Enteric anendocrinosis attributable to a novel Neurogenin-3 variant

Congenital diarrhea and enteropathies (CODEs) are a group of monogenic disorders that often present with severe diarrhea in the first weeks of life. Enteric anendocrinosis (EA), an extremely rare cause of CODE, is characterized by a marked reduction of intestinal enteroendocrine cells (EC). EA is associated with recessively inherited variants in Neurogenin-3 (NEUROG3) gene. Here we investigate a case of a male infant who presented with mysterious severe malabsorptive diarrhea since birth. Thorough clinical assessments and laboratory tests were successful to exclude the majority of differential diagnosis categories. However, the patient's diagnosis was not established until the genetic test using whole-exome sequencing (WES) was performed. We identified a novel homozygous missense disease-causing variant (DCV) in NEUROG3 (c.413C>G, p.Thr138Arg). Moreover, molecular dynamic simulation analysis showed that (p.Thr138Arg) led to a global change of the NEUROG3 orientation affecting its DNA binding capacity. To the best of our knowledge, this is the first time to apply WES to reach a differential diagnosis of patients with CODEs. Our study not only expands our knowledge about NEUROG3 variants and their clinical consequences but also proves that WES is a very effective tool for the diagnosis of CODEs. This might be of value in early diagnosis of diseases and prenatal CODEs detection.     

Serotonin-induced increase in cAMP in ganglia isolated from the myenteric plexus of the guinea pig small intestine: Mediation by a novel 5-HT receptor

Serotonin (5-HT) is a mediator (through 5-HT_1P receptors) of slow EPSPs in myenteric ganglia of the small intestine. The effect of 5-HT can be mimicked by elevating cAMP; therefore, we tested the hypothesis that the slow EPSP-like response to 5-HT is cAMP-mediated. Guinea pig gut was enzymatically dissociated; myenteric ganglia remained intact and were collected by filtration. Neurons in the isolated ganglia retained their ability to manifest the slow EPSP-like response to 5-HT. Exposure to 5-HT raised the ganglionic level of cAMP (ED₅₀ 0.3 μM). This effect was not antagonized by the 5-HT_1P antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (100.0 μM), or mimicked by the 5-HT_1P agonist, 5-hydroxyindalpine (10.0 μM). Increases in cAMP were also evoked by the 5-HT₁ agonist, 5-carboxyamidotryptamine (10.0 μM), the 5-HT₂ agonist, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 1.0–10.0 μM), and by the 5-HT₄ agonists, renzapride (1.0–10.0 μM) and 5-methoxytryptamine (1.0–10.0 μM); however, neither the 5-HT₁/5-HT₂ antagonists, spiperone, methysergide, and methiothepin, nor the 5-HT₄ antagonist, tropisetron (ICS 205–930; 10.0 μM), were able to inhibit the rise in cAMP evoked by these compounds or by 5-HT (0.1–10.0 μM). The 5-HT-evoked elevation of cAMP was antagonized by ketanserin (10.0 μM), which also blocked the effects of 5-methoxytryptamine and DOI, but not those of renzapride. The effective concentration of DOI, however, was higher than that needed for activation of 5-HT₂ receptors, and Northern analysis using a cDNA probe encoding the rat 5-HT₂ receptor failed to reveal the presence of 5-HT₂ mRNA in myenteric ganglia, although it hybridizes with mRNA of the right size in the guinea pig brain. Compounds that failed to change levels of cAMP or to antagonize the action of 5-HT included 8-hydroxy-di-n-propylamino tetralin, R58639, R88226, and sumatriptan. It is concluded that the receptor responsible for the 5-HT-induced rise in cAMP in ganglia isolated from the guinea pig myenteric plexus is not a known subtype of 5-HT receptor. Since the pharmacology of this novel receptor is different from that of the slow EPSP-like response to 5-HT, the receptor probably does not mediate the slow EPSP. © 1993 Wiley-Liss, Inc.     

Mesenteric and omental cysts: An ultrasonographic and clinical study of 15 patients

The clinical and ultrasonographic (US) features of 15 cases of mesenteric or omental cyst are herein described. This series included seven male and eight female patients, whose age ranged from 2–89 years. Correct clinical diagnosis was made in two children only, but preoperative US examination accurately demonstrated the lesion in 11 of 13 patients (85%). These cystic lesions usually had a thin wall, internal septations, and fluid content with sedimentation. Enteric duplication cysts had a relatively thick wall merging with the muscle layer of bowel loop, and multiloculation was noted mainly with cystic lymphangiomas or pseudocysts. The diagnostic and surgical management of these lesions are briefly reviewed and their US appearance is illustrated.     

Adaptation of the disector method to rare small organelles in TEM sections exemplified by counting synaptic bodies in the rat pineal gland

The disector is the only objective method for quantifying particles of variable size in a given volume. With this method, cell organelles are identified on adjacent sections, but only those present in one section are counted. When counting extremely rare structures in transmission electron microscope sections (physical disector), the usual procedure of counting on electron micrographs is limited for economic reasons (e.g. micrographs highly outnumbering the investigated structures). Hence, to apply this unbiased stereological method, a modification of the physical disector concerning 3 aspects has been developed. (1) The prerequisite of screening large corresponding tissue areas (here ∼65000 μm²) was fulfilled by examining tissue areas along the edges of ultrathin sections. (2) The size of the counting frame was determined by measuring the lengths of the section margins (minus a guard area) by means of a Morphomat. This value was multiplied by the width of the investigated tissue zone, corresponding to the diameter of the electron microscope viewing screen. (3) Disector counting was carried out simultaneously on both sections (bidirectional disector) to improve efficiency. In the present study tiny synaptic bodies (SBs) were quantitated by disector in a rat pineal gland, yielding ∼30 SBs/1000 μm³. By contrast, single section profile counts of SBs amounted to 90 SBs/20000 μm². Since the presently described adaptation of the disector is time-consuming, it is proposed to determine a proportion factor allowing to estimate number of structures per volume based on single section profile counts. This would decrease the evaluation time by more than 50%.     

嘉兴外来人员生活环境与肠道传染病相关知信行现况调查分析

目的了解城郊结合部外来人员聚集地生活环境现况和霍乱等肠道传染病健康知信行情况,进一步明确肠道传染病防治健康教育与健康促进工作的重点开展方向。方法采用随机抽样的方法,运用统一设计的调查问卷对嘉兴市5县2区城郊结合部等外来人员聚集地进行流行病学调查。结果93.06%的调查对象以租房为主,90.61%的生活用水来源于自来水,9.25%用井水;78.61%的人知道霍乱传播途径,21.53%的人知道霍乱主要临床症状,28.03%的人知道肠道传染病全部预防措施;77.31%的知识来源于电视,其他顺位依次为书刊、广播、家人或朋友处;希望获得宣传资料形式:传单占46.68%,墙报占31.21%。结论外来人员租房人数居多,有部分人使用井水;肠道传染病相关知识了解的较少。应采取多部门齐抓共管来改善外来人员聚集地的生活环境,因地制宜地对外来人员开展有针对性的预防肠道传染病健康教育。     

Characterisation of pre- and post-synaptic α-adrenoceptors in modulation of the rat ileum longitudinal and circular muscle activities

Previous study has shown that α_2D-adrenoceptors are involved in modulation of peristalsis in the rat ileum. The aim of the present study was to determine the tissue location of α-adrenoceptors in the rat ileum by using a recently devised method. The pre-synaptic α-adrenoceptors were characterised by measuring the potencies of agonists to inhibit transmurally-evoked (1 ms pulses, 10 Hz, 8-10 s trains) contractions of the longitudinal and circular muscles and the affinities of antagonists. Post synaptic α-adrenoceptors were identified by screening agonists and antagonists in carbachol-contracted tissues. In the circular muscle the order of potencies for inhibiting transmurally-induced contraction was: clonidine ≥ oxymetazoline ≥ UK 14,304 ≥ guanfacine > talipexole > phenylephrine > azepexole. The potency ratios relative to clonidine correlated to those previously derived using the rat ileum peristaltic reflex preparation. Most of the α-adrenoceptor agonists, however, caused only small inhibitions of the longitudinal muscle contraction in response to transmural stimulation, except phenylephrine and azepexole. RX 821002, yohimbine, rauwolscine, BRL 44408, phentolamine, idazoxan, ARC 239, and prazosin inhibited the effect of clonidine on the circular muscle response with apparent pK_B values best correlated with pK_B or pK_i values derived from the rat ileum peristaltic reflex preparation and other tissues known to have the α_2D-subtype. The rank order of potencies at inhibiting carbachol-induced responses of both muscle layers was: phenylephrine ≥ oxymetazoline > clonidine ≥ talipexole > azepexole >> guanfacine. UK 14,304 was inactive up to 10 μM. The EC₅₀ value of each agonist on the longitudinal muscle was not significantly different to the corresponding value on the circular muscle. Prazosin was more potent than yohimbine at inhibiting the relaxant effect of phenylephrine in both muscle layers of carbachol-contracted tissues. It is concluded that the recently identified α_2D-adrenoceptors of the rat ileum are located on cholinergic neurons controlling circular muscle contraction. The study also demonstrated the presence of postsynaptic α₁-adrenoceptors involved in mediating relaxation in both muscle layers. Received: 4 November 1996 / Accepted: 10 April 1997     

The omptin family of enterobacterial surface proteases/adhesins: from housekeeping in Escherichia coli to systemic spread of Yersinia pestis

The omptins are a family of enterobacterial surface proteases/adhesins that share high sequence identity and a conserved beta-barrel fold in the outer membrane. The omptins are multifunctional, and the individual omptins exhibit differing virulence-associated functions. The Pla plasminogen activator of Yersinia pestis contributes by several mechanisms to bacterial invasiveness and the systemic, uncontrolled proteolysis in plague. Pla proteolytically activates the human proenzyme plasminogen and inactivates the antiprotease alpha2-antiplasmin, and its binding to laminin localizes the uncontrolled plasmin activity onto basement membranes. These properties enhance bacterial migration through tissue barriers. Pla also degrades circulating complement proteins and functions in bacterial invasion into human epithelial cells. PgtE of Salmonella enterica and OmpT of Escherichia coli have been shown to degrade cationic antimicrobial peptides from epithelial cells or macrophages. PgtE and SopA of Shigella flexneri appear important in the intracellular phases of salmonellosis and shigellosis, whereas functions of OmpT have mainly been associated with protein degradation in E. coli cells. The differing virulence roles and functions have been attributed to minor sequence variations at the surface-exposed regions important for substrate recognition, to the dependence of omptin functions on lipopolysaccharide, and to the different regulation of omptin expression.     

Repeated immunization with plasmid DNA formulated in poly(lactide-co-glycolide) microparticles is well tolerated and stimulates durable T cell responses to the tumor-associated antigen cytochrome P450 1B1

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation. Immunization of HLA-A2/Kb transgenic mice with human CYP1B1 encoding plasmid DNA formulated in poly(lactide-co-glycolide) (PLG) microparticles elicits CD8+ T cells that respond to human CYP1B1-positive target cells. The duration of the immune response, the effect on the immune response of multiple injections, and the safety of repeated injections were studied. These results show that the PLG-encapsulated DNA therapeutic elicits durable immune responses to CYP1B1, the responses are dependent on repeat immunization, and that the formulation is well tolerated.     

Effects of noradrenaline and somatostatin on basal and stimulated mucosal ion transport in the guinea-pig small intestine

Summary Noradrenaline (NA) and somatostatin (SOM) stimulate intestinal water and ion absorption and are found in mucosal nerve fibres and nerve terminals in submucous ganglia of the guinea-pig small intestine. As the main projection of submucous neurons is to the mucosa, NA and SOM might alter mucosal transport either by a direct effect on the epithelium or indirectly, by affecting submucous neurons. In this study these two possible sites of action of NA and SOM have been investigated in mucosa-submucosa preparations of guinea-pig ileum. In addition, the actions of NA and SOM on the secretory responses caused by stimulation of different populations of submucous neurons have been studied. The stimulants of secretion used were a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10^–5 M), 5-hydroxytryptamine (5-HT, 10^–7 M) and electrical field stimulation (EFS), which activate cholinergic, noncholinergic and mixed populations of submucous secretomotor neurons, respectively.Segments of intestine were dissected free of external muscle and myenteric plexus and mounted in Ussing chambers. Short-circuit current (I _sc) was measured as an indication of net active ion transport across the tissue. NA (10^–8 M) and SOM (>10^–10 M) each caused a decrease in I _sc, indicating a net increase in ion absorption. The NA response was abolished and the magnitude of the SOM response was reduced to 20% by tetrodotoxin (10^–7 M). DMPP, 5-HT and EFS each stimulated nerves that increased I _sc and each of these responses was significantly diminished by NA and SOM; for both NA and SOM the decrease in the DMPP response was significantly greater than the decrease observed in the response to carbachol (10^–6 M). Phentolamine (10^–6 M) abolished all of the effects of NA but caused no change in the SOM effects. These studies have shown that NA and SOM cause similar changes in net ion transport, that their actions are primarily on submucous secretomotor neurons and that NA and SOM can diminish the responses to stimulation of both cholinergic and noncholinergic submucous neurons.In this tissue it is also known that SOM coexists with NA in noradrenergic nerve terminals in the submucosa. However, when applied together, NA and SOM caused no greater decrement in the carbachol and 5-HT responses than would be predicted by adding the separate effects of NA and SOM. Hence there was no obvious interaction between NA and SOM effects on mucosal transport.     

Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract

Summary Eight smooth muscle preparations from the stomach, small intestine and large intestine of the guinea-pig were used to compare apamin's actions in reducing the effectiveness of transmission from enteric inhibitory nerves and in reducing responses to inhibitory agonists ,-methylene ATP, VIP and isoprenaline. The effects of apamin on inhibitory reflexes in the ileum and colon were also evaluated. Apamin had little or no effect on responses to VIP and isoprenaline in any region, but consistently and substantially reduced responses to ,-methylene ATP. Responses to stimulation of enteric inhibitory neurons were substantially reduced by apamin in the antrum circular muscle, ileum longitudinal and circular muscle, taenia coli and distal colon longitudinal muscle, but it was ineffective in the fundus circular muscle, proximal colon longitudinal muscle and distal colon circular muscle. It caused a small reduction of the relaxation of the ileal circular muscle caused reflexly by distension, but did not modify the similar descending inhibitory reflex in the circular muscle of the colon. It is concluded that apamin can be used to distinguish two types of non-noradrenergic transmission from enteric inhibitory nerves to gastrointestinal muscle. Furthermore, neither VIP nor ATP can be the sole transmitter chemical released from enteric inhibitory neurons throughout the gastrointestinal tract.