gave similar M1r-IR patterns and M1r-IR was abolished upon antibody preabsorption. M1r-IR was present on cholinergic and nNOS-IR nerve cell bodies in both guinea pig and human myenteric neurons. Presynaptic M1r-IR was present on NOS-IR and VAChT-IR nerve fibres in the circular muscle in the human colon. In the submucosal ganglia, M1r-IR was present on a population of neurons that contained cChAT-IR, but did not contain NPY-IR or calretinin-IR. M1r-IR was present on endothelial cells of blood vessels in the submucosal plexus. The localisation of M1r-IR in the guinea pig and human ENS shown in this study agrees with physiological studies. M1r-IR in cholinergic and nitrergic neurons and nerve fibres indicate that M1rs have a role in both cholinergic and nitrergic transmission. M1r-IR present in submucosal neurons suggests a role in mediating acetylcholine's effect on submucosal sensory and secretomotor/vasodilator neurons. M1r-IR present on blood vessel endothelial cells suggests that M1rs may also mediate acetylcholine's direct effect on vasoactivation.

Characterization of the rat INSL3 receptor.

Human LGR8, initially discovered as a low-affinity relaxin receptor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full-length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interestingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrated in the gubernaculum at the time of testis descent and in the testis associated with germ cells.

Immunohistochemical localization of substance P NK1 receptor in guinea pig distal colon.

BACKGROUND: Neurokinin receptors facilitate tachykinin mediated intestinal motility and secretion. Distribution of Substance P (SP) neurokinin 1 receptor (NK1r) immunoreactivity (IR) has been previously characterized in guinea pig ileum, but not colon. This study localizes NK1rs in guinea pig distal colon. METHODS: Neurons were double labelled for NK1r and either acetylcholine transferase (ChAT), calbindin (calb), neuropeptide Y (NPY), nitric oxide synthase (NOS) or SP. The NK1r endocytosis was induced by 10(-5) mol L(-1) SP, septide, [SarMet] SP or neurokinin A. RESULTS: In guinea pig distal colon, NK1r-IR was present on 70% of submucosal neurons. Sixty-threepercent of the NK1r-IR submucosal neurons were ChAT-IR, 16% calb/SP-IR, 19% NPY-IR and 14% NOS-IR neurons. The NK1r-IR was present on 5% of myenteric neurons. Of these 63% were ChAT-IR, 16% calb-IR neurons and 25% NOS-IR. The NK1rs were also on myenteric plexus interstitial cells of Cajal and on circular muscle. CONCLUSION: In guinea pig distal colon, NK1rs were on 70% of submucosal neurons including all three secretomotor neuron subtypes and sensory neurons, suggesting NK1rs have a major role in neuronal control of mucosal reflexes. The NK1rs were on few myenteric neurons but were dense on muscle cells, suggesting NK1rs affect motility through neuro-muscular rather than neuro-neuronal transmission.

Is Mullerian-inhibiting substance a circulating hormone in the chick-quail chimera?

Mullerian-inhibiting substance (MIS) is a glycoprotein from the fetal testis which causes regression of the embryonic Mullerian duct. It was thought to be a locally acting agent, because in the true hermaphrodite, a Fallopian tube remains on the side contralateral to that bearing a testis, but is absent on the side adjacent to the testis. To test whether Mullerian duct regression could occur at a distant site, the chick-quail chimera was used. Chick embryos were maintained in shell-less culture from 3-14 days of incubation. At 7-9 days of incubation, a chick/quail chimera was created by grafting a quail Mullerian duct into the eye of the chick. Three or four days later, the eye was enucleated and histologically examined using the Feulgen reaction or a modification of this technique. Under these conditions, the quail cell nuclei could be readily identified, allowing absolute identification of the cells around the graft. Twenty-three female chick hosts received grafts; of the 17 grafts recovered, 16 were developing normally. In 16 male chicks receiving grafts, 10 Mullerian ducts were recovered, with 9 of these showing clear signs of regression, such as basement membrane dissolution, condensation of mesenchyme, diminution of epithelial tube size, and thinning of mesenchymal cuff. These results suggest that MIS reached the quail duct in the eye and was functionally active. This model suggests that MIS may be a true endocrine testicular secretion.

Pretreatment of chick embryos with estrogen in ovo prevents mullerian duct regression in organ culture.

Diethylstilbestrol (DES) feminizes chick embryos in ovo, preventing regression of the Mullerian ducts (MDs). The feminized testis, however, despite its ovarian-like appearance, continues to produce Mullerian inhibiting substance (MIS). This study was designed to test whether exogenous DES preserved the MD against endogenous MIS by affecting the target organ (feminization). DES (0.1 mg) was injected into the air sacs of 5-day-old chick embryos. Testes were obtained from 15-day-old chick embryos and MDs from 8-day-old chick embryos and combined in organ culture for 72 h. MD regression was examined macroscopically and microscopically after 3 days in vitro in medium without DES (group A: n = 155) and with 10(-6) M DES (group B: n = 169). Four types of coculture were set up as follows: 1) Control testis with control MD; 2) DES-treated testis with DES-treated MD; 3) Control testis with DES-treated MD; 4) DES-treated testis with control MD. In medium without DES (group A), MD regression was inhibited significantly when the duct was pretreated with DES [2) and 3) vs. 1) and 4)]. The pretreated testis produced slightly less regression [4) vs. 1)], but this was not significant. Previous studies in ovo had suggested that estrogen prevented MD regression directly as well as causing feminization of the testis, because MIS secretion was not inhibited from the feminized gonad. These results show that pretreatment of the MD with DES blocks regression in vitro by a normal testis, confirming that the primary site of action of estrogen is on the duct itself.

Testicular descent. III. The neonatal mouse gubernaculum shows rhythmic contraction in organ culture in response to calcitonin gene-related peptide.

The effects of calcitonin gene-related peptide (CGRP) and CGRP 8-37 on the neonatal mouse gubernaculum were examined in organ culture, with the aim of seeing whether CGRP has a direct effect on the gubernaculum. A total of 440 gubernacula were studied. Two hundred and fifty gubernacula were treated with CGRP in concentrations ranging from 0-714 nM/liter. With increasing doses of CGRP the percentage of gubernacula showing vigorous contraction increased from 18-50%. The total percentage of gubernacula showing any form of contraction increased from 76-96%. One hundred and fifty gubernacula were exposed to the CGRP analog CGRP 8-37. Increasing concentrations of CGRP 8-37 from 179-714 nM/liter decreased the rate of vigorous contraction from 18-4%. The percentage of gubernacula showing any degree of contraction decreased from 76-14%. Forty gubernacula removed from testicular feminization (TFM) mice were exposed to varying concentrations of CGRP. In the absence of exogenous CGRP no contractility was observed. By contrast, in the presence of CGRP the gubernacula showed vigorous contractility increasing from 38-90%. The total number of gubernacula showing contraction increased from 75-100%. These studies demonstrated that the neonatal mouse gubernaculum exhibits a high level of endogenous contractility, which can be enhanced in a dose responsive manner with exogenous CGRP. CGRP 8-37 caused a dose responsive inhibition. The androgen-insensitive gubernaculum from the TFM mouse showed no endogenous contraction, but on exposure to CGRP showed an enhanced rate of contractility. These results are consistent with the hypothesis that androgens may control gubernacular migration indirectly via release of CGRP from the genitofemoral nerve in the inguinoscrotal region. The failure of gubernacular motility in vitro and migration in vivo in the TFM mouse may indicate lack of CGRP release from the genitofemoral nerve.

Testicular descent. II. Ontogeny and response to denervation of calcitonin gene-related peptide receptors in neonatal rat gubernaculum.

The gubernaculum guides inguino-scrotal testicular descent by migrating into the scrotum ahead of the testis. Calcitonin gene-related peptide (CGRP) is present in the genitofemoral nerve of the neonatal rat and stimulates gubernacular motility in vitro. In a previous study in vitro autoradiography demonstrated a distinctive distribution of binding sites for CGRP over the developing cremasteric muscle in the gubernaculum. Binding analysis using computerized densitometry revealed a single class of sites. This study aimed to characterize the ontogeny of CGRP receptors in the gubernaculum and their response to denervation. Gubernacular sections from neonatal male rats were incubated with [125I]human CGRP as well as a variety of unlabeled neuropeptides. The expression of CGRP receptors culminates during the first week after birth, when gubernacular migration actually occurs. Significantly higher binding capacities were found in the denervated gubernacula compared with those in controls, which suggests an upregulation of CGRP receptors as a result of the genitofemoral nerve denervation. These results are consistent with the hypothesis that CGRP released from the nerve acts directly on the developing cremaster via its own receptors, which have not been described previously in this tissue.

Specific estradiol binding in embryonic Mullerian ducts: a potential modulator of regression in the male and female chick.

The total content of putative estradiol-specific cytosolic type I and nuclear type I and II estradiol-specific binders was measured in 8- and 9-day-old male and female chick embryo Mullerian ducts. Cytosolic and nuclear type I estradiol-specific binding levels were similar in males and females, and no significant differences were noted among right vs. left and 8-day-old vs. 9-day-old embryo Mullerian ducts. The levels of nuclear type I estradiol binder were consistently higher than the cytosolic type I binder, but this difference was not significant. Nuclear type II estradiol-specific binding, however, was significantly higher in the left Mullerian ducts of both male and female embryos. The significance of these findings in relation to the regression of Mullerian ducts in male and female chick embryos is discussed.

Cyclic adenosine 3',5'-monophosphate modulation of mullerian duct regression.

(Bu)2cAMP inhibits the action of testicular Mullerian inhibiting substance (MIS) in vitro, but it is unknown whether the intracellular nucleotide diminishes production of MIS by the testis or interferes with its action at the Mullerian duct. When added to the 14 1/2-day old rat embryo Mullerian duct in organ culture, (Bu)2cAMP (0.1 or 1.0 mM) inhibited regression caused by biologically active exogenous MIS fractions, as well as that produced by the fetal testis (1.0 mM). Dibutyryl cyclic guanosine 3',5'-monophosphate was ineffective against exogenous MIS fractions or the fetal testis. The phosphodiesterase inhibitor, methyl-isobutyl-xanthine, had the same inhibitory effect on the MIS-fraction (0.1 or 1.0 mM), as well as against MIS secreted from the fetal testis (1.0 mM). Theophylline at 1.0 mM inhibited the action of the MIS fraction. The presence of (Bu)2cAMP in the medium was required for at least the first 24 h of the 72-h incubation to significantly inhibit MIS action. These results indicate that intracellular cAMP inhibits the action of MIS at the Mullerian duct itself by a potentially reversible change in the cells. We speculate that cAMP may act by altering the state of differentiation in the cells, perhaps by mediating phosphorylation of intracellular (and extracellular) proteins.

Androgen stimulation of nucleotide pyrophosphatase during mullerian duct regression.

Mullerian inhibiting substance (MIS) causes regression of the embryonic Mullerian duct. In the fetal rat urogenital ridge, extracellular nucleotide pyrophosphatase (NPPase) can be detected by histochemical staining on the regressing male Mullerian duct, with no corresponding enzyme localization on developing female Mullerian ducts. In vivo results in male embryos can be confirmed in vitro by incubating 14.5-day-gestation female urogenital ridges with MIS and testosterone for 72 h before enzyme localization. Since the addition of testosterone to MIS is obligatory to detect NPPase activity in vitro, and certain steroids enhance Mullerian duct regression, additional steroids were tested in vitro alone or in combination with MIS for their abilities to stimulate NPPase. NPPase induction occurred only with the combinations of MIS and testosterone or MIS and medroxyprogesterone acetate. Neither MIS alone nor any steroid used alone stimulated NPPase activity. The effect of exogenous NPPase added alone to the developing urogenital ridge was also assessed. Incubation of the female urogenital ridge for 72 h with exogenous NPPase caused marked hyperplasia of the Mullerian duct epithelial cells and early mesenchymal cell condensation, without the basement membrane breakdown normally seen in regression. Since NPPase activity is present in the Mullerian duct only during regression, these findings suggest that MIS and fetal androgens are synergistically modulating the activity of this enzyme. Its role in the Mullerian duct, as suggested by its cytological effects, may be to stimulate cellular responses before migration during regression.

Serum levels of mullerian inhibiting substance in boys throughout puberty and in the first two years of life.

Serum levels of Mullerian inhibiting substance (MIS) have been measured in 91 boys throughout normal pubertal development. MIS levels fell sharply after pubertal stage 1 and were mostly undetectable at pubertal stage 6. The relationship between MIS concentration and pubertal stage was similar when compared with age. Seven patients with precocious puberty and 12 with delayed puberty were also investigated and found to have MIS levels consistent with their degree of pubertal development. Precocious puberty was associated with MIS levels that were abnormally low for age, while delayed puberty resulted in persistence of high MIS levels. Serum MIS levels were also measured in 29 boys less than 2 yr of age undergoing minor surgery. High levels were found throughout this time period, which is consistent with previous reports. MIS levels appear to be inversely related to levels of gonadotropins, steroids, and inhibin, which fall in the first 2 yr of life and rise throughout puberty.

Serum levels of müllerian inhibiting substance in boys from birth to 18 years, as determined by enzyme immunoassay.

An enzyme immunoassay was set up with the aim of determining the serum levels of Müllerian inhibiting substance (MIS) during childhood. A monoclonal antibody against purified bovine MIS was combined with a polyclonal antibody against recombinant human MIS to make a sandwich assay. This assay detected MIS in human serum within the following criteria. Ninety-eight boys, aged between birth and 18 yr, who had been admitted to the Royal Children's Hospital, were included. MIS levels were measured in samples taken for biochemical screening of unrelated disorders. MIS was detected in the serum up to 16 yr of age, but was low beyond 12 yr and undetectable at 18 yr. High MIS levels were found at 4-12 months, consistent with MIS having an important function at this time. Germ cells undergo an important transformation from gonocytes to spermatogonia at the same time as the MIS levels peak, suggesting a possible function for MIS.