Fate of swallowed interleukin 8 and TNFα, and effect on the Wistar rat gut

To evaluate the passage of cytokines through the gastrointestinal tract, we investigated the digestion of interleukin-8 (IL-8) and tumour necrosis factor α (TNFα), in vitro and in vivo, and their propensity to induce intestinal inflammation. We serially immuno-assayed IL-8 and TNFα solutions co-incubated with each of three pancreatin preparations at pH 4.5 and pH 8. We gavaged IL-8, TNFα and marker into 15 Wistar rats, and measured their faecal cytokine concentrations by ELISA and histologically examined their guts. IL-8 immunoreactivity was extinguished by all pancreatin preparations after 1 h of incubation at 37 °C. TNFα concentration progressively fell from 1 to 4 h with all enzyme preparations. Buffer control samples maintained their cytokine concentrations throughout incubation. No IL-8 or TNFα was detected in any rat faecal pellets. There was no significant proinflammatory effect of the gavaged cytokines on rat intestine. IL-8 and TNFα in aqueous solution could well be fully digested in the CF gut when transit time is normal and exogenous enzymes are provided, although cytokines swallowed in viscous sputum may be protected from such digestion

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

Lavage treatment of distal intestinal obstruction syndrome in children with cystic fibrosis

The efficacy, adverse reactions, and long-term effects of intestinal lavage treatment with a balanced electrolyte solution (Golytely) was evaluated in patients with cystic fibrosis and distal intestinal obstruction syndrome. Twenty-two patients with cystic fibrosis (mean age 21.8 years, range 14 to 34 years, 15 boys or men) who sough medical attention because of abdominal pain and a mass in the right iliac fossa received Golytely, 5.6 ± 1.9 L (mean ± 1 SD), either orally (n = 14) or via nasogastric tube (n = 8) during 5.6 ± 2.4 hours. No serious side effects occurred. Serum electrolyte values remained within normal limits. Body weight did not change significantly. Minor adverse reactions included bloating (n = 12), nausea (n = 8), vomiting (n = 1), and chills (n = 3). All but one patient reported impressive relief of symptoms and remained pain free for an average of 3 months (range 1 to 19 months). Symptoms of abdominal pain and radiologic signs of fecal impaction assessed before and after lavage both decreased significantly (P < .0001). During follow-up (mean 15.2 months, range 4 to 26 months), 11 patients required a total of 38 (range one to nine) additional doses of Golytely. Seven patients drank the solution at home (21 treatments); only two patients chose a nasogastric tube. In ten patients with symptoms of recurrent distal intestinal obstruction syndrome prior to institution of therapy, duration of hospitalization was significantly reduced by this treatment (5.1 ± 7.6 v 2.3 ± 6.3 hospital days per annum, P < .02). It is concluded that intestinal lavage is a well-accepted, safe, and effective therapy for distal intestinal obstruction syndrome in patients with cystic fibrosis.

Treatment of distal intestinal obstruction syndrome in cystic fibrosis with a balanced intestinal lavage solution

Conventional treatment of distal intestinal obstruction syndrome (DIOS) with high doses of pancreatic enzymes, mucolytic agents, and enemas is neither predictably effective nor rapid in action. In 6 cystic fibrosis patients with DIOS a balanced, non-absorbable intestinal lavage solution produced clinical and radiological improvement and striking improvement in DIOS scores. It is suggested that a balanced intestinal lavage solution should be considered as an alternative treatment for DIOS in patients with cystic fibrosis.

Neonatal intestinal obstruction associated with oral calcium supplementation

A case report of a 920 g infant developing a small intestinal obstruction following therapy for congestive cardiac failure is presented. Although the causation was thought to be milk curd obstruction, subsequent analysis revealed high concentration of calcium and phosphate in the stools. The possible pathogenesis is discussed in relation to the inspissated milk syndrome.

Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOM™ assay data using the Fisher-Kolmogorov model

Background: Standard methods for quantifying IncuCyte ZOOM™ assays involve measurements that quantify how rapidly the initially-vacant area becomes re-colonised with cells as a function of time. Unfortunately, these measurements give no insight into the details of the cellular-level mechanisms acting to close the initially-vacant area. We provide an alternative method enabling us to quantify the role of cell motility and cell proliferation separately. To achieve this we calibrate standard data available from IncuCyte ZOOM™ images to the solution of the Fisher-Kolmogorov model. Results: The Fisher-Kolmogorov model is a reaction-diffusion equation that has been used to describe collective cell spreading driven by cell migration, characterised by a cell diffusivity, D, and carrying capacity limited proliferation with proliferation rate, λ, and carrying capacity density, K. By analysing temporal changes in cell density in several subregions located well-behind the initial position of the leading edge we estimate λ and K. Given these estimates, we then apply automatic leading edge detection algorithms to the images produced by the IncuCyte ZOOM™ assay and match this data with a numerical solution of the Fisher-Kolmogorov equation to provide an estimate of D. We demonstrate this method by applying it to interpret a suite of IncuCyte ZOOM™ assays using PC-3 prostate cancer cells and obtain estimates of D, λ and K. Comparing estimates of D, λ and K for a control assay with estimates of D, λ and K for assays where epidermal growth factor (EGF) is applied in varying concentrations confirms that EGF enhances the rate of scratch closure and that this stimulation is driven by an increase in D and λ, whereas K is relatively unaffected by EGF. Conclusions: Our approach for estimating D, λ and K from an IncuCyte ZOOM™ assay provides more detail about cellular-level behaviour than standard methods for analysing these assays. In particular, our approach can be used to quantify the balance of cell migration and cell proliferation and, as we demonstrate, allow us to quantify how the addition of growth factors affects these processes individually.

Biofilm formation by multidrug resistant Escherichia coli ST131 is dependent on type 1 fimbriae and assay conditions.

Escherichia coli sequence type 131 (ST131) have emerged as a pandemic lineage of important multidrug resistant pathogens worldwide. Despite many studies examining the epidemiology of ST131, only a few studies to date have investigated the capacity of ST131 strains to form biofilms. Some of these studies have reported contrasting findings, with no specific ST131 biofilm-promoting factors identified. Here we examined a diverse collection of ST131 isolates for in vitro biofilm formation in different media and assay conditions, including urine from healthy adult women. We found significant differences among strains and assay conditions, which offers an explanation for the contrasting findings reported by previous studies using a single condition. Importantly, we showed that expression of type 1 fimbriae is a critical determinant for biofilm formation by ST131 strains and that inhibition of the FimH adhesin significantly reduces biofilm formation. We also offer direct genetic evidence for the contribution of type 1 fimbriae in biofilm formation by the reference ST131 strain EC958, a representative of the clinically dominant H30-Rx ST131 subgroup. This is the first study of ST131 biofilm formation in biologically relevant conditions and paves the way for the application of FimH inhibitors in treating drug resistant ST131 biofilm infections.

Pheo-type: A diagnostic gene-expression assay for the classification of pheochromocytoma and paraganglioma

Context: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. Objective: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. Design: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. Results: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1–3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. Conclusions: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.