Diagnostic value of confocal endomicroscopy in celiac disease.

BACKGROUND: Improved endoscopic screening with targeted biopsies might enhance diagnostic yield in celiac disease. Confocal endomicroscopy (CEM) allows high-resolution in vivo histological analysis. We compared the endomicroscopic findings during ongoing endoscopy with the histological findings graded according to the Marsh classification. METHODS: Twenty-four patients with celiac disease and six patients with celiac disease that was refractory on a gluten-free diet were examined using CEM. The duodenal mucosa was evaluated by CEM and by conventional histological analysis in respect of villous atrophy, crypt hyperplasia, and increased numbers of intraepithelial lymphocytes (IELs > 40 / 100 enterocytes). The CEM results were assessed as to sensitivity, specificity, and interobserver variability. A Marsh classification score determined by CEM was compared to that obtained by histology. Thirty patients undergoing routine upper gastrointestinal endoscopy were used as controls. RESULTS: Conventional histology showed villous atrophy and crypt hyperplasia in 23 and increased numbers of IELs in 27 of the 30 patients with celiac disease. With CEM, villous atrophy, crypt hyperplasia, and increased IELs were respectively identified in 17, 12, and 22 of the 30 patients. The agreement of the findings on CEM with those of conventional histology was good in relation to villous atrophy (sensitivity 74 %) and increased numbers of IELs (sensitivity 81 %), but inadequate in relation to crypt hyperplasia (sensitivity 52 %). The kappa values for determination of interobserver variability were 0.90 for villous atrophy, 1.00 for crypt hyperplasia, and 0.84 for IEL detection. In the 30 control patients, normal duodenal architecture was found by both histology and endomicroscopy, indicating an overall specificity of 100 %. CONCLUSION: The assessment of duodenal histology by CEM in patients with celiac disease is sensitive and specific in determining increased numbers of IELs and villous atrophy, but insufficient in respect of crypt hyperplasia. For routine use of CEM in patients with celiac disease, the technique would need to be improved.

In vivo diagnosis of acute intestinal graft-versus-host disease by confocal endomicroscopy.

BACKGROUND AND STUDY AIMS: Conventional histology with hematoxylin and eosin (H&E) staining is the accepted standard for diagnosing acute intestinal graft-versus-host disease (GvHD). Confocal endomicroscopy (CEM) is a noninvasive method that allows in vivo histology to be performed during endoscopy. The aim of this study was to evaluate CEM for the diagnosis of acute intestinal GvHD. PATIENTS AND METHODS: This observational pilot study conducted between September 2006 and August 2008 included patients with acute diarrhea after stem cell transplantation, infectious diarrhea, or active ulcerative colitis. CEM (EC-3870CIFK, Pentax, Tokyo, Japan) was performed after intravenous injection of fluorescein 10% and topical application of acriflavine 0.05%. RESULTS: A total of 35 patients with acute diarrhea after stem cell transplantation were examined. In 16 patients, CEM and histology showed no evidence of GvHD. In 14/19 patients with histologically confirmed GvHD, the diagnosis could already be established by CEM during ongoing endoscopy. In GvHD grade IV, near complete destruction of the colonic crypts ("flat mucosa") was visible. Control patients with infectious colitis (N = 15) or ulcerative colitis (N = 15) displayed inflammatory changes but no evidence of GvHD. Altogether, sensitivity of CEM was 74% and specificity was 100 %. CONCLUSIONS: CEM improves rapid diagnosis of acute intestinal GvHD with high accuracy while performing endoscopy. Platelet transfusions and unnecessary biopsy acquisition can be avoided once acute intestinal GvHD has been diagnosed in vivo.

Tricellulin is regulated via interleukin-13-receptor α2, affects macromolecule uptake, and is decreased in ulcerative colitis.

In the two inflammatory bowel diseases, ulcerative colitis (UC) and Crohn's disease (CD), altered expression of tight junction (TJ) proteins leads to an impaired epithelial barrier including increased uptake of luminal antigens supporting the inflammation. Here, we focused on regulation of tricellulin (Tric), a protein of the tricellular TJ essential for the barrier against macromolecules, and hypothesized a role in paracellular antigen uptake. We report that Tric is downregulated in UC, but not in CD, and that its reduction increases the passage of macromolecules. Using a novel visualization method, passage sites were identified at TJ regions usually sealed by Tric. We show that interleukin-13 (IL-13), beyond its known effect on claudin-2, downregulates Tric expression. These two effects of IL-13 are regulated by different signaling pathways: The IL-13 receptor α1 upregulates claudin-2, whereas IL-13 receptor α2 downregulates Tric. We suggest to target the α2 receptor in future developments of therapeutical IL-13-based biologicals.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

This corrects the article DOI: 10.1038/mi.2017.66.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

Campylobacter jejuni is the most prevalent cause of foodborne bacterial enteritis worldwide. Patients present with diarrhea and immune responses lead to complications like arthritis and irritable bowel syndrome. Although studies exist in animal and cell models, we aimed at a functional and structural characterization of intestinal dysfunction and the involved regulatory mechanisms in human colon. First, in patients' colonic biopsies, sodium malabsorption was identified as an important diarrheal mechanism resulting from hampered epithelial ion transport via impaired epithelial sodium channel (ENaC) ß- and γ-subunit. In addition, barrier dysfunction from disrupted epithelial tight junction proteins (claudin-1, -3, -4, -5, and -8), epithelial apoptosis, and appearance of lesions was detected, which cause leak-flux diarrhea and can perpetuate immune responses. Importantly, these effects in human biopsies either represent direct action of Campylobacter jejuni (ENaC impairment) or are caused by proinflammatory signaling (barrier dysfunction). This was revealed by regulator analysis from RNA-sequencing (cytometric bead array-checked) and confirmed in cell models, which identified interferon-γ, TNFα, IL-13, and IL-1ß. Finally, bioinformatics' predictions yielded additional information on protective influences like vitamin D, which was confirmed in cell models. Thus, these are candidates for intervention strategies against C. jejuni infection and post-infectious sequelae, which result from the permissive barrier defect along the leaky gut.

High prevalence and functional effects of serum antineuronal antibodies in patients with gastrointestinal disorders.

BACKGROUND: Antineuronal antibodies can be associated with both gastrointestinal (GI) and brain disorders. For example, antibodies against the potassium channel subunit dipeptidyl-peptidase-like protein-6 (DPPX) bind to neurons in the central nervous system (CNS) and myenteric plexus and cause encephalitis, commonly preceded by severe unspecific GI symptoms. We therefore investigated the prevalence of antineuronal antibodies indicative of treatable autoimmune CNS etiologies in GI patients. METHODS: Serum samples of 107 patients (Crohn's disease n = 42, ulcerative colitis n = 16, irritable bowel syndrome n = 13, others n = 36) and 44 healthy controls were screened for anti-DPPX and further antineuronal antibodies using immunofluorescence on rat brain and intestine and cell-based assays. Functional effects of high-titer reactive sera were assessed in organ bath and Ussing chamber experiments and compared to non-reactive patient sera. KEY RESULTS: Twenty-one of 107 patients (19.6%) had antibodies against the enteric nervous system, and 22 (20.6%) had anti-CNS antibodies, thus significantly exceeding frequencies in healthy controls (4.5% each). Screening on cell-based assays excluded established antienteric antibodies. Antibody-positive sera were not associated with motility effects in organ bath experiments. However, they induced significant, tetrodotoxin (TTX)-insensitive secretion in Ussing chambers compared to antibody-negative sera. CONCLUSIONS & INFERENCES: Antineuronal antibodies were significantly more frequent in GI patients and associated with functional effects on bowel secretion. Future studies will determine whether such antibodies indicate patients who might benefit from additional antibody-directed therapies. However, well-characterized encephalitis-related autoantibodies such as against DPPX were not detected, underlining their rarity in routine cohorts.

Excitation energy transport and trapping in concentrated solid solutions of flavomononucleotide.

Excitation energy transport and trapping is studied for monomer-fluorescent dimer system of flavomononucleotide (FMN) in polyvinyl alcohol films (PVA). It is shown that the theory neglecting reverse energy transfer (RET) from dimers to monomers does not allow for the explanation of concentration quenching and concentration depolarization results presented herein. Much better agreement has been obtained using generalized energy transport theory in which fluorescent dimers are treated as imperfect traps for excitation energy. Such parameters like the dimer quantum yield and its emission anisotropy are estimated.

Spectroscopic manifestations of flavomononucleotide dimers in polyvinyl alcohol films.

Absorption and fluorescence spectra of flavomononucleotide (FMN) in polyvinyl alcohol films (PVA) over a very wide concentration range are investigated. The dimerization constant as well as the pure monomer and dimer spectra are calculated and the structural parameters of FMN dimer are established. Excitation wavelength and temperature dependencies of FMN/PVA fluorescence spectra for different FMN concentrations were carried out. These measurements together with those of absorption reveal that dimers are imperfect traps for excitation energy and that the energy transfer can occur both in forward and in reverse direction. Moreover, it was shown that the observed temperature changes in fluorescence spectra may be qualitatively explained by the effect of inhomogeneous broadening of FMN energy levels and by the presence of fluorescent dimers.

Apoptosis and intestinal barrier function.

The signal transduction pathways of the induction of apoptosis in the gastrointestinal tract have in part been discovered. However, almost nothing is known about the functional influence of apoptotic signals on intestinal barrier function. In this study the effect of camptothecin-induced apoptosis in HT-29/B6 monolayers and the influence of apoptosis on epithelial barrier function were characterized. We demonstrated that camptothecin causes a decrease of transepithelial resistance and an increase in fluxes of the paracellular marker [3H]mannitol. Camptothecin increased the apoptotic rate and the conductance of single-cell apoptosis as measured by the conductance scanning technique. We conclude that in our model of HT-29/B6 cells camptothecin is a potent inductor of apoptosis that causes significant barrier defects measured by the Ussing chamber technique and the conductance scanning technique. Based on these results we are able to investigate the effect of other cytokines--TGF-beta, for instance, and its role in apoptotic conditions.

Investigations on the fluorescence concentration quenching of flavomononucleotide in glycerine-water solutions.

We measured concentration changes of the fluorescence quantum yield eta/eta 0 of flavomononucleotide (FMN) in glycerine-water solutions of various viscosities. The results obtained show that FMN dimers are traps for the exciting light energy as well as the energy transferred non-radiatively. Very good agreement between the experimental and theoretical eta/eta 0 results was obtained. It has been shown that in the investigated solutions non-radiative energy transfer from monomers to dimers takes place, preceded by the migration of energy. It has been stated that no monomer quenching occurs in these solutions. The contribution of the individual fluorescence concentration quenching mechanisms has been determined.

The influence of fluorescence concentration quenching on the emission anisotropy of flavins in glycerine-water solutions.

The measurements of the emission anisotropy r/r0 of flavomononucleotide (FMN) within a range of concentrations of 10(-5)-10(-1) mol/L in glycerine-water solutions of different viscosities--0.056 Pa/s (system I) and 0.256 Pa/s (system II) have been carried out. In the range of high concentrations the repolarization effect due to the sharp drop of the quantum yield has been observed. The experimental results have been compared with theoretical expressions evaluated by taking into account both concentration and rotation depolarization. A good agreement on the values of the theoretical parameters obtained from independent measurements has been found. It has been stated that in the investigated systems the excitation energy transfer may be treated as a Markov process.

Diffuse gastric cancer with peritoneal carcinomatosis can mimic Crohn's disease.

In some cases the diagnosis of gastric cancer is difficult and the endoscopic presentation may be misleading. Diffuse type gastric carcinoma with peritoneal metastasis may present primarily with abdominal pain, colonic infiltration and/or diarrhea, thus other differential diagnoses like Crohn's disease (CD) may be considered at first. Therefore intensive diagnostic work-up is important. We report two cases of gastric cancer with ascites due to peritoneal carcinomatosis who were first diagnosed as CD. The patients were hospitalized in different institutions for weight loss, abdominal pain and nausea. The first colonoscopy, upper endoscopy with multiple biopsies and ascites puncture were negative for malignant disease, but macroscopic lesions resembling CD were described. Both patients were released on a prednisolone-based treatment for suspected CD. They presented to our hospital for further evaluation due to persistent symptoms. Neither lower nor upper endoscopy were suggestive of CD and endoscopic ultrasound was suspicious of malignancy in one case. Histology was diagnostic and showed gastric infiltration by a poorly differentiated adenocarcinoma. Diffuse type gastric cancer (gastric linitis plastica) with peritoneal metastasis may mimic certain clinical, endoscopic and CT imaging features of CD. Repeated biopsies and endoscopic investigations are often necessary to confirm a malignant process, especially in case of an inconclusive clinical and endoscopic picture. Endoscopic ultrasound may be useful to evaluate the risk of malignancy in patients with macroscopic suspicion of malignancy and negative biopsies.

[Neuroendocrine colorectal tumors. Surgical and endoscopic treatment]. / Neuroendokrine kolorektale Tumoren. Chirurgische und endoskopische Therapieverfahren.

The incidence of colorectal neuroendocrine tumors (NETs) is rising in developed countries primarily as a result of increased incidental detection by endoscopy and probably also due to a more adequate diagnosis according to the WHO classification. Less than 1% of colorectal NETs produce serotonin so that such tumors are practically never associated with a hormonal carcinoid syndrome. An exact clinico-pathological staging is of paramount importance for the therapeutic strategy and comprises the classification of the tumor type (well or poorly differentiated) and the assessment of established prognostic risk factors (depth of infiltration, vascular invasion, lymph node and distant metastases). Poorly differentiated colorectal NETs often present in an advanced, metastatic state, where surgical therapy is basically palliative. Well-differentiated tumors larger than 2 cm have a high risk of metastatic spread and should be treated as adenocarcinomas by radical oncological surgical resection. This applies to the majority of colon NETs. Tumors smaller than 1 cm, mainly locacted in the rectum, only rarely metastasize and are usually accessible for endoscopic treatment or transanal local surgery. Tumors between 1 and 2 cm in size have an uncertain prognosis and additional risk factors and co-morbidities of the patient have to be considered for a suitable, multidisciplinary therapeutic decision.

Confocal neurolasermicroscopy in human brain - perspectives for neurosurgery on a cellular level (including additional comments to this article).

BACKGROUND: During neurosurgery intraoperative imaging of vital neural structures on a cellular level would facilitate the development of new strategies for diagnosis and treatment. In vivo imaging would permit the detection of the tumour centre and infiltration zone. With targeted biopsies the lesion of interest could be determined before performing the biopsy, facilitating the final pathological diagnosis. In this study we present confocal neurolasermicroscopy as a new method in neurosurgery. METHODS: A miniaturised confocal neurolasermicroscope (NLM) was used ex vivo immediately after tumour resection of glioblastoma multiforme (GBM). NLM was performed with subcellular magnification up to a tissue depth of 100 microm. NLM images were compared to conventional histological images of the same tumour. RESULTS: The application of the method in nine patients allowed adequate diagnosis of a malignant glioma fulfilling the WHO criteria when compared to conventional histology. In one patient with glioblastoma multiforme NLM allowed the correct diagnosis of GBM to be made, demonstrating the high mitotic rate and cell pleomorphy of the tumour cells. Additional characteristics such as pleomorphic cells, mitotic figures, fibrillary matrix and the distinction between tumour centre and infiltration zone could be shown. CONCLUSIONS: NLM is a tool which could be adapted for neurosurgical intraoperative applications with the potential to diagnose tumours and recognise the tumour centre and infiltration zone in vivo. Further applications of NLM to characterise subcellular structures and vascular architecture are possible.

Occurrence of adenomas in the pouch and small intestine of FAP patients after proctocolectomy with ileoanal pouch construction.

PURPOSE: Proctocolectomy with ileoanal pouch construction is the standard therapy for patients with familial adenomatous polyposis coli (FAP) to prevent the genesis of colorectal carcinomas. In our patient population, we observed the postoperative development of adenomas not only in the pouch but also in the remaining small intestine. The exact incidence of these ileal polyps is still unknown, since the diagnostic possibilities of examining the small intestine are limited. METHODS: We performed wireless capsule endoscopy (CE) in patients who developed postoperative pouch adenomas (PA) to record the simultaneous occurrence of small bowel adenomas and PA. We operated on 46 patients with FAP (m:f 17:10, age 33 +/- 9 years). Thirty-five patients underwent proctocolectomy with ileoanal pouch creation. Pouch endoscopy was performed in regular intervals at 3 months and then annually after proctocolectomy. Capsule endoscopy was additionally carried out in all patients with PA. RESULTS: Ileal PA occurred in 22.8% (n = 8) of the patients with proctocolectomy (n = 35) after a mean of 5 years after surgery. Eight PA patients (all with PA) also had adenomas in the small intestine diagnosed by CE. CONCLUSIONS: Since jejunal and ileal adenomas occur in all patients with PA, we recommend regular follow-up examinations, which include pouch endoscopy at 3 months and annually after surgery in the presence of PA after proctocolectomy and pouch creation. On the basis of our observations, we recommend adding CE or double-balloon enteroscopy to the follow-up examination.

Direct and reverse energy transport in systems of monomers and imperfect traps: Monte Carlo simulations.

A Monte Carlo simulation of the concentration dependence of the fluorescence quantum yield ŋM and emission anisotropyr M of a system containing dye molecules in the form of monomers M and clusters T (statistical pairs and trimers) playing the role of the imperfect traps for nonradiative excitation energy transfer (NET) has been carried out. The simulation has been made for determined values of Förster critical distancesR 0 (MM) andR 0 (MT) and for several values ofR 0 (TM) andR 0 (TT) , assuming that the energy may be transferred from M(*) to T as well as from T(*) to M (reverse nonradiative energy transfer, RNET). It was shown that the RNET process in the range of high concentrations may strongly change the values ofr M as well as those of ŋM. For emission anisotropyr M an effect of repolarization was observed which decreases rapidly with increasingR 0 (TM) andR 0 (TT) . A very good agreement between the simulation results of ŋM and the theoretical model with no adjustable parameters was found. In the model, the RNET process and influence of correlation between active molecules on energy migration among monomers were taken into account.

Effects of prolactin and hCG treatment on luteal activity and the conceptus during delayed implantation in Schreibers' long-fingered bat (Miniopterus schreibersii).

In Schreibers' long-fingered bat (Miniopterus schreibersii) from South Africa, implantation is delayed from May to August during which time the corpus luteum is inactive and plasma progesterone and LH concentrations low. Treatment of pregnant long-fingered bats with exogenous prolactin for 10 days in early delayed implantation (May) resulted in changes in luteal ultrastructure that were consistent with increased steroidogenesis, high plasma progesterone concentrations and the initiation of implantation. Treatment of bats with hCG for 10 days in early delayed implantation induced similar changes in luteal ultrastructure and plasma progesterone concentrations, but did not initiate implantation. Treatment with exogenous progesterone during early delayed implantation had no influence on the corpus luteum or the conceptus. We conclude that prolactin is the most important pituitary hormone in the control of delayed implantation, although the route via which it induces implantation is unknown.

Patterns of Leydig cell and LH gonadotroph activity, and plasma testosterone concentrations in the seasonally reproducing Schreibers' long-fingered bat (Miniopterus schreibersii).

Spermatogenesis in Schreibers' long-fingered bat from approximately 33 degrees S in South Africa was seasonal, and occurred in the 3 months (February-April) preceding ovulation. The ultrastructure of the Leydig cells indicated a period of increased steroidogenesis at this time, and plasma testosterone concentrations were elevated from March to May (10.3 ng/ml). The reproductive accessory glands were secretorily active between March and May, and copulation occurred at the end of this period of activity. Changes in LH-beta immunoreactivity suggest that the LH gonadotrophs were secretorily active 1 month before the onset of spermatogenesis and that peak activity coincided with peak plasma testosterone concentrations, spermiogenesis and spermiation. During winter (May-August) there was no reproductive activity and the bats remained active, only entering prolonged periods of torpor during particularly cold spells. A secondary elevation in plasma testosterone concentration, during reproductive inactivity (October; 9.3 ng/ml), was not accompanied by any change in Leydig cell ultrastructure, and the biological significance of this peak is unknown. Such synchronous activity of the pituitary, Leydig cells, seminiferous epithelium and accessory glands is associated with the typical reproductive cycle of long-fingered bats in which copulation and fertilization are restricted to a brief period at the end of summer, and in which neither sperm storage nor a prolonged period of copulation occur.