Validation of Morphometric Analyses of Small-Intestinal Biopsy Readouts in Celiac Disease

Background

Assessment of the gluten-induced small-intestinal mucosal injury remains the cornerstone of celiac disease diagnosis. Usually the injury is evaluated using grouped classifications (e.g. Marsh groups), but this is often too imprecise and ignores minor but significant changes in the mucosa. Consequently, there is a need for validated continuous variables in everyday practice and in academic and pharmacological research.

Methods

We studied the performance of our standard operating procedure (SOP) on 93 selected biopsy specimens from adult celiac disease patients and non-celiac disease controls. The specimens, which comprised different grades of gluten-induced mucosal injury, were evaluated by morphometric measurements. Specimens with tangential cutting resulting from poorly oriented biopsies were included. Two accredited evaluators performed the measurements in blinded fashion. The intraobserver and interobserver variations for villus height and crypt depth ratio (VH:CrD) and densities of intraepithelial lymphocytes (IELs) were analyzed by the Bland-Altman method and intraclass correlation.

Results

Unevaluable biopsies according to our SOP were correctly identified. The intraobserver analysis of VH:CrD showed a mean difference of 0.087 with limits of agreement from −0.398 to 0.224; the standard deviation (SD) was 0.159. The mean difference in interobserver analysis was 0.070, limits of agreement −0.516 to 0.375, and SD 0.227. The intraclass correlation coefficient in intraobserver variation was 0.983 and that in interobserver variation 0.978. CD3⁺ IEL density countings in the paraffin-embedded and frozen biopsies showed SDs of 17.1% and 16.5%; the intraclass correlation coefficients were 0.961 and 0.956, respectively.

Conclusions

Using our SOP, quantitative, reliable and reproducible morphometric results can be obtained on duodenal biopsy specimens with different grades of gluten-induced injury. Clinically significant changes were defined according to the error margins (2SD) of the analyses in VH:CrD as 0.4 and in CD3⁺-stained IELs as 30%.     

Following Mitochondrial Footprints through a Long Mucosal Path to Lung Cancer

Background

Mitochondrial DNA (mtDNA) mutations are reported in different tumors. However, there is no information on the temporal development of the mtDNA mutations/content alteration and their extent in normal and abnormal mucosa continuously exposed to tobacco smoke in lung cancer patients.

Methodology

We examined the pattern of mtDNA alteration (mtDNA mutation and content index) in 25 airway mucosal biopsies, corresponding tumors and normal lymph nodes obtained from three patients with primary lung cancers. In addition, we examined the pattern of mtDNA mutation in corresponding tumors and normal lymph nodes obtained from eight other patients with primary lung cancers. The entire 16.5 kb mitochondrial genome was sequenced on Affymetrix Mitochip v2.0 sequencing platform in every sample. To examine mtDNA content index, we performed real-time PCR analysis.

Principal Findings

The airway mucosal biopsies obtained from three lung cancer patients were histopathologically negative but exhibited multiple clonal mtDNA mutations detectable in the corresponding tumors. One of the patients was operated twice for the removal of tumor from the right upper and left lower lobe respectively within a span of two years. Both of these tumors exhibited twenty identical mtDNA mutations. MtDNA content increased significantly (P<0.001) in the lung cancer and all the histologically negative mucosal biopsies except one compared to the control lymph node.

Conclusions/Significance:

Our results document the extent of massive clonal patches that develop in lifetime smokers and ultimately give rise to clinically significant cancers. These observations shed light on the extent of disease in the airway of smokers traceable through mtDNA mutation. MtDNA mutation could be a reliable tool for molecular assessment of respiratory epithelium exposed to continuous smoke as well as disease detection and monitoring. Functional analysis of the pathogenic mtDNA mutations may be useful to understand their role in lung tumorigenesis.     

Phenylbutyrate counteracts Shigella mediated downregulation of cathelicidin in rabbit lung and intestinal epithelia: a potential therapeutic strategy

Background

Cathelicidins and defensins are endogenous antimicrobial peptides (AMPs) that are downregulated in the mucosal epithelia of the large intestine in shigellosis. Oral treatment of Shigella infected rabbits with sodium butyrate (NaB) reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia.

Aims

To develop novel regimen for treating infectious diseases by inducing innate immunity, we selected sodium 4-phenylbutyrate (PB), a registered drug for a metabolic disorder as a potential therapeutic candidate in a rabbit model of shigellosis. Since acute respiratory infections often cause secondary complications during shigellosis, the systemic effect of PB and NaB on CAP-18 expression in respiratory epithelia was also evaluated.

Methods

The readouts were clinical outcomes, CAP-18 expression in mucosa of colon, rectum, lung and trachea (immunohistochemistry and real-time PCR) and release of the CAP-18 peptide/protein in stool (Western blot).

Principal findings

Significant downregulation of CAP-18 expression in the epithelia of rectum and colon, the site of Shigella infection was confirmed. Interestingly, reduced expression of CAP-18 was also noticed in the epithelia of lung and trachea, indicating a systemic effect of the infection. This suggests a causative link to acute respiratory infections during shigellosis. Oral treatment with PB resulted in reduced clinical illness and upregulation of CAP-18 in the epithelium of rectum. Both PB and NaB counteracted the downregulation of CAP-18 in lung epithelium. The drug effect is suggested to be systemic as intravenous administration of NaB could also upregulate CAP-18 in the epithelia of lung, rectum and colon.

Conclusion

Our results suggest that PB has treatment potential in human shigellosis. Enhancement of CAP-18 in the mucosal epithelia of the respiratory tract by PB or NaB is a novel discovery. This could mediate protection from secondary respiratory infections that frequently are the lethal causes in dysentery.     

Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients

Background

The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited.

Objectives

To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp.

Methods

Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above.

Results

TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1β in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls.

Conclusion

Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.     

Downregulation of peroxisome proliferator-activated receptors (PPARs) in nasal polyposis

Background

Peroxisome proliferator-activated receptor (PPAR) α, βδ and γ are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested.

Methods

Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification.

Results

mRNA expression of PPARα, PPARβδ, PPARγ was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARα and PPARγ than normal nasal mucosa and these levels were, for PPARγ, further reduced following steroid treatment. PPARγ immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARγ immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment.

Conclusion

The down-regulation of PPARγ, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARγ might be of importance in long standing inflammations.     

Phasic Contractions in Urinary Bladder from Juvenile versus Adult Pigs

Aims

Alterations in properties of the bladder with maturation are relevant physiologically and pathophysiologically. The aim of this study was to investigate alterations in bladder properties with maturation in juvenile vs. adult pig, focussing on differences between layers of the bladder wall (mucosa vs. detrusor) and the presence and functional contribution of interstitial cells (ICs).

Methods

Basal and cholinergic-induced phasic contractions (PCs) in mucosal and denuded-detrusor strips from juvenile and adult pigs were assessed. Expression of c-kit, a marker of ICs, was investigated in the mucosa and the detrusor layers of the pig bladder. The functional role of ICs in mediating PCs was examined using imatinib.

Results

Mucosal strips from juvenile and adult pig bladders demonstrated basal PCs whilst denuded-detrusor strips did not. PCs of mucosal strips from juvenile pigs were significantly greater than those from adult bladders. Immunoreactivity for c-kit was detected in mucosa and detrusor layers of pig bladder. Histological studies demonstrated a distinct layer of smooth muscle between the urothelium and bladder detrusor, termed the muscularis mucosa. Imatinib was only effective in inhibiting PCs in mucosal strips from juvenile pigs. Imatinib inhibited the carbachol-induced PCs of both juvenile and adult denuded-detrusor strips, although strips from juvenile bladders demonstrated a trend towards being more sensitive to this inhibition.

Conclusions

We confirm the presence of c-kit positive ICs in pig urinary bladder. The enhanced PCs of mucosal strips from juvenile animals could be due to altered properties of ICs or the muscularis mucosa in the bladders of these animals.     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

CD98 Positive Eosinophils Contribute to T Helper 1 Pattern Inflammation

Background and Aims

The pathogenesis of inflammatory bowel disease (IBD) has not been fully understood yet. Eosinophils (Eo) are one type of the major proinflammatory cells of the chronic inflammation in the intestine. CD98 is involved in the pathogenesis of a number of inflammations. This study aims to elucidate the role of CD98⁺ Eos in the initiation of intestinal inflammation.

Methods

The colon biopsies were collected from 60 patients with IBD. The expression of CD98 in the biopsies was examined by immunohistochemistry. The serum levels of the flagellin (FGN) antibody and Eo-derived mediators in the culture supernatants were assessed by enzyme-linked immunosorbent assay. The role of FGN on Eo activation was examined in a cell culture model. The role of FGN in the induction of colitis was observed in a mouse model.

Results

Compared to normal controls, the frequency of CD98⁺ Eos was markedly increased in the IBD colon mucosa. FGN were detected in the colon biopsies and in the sera of IBD patients. Exposure to FGN induced the expression of galectin 3 (the ligand of CD98) in dendritic cells. The exposure to galectin 3 activated the CD98⁺ Eos. After treatment with FGN intrarectally, mice with eosinophilia showed severe inflammation in the colon.

Conclusions

The interaction of galectin 3 and CD98 can induce Eos to release chemical mediators that contributes to the initiation of the intestinal inflammation.     

Up-regulation of Toll-like receptors 2, 3 and 4 in allergic rhinitis

Background

Toll-like receptors enable the host to recognize a large number of pathogen-associated molecular patterns such as bacterial lipopolysaccharide, viral RNA, CpG-containing DNA and flagellin. Toll-like receptors have also been shown to play a pivotal role in both innate and adaptive immune responses. The role of Toll-like receptors as a primary part of our microbe defense system has been shown in several studies, but their possible function as mediators in allergy and asthma remains to be established. The present study was designed to examine the expression of Toll-like receptors 2, 3 and 4 in the nasal mucosa of patients with intermittent allergic rhinitis, focusing on changes induced by exposure to pollen.

Methods

27 healthy controls and 42 patients with seasonal allergic rhinitis volunteered for the study. Nasal biopsies were obtained before and during pollen season as well as before and after allergen challenge. The seasonal material was used for mRNA quantification of Toll-like receptors 2, 3 and 4 with real-time polymerase chain reaction, whereas specimens achieved in conjunction with allergen challenge were used for immunohistochemical localization and quantification of corresponding proteins.

Results

mRNA and protein representing Toll-like receptors 2, 3 and 4 could be demonstrated in all specimens. An increase in protein expression for all three receptors could be seen following allergen challenge, whereas a significant increase of mRNA only could be obtained for Toll-like receptor 3 during pollen season.

Conclusion

The up-regulation of Toll-like receptors 2, 3 and 4 in the nasal mucosa of patients with symptomatic allergic rhinitis supports the idea of a role for Toll-like receptors in allergic airway inflammation.     

Endoscopic Biopsy in Gastrointestinal Neuroendocrine Neoplasms: A Retrospective Study

Background

Gastrointestinal neuroendocrine neoplasms (GI-NENs) are often located in the deep mucosa or submucosa, and the efficacy of endoscopic biopsy for diagnosis and treatment of GI-NENs is not fully understood.

Objective

The current study analyzed GI-NENs, especially those diagnosed pathologically and resected endoscopically, and focused on the biopsy and cold biopsy forceps polypectomy (CBP) to analyze their roles in diagnosing and treating GI-NENs.

Methods

Clinical data of all GI-NENs were reviewed from January 2006 to March 2012. Histopathology was used to diagnose GI-NENs, which were confirmed by immunohistochemistry.

Results

67.96% GI-NENs were diagnosed pathologically by endoscopy. Only 26.21% were diagnosed pathologically by biopsies before treatment. The diagnostic rate was significantly higher in polypoid (76.47%) and submucosal lesions (68.75%), than in ulcerative lesions (12.00%). However, biopsies were only taken in 56.31% patients, including 51.52% of polypoid lesions, 35.56% of submucosal lesions and 100.00% of ulcerative lesions. Endoscopic resection removed 61.76% of GI-NENs, including six by CBP, 14 by snare polypectomy with electrocauterization, 28 by endoscopic mucosal resection (EMR) and 15 by endoscopic submucosal dissection (ESD). 51.52% polypoid GI-NENs had infiltrated the submucosa under microscopic examination. CBP had a significantly higher rate of remnant (33.33%) than snare polypectomy with electrocauterization, EMR and ESD (all 0.00%).

Conclusions

Biopsies for all polypoid and submucosal lesions will improve pre-operative diagnosis. The high rate of submucosal infiltration of polypoid GI-NENs determined that CBP was inadequate in the treatment of GI-NENs. Diminutive polypoid GI-NENs that disappeared after CBP had a high risk of remnant and should be closely followed up over the long term.     

Evidence for local dendritic cell activation in pulmonary sarcoidosis

Background

Sarcoidosis is a granulomatous disease characterized by a seemingly exaggerated immune response against a difficult to discern antigen. Dendritic cells (DCs) are pivotal antigen presenting cells thought to play an important role in the pathogenesis. Paradoxically, decreased DC immune reactivity was reported in blood samples from pulmonary sarcoidosis patients. However, functional data on lung DCs in sarcoidosis are lacking. We hypothesized that at the site of disease DCs are mature, immunocompetent and involved in granuloma formation.

Methods

We analyzed myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in broncho-alveolar lavage (BAL) and blood from newly diagnosed, untreated pulmonary sarcoidosis patients and healthy controls using 9-color flowcytometry. DCs, isolated from BAL using flowcytometric sorting (mDCs) or cultured from monocytes (mo-DCs), were functionally assessed in a mixed leukocyte reaction with naïve allogeneic CD4+ T cells. Using Immunohistochemistry, location and activation status of CD11c⁺DCs was assessed in mucosal airway biopsies.

Results

mDCs in BAL, but not in blood, from sarcoidosis patients were increased in number when compared with mDCs from healthy controls. mDCs purified from BAL of sarcoidosis patients induced T cell proliferation and differentiation and did not show diminished immune reactivity. Mo-DCs from patients induced increased TNFα release in co-cultures with naïve allogeneic CD4⁺ T cells. Finally, immunohistochemical analyses revealed increased numbers of mature CD86⁺ DCs in granuloma-containing airway mucosal biopsies from sarcoidosis patients.

Conclusion

Taken together, these finding implicate increased local DC activation in granuloma formation or maintenance in pulmonary sarcoidosis.     

Colonoscopic Findings in Peruvian Patients with Chronic Diarrhea

Objective

To report the colonoscopic and pathological findings in patients with chronic diarrhea from a gastroenterology unit during approximately 3 years in a general teaching hospital located in Lima-Peru.

Materials and Methods

Patients with chronic diarrhea as the motive for colonoscopy from March 2008 to December 2010 were selected from the colonoscopy report computerized database. Colonoscopic findings were registered. Biopsies taken during the procedure were prospectively reviewed.

Results

226 patients were included, of which 162 (71.7%) had a colon biopsy available. The average age of the patients was 53.6±16.36. 85.8% of patients were reported to have a normal colon. 14.8% of patients were found to have a normal colonic mucosa or mucosal edema, 35.8% of patients had lymphocytic colitis and 28.4% had paucicelular colitis.

Conclusions

The majority of colonoscopies were reported with unremarkable macroscopic findings. Lymphocytic colitis was unusually frequent compared to previous reports.     

Endobronchial Mucosa Invasion Predicts Survival in Patients with Small Cell Lung Cancer

Background

Current staging system for small cell lung cancer (SCLC) categorizes patients into limited- or extensive-stage disease groups according to anatomical localizations. Even so, a wide-range of survival times has been observed among patients in the same staging system. This study aimed to identify whether endobronchial mucosa invasion is an independent predictor for poor survival in patients with SCLC, and to compare the survival time between patients with and without endobronchial mucosa invasion.

Methods

We studied 432 consecutive patients with SCLC based on histological examination of biopsy specimens or on fine-needle aspiration cytology, and received computed tomography and bone scan for staging. All the enrolled patients were assessed for endobronchial mucosa invasion by bronchoscopic and histological examination. Survival days were compared between patients with or without endobronchial mucosa invasion and the predictors of decreased survival days were investigated.

Results

84% (364/432) of SCLC patients had endobronchial mucosal invasion by cancer cells at initial diagnosis. Endobronchial mucosal involvement (Hazard ratio [HR], 2.01; 95% Confidence Interval [CI], 1.30–3.10), age (HR, 1.04; 95% CI, 1.03–1.06), and extensive stage (HR, 1.39; 95% CI, 1.06–1.84) were independent contributing factors for shorter survival time, while received chemotherapy (HR, 0.32; 95% CI, 0.25–0.42) was an independent contributing factor better outcome. The survival days of SCLC patients with endobronchial involvement were markedly decreased compared with patients without (median 145 vs. 290, p<0.0001). Among SCLC patients of either limited (median 180 vs. 460, p<0.0001) or extensive (median 125 vs. 207, p<0.0001) stages, the median survival duration for patients with endobronchial mucosal invasion was shorter than those with intact endobronchial mucosa, respectively.

Conclusion

Endobronchial mucosal involvement is an independent prognostic factor for SCLC patients and associated with decreased survival days.     

Correlation of LMP10 Expression and Clinical Outcome in Human Papillomavirus (HPV) Positive and HPV-Negative Tonsillar and Base of Tongue Cancer

Aim

To examine LMP10 expression and its possible impact on clinical outcome in human papillomavirus (HPV) positive and HPV-negative tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC).

Background

Outcome is better in HPV-positive TSCC and BOTSCC compared to matching HPV-negative tumours, with roughly 80% vs. 40% 5-year disease free survival (DFS) with less aggressive treatment than today’s chemoradiotherapy. Since current treatment often results in harmful side effects, less intensive therapy, with sustained patient survival would be an attractive alternative. However, other markers together with HPV status are necessary to select patients and for this purpose LMP10 expression is investigated here in parallel to HPV status and clinical outcome.

Materials and Methods

From 385 patients diagnosed between 2000 and 2007 at the Karolinska University Hospital, 278 formalin fixed paraffin embedded TSCC and BOTSCC biopsies, with known HPV DNA status, were tested for LMP10 nuclear and cytoplasmic expression (fraction of positive cells and staining intensity). The data was then correlated to clinical outcome.

Results

An absent/low compared to a moderate/high LMP10 nuclear fraction of positive cells was correlated to a better 3-year DFS in the HPV-positive group of patients (log-rank p = 0.005), but not in the HPV-negative group. In the HPV-negative group of patients, in contrast to the HPV-positive group, moderate/high LMP10 cytoplasmic fraction and weak/moderate/high LMP10 cytoplasmic intensity correlated to a better 3-year DFS (p = 0.003 and p = 0.001) and 3-year overall survival (p = 0.001 and 0.009).

Conclusion

LMP10 nuclear expression in the HPV-positive group and LMP10 cytoplasmic expression in the HPV-negative group of patients correlated to better clinical outcome.     

A Mouse Model of Otitis Media Identifies HB-EGF as a Mediator of Inflammation-Induced Mucosal Proliferation

Objective

Otitis media is one of the most common pediatric infections. While it is usually treated without difficulty, up to 20% of children may progress to long-term complications that include hearing loss, impaired speech and language development, academic underachievement, and irreversible disease. Hyperplasia of middle ear mucosa contributes to the sequelae of acute otitis media and is of important clinical significance. Understanding the role of growth factors in the mediation of mucosal hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae.

Methods

From a whole genome gene array analysis of mRNA expression during acute otitis media, we identified growth factors with expression kinetics temporally related to hyperplasia. We then tested these factors for their ability to stimulate mucosal epithelial growth in vitro, and determined protein levels and histological distribution in vivo for active factors.

Results

From the gene array, we identified seven candidate growth factors with upregulation of mRNA expression kinetics related to mucosal hyperplasia. Of the seven, only HB-EGF (heparin-binding-epidermal growth factor) induced significant mucosal epithelial hyperplasia in vitro. Subsequent quantification of HB-EGF protein expression in vivo via Western blot analysis confirmed that the protein is highly expressed from 6 hours to 24 hours after bacterial inoculation, while immunohistochemistry revealed production by middle ear epithelial cells and infiltrating lymphocytes.

Conclusion

Our data suggest an active role for HB-EGF in the hyperplasia of the middle ear mucosal epithelium during otitis media. These results imply that therapies targeting HB-EGF could ameliorate mucosal growth during otitis media, and thereby reduce detrimental sequelae of this childhood disease.     

Testing Stem Cell Therapy in a Rat Model of Inflammatory Bowel Disease: Role of Bone Marrow Stem Cells and Stem Cell Factor in Mucosal Regeneration

Background

The gastrointestinal (GI) mucosal cells turnover regularly under physiological conditions, which may be stimulated in various pathological situations including inflammation. Local epithelial stem cells appear to play a major role in such mucosal renewal or pathological regeneration. Less is clear about the involvement of multipotent stem cells from blood in GI repair. We attempted to explore a role of bone marrow mesenchymal stromal cells (BMMSCs) and soluble stem cell factor (SCF) in GI mucosa regeneration in a rat model of inflammatory bowel diseases (IBD).

Methods

BMMSCs labelled with the fluorescent dye PKH26 from donor rats were transfused into rats suffering indomethacin-induced GI injury. Experimental effects by BMMSCs transplant and SCF were determined by morphometry of intestinal mucosa, double labeling of PKH26 positive BMMSCs with endogenous proliferative and intestinal cell markers, and western blot and PCR analyses of the above molecular markers in the recipient rats relative to controls.

Results

PKH26 positive BMMSCs were found in the recipient mucosa, partially colocalizing with the proliferating cell nuclear antigen (PCNA), Lgr5, Musashi-1 and ephrin-B3. mRNA and protein levels of PCNA, Lgr5, Musashi-1 and ephrin-B3 were elevated in the intestine in BMMSCs-treated rats, most prominent in the BMMSCs-SCF co-treatment group. The mucosal layer and the crypt layer of the small intestine were thicker in BMMSCs-treated rats, more evident in the BMMSCs-SCF co-treatment group.

Conclusion

BMMSCs and SCF participate in but may play a synergistic role in mucosal cell regeneration following experimentally induced intestinal injury. Bone marrow stem cell therapy and SCF administration may be of therapeutic value in IBD.     

Immunoglobulin free light chains are increased in hypersensitivity pneumonitis and idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF), a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP), a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs) are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF.

Methods

In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry.

Results

FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung.

Conclusion

These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases.     

Role of Toll Like Receptors in Irritable Bowel Syndrome: Differential Mucosal Immune Activation According to the Disease Subtype

Background

The irritable bowel syndrome (IBS) is a functional gastrointestinal disorder whose pathogenesis is not completely understood. Its high prevalence and the considerable effects on quality of life make IBS a disease with high social cost. Recent studies suggest that low grade mucosal immune activation, increased intestinal permeability and the altered host-microbiota interactions that modulate innate immune response, contribute to the pathophysiology of IBS. However, the understanding of the precise molecular pathophysiology remains largely unknown.

Methodology and Findings

In this study our objective was to evaluate the TLR expression as a key player in the innate immune response, in the colonic mucosa of IBS patients classified into the three main subtypes (with constipation, with diarrhea or mixed). TLR2 and TLR4 mRNA expression was assessed by real time RT-PCR while TLRs protein expression in intestinal epithelial cells was specifically assessed by flow cytometry and immunofluorescence. Mucosal inflammatory cytokine production was investigated by the multiplex technology. Here we report that the IBS-Mixed subgroup displayed a significant up-regulation of TLR2 and TLR4 in the colonic mucosa. Furthermore, these expressions were localized in the epithelial cells, opening new perspectives for a potential role of epithelial cells in host-immune interactions in IBS. In addition, the increased TLR expression in IBS-M patients elicited intracellular signaling pathways resulting in increased expression of the mucosal proinflammatory cytokines IL-8 and IL1β.

Conclusions

Our results provide the first evidence of differential expression of TLR in IBS patients according to the disease subtype. These results offer further support that microflora plays a central role in the complex pathophysiology of IBS providing novel pharmacological targets for this chronic gastrointestinal disorder according to bowel habits.     

Change in DNA Methylation Patterns of SLC6A4 Gene in the Gastric Mucosa in Functional Dyspepsia

Background

The neurochemical serotonin (5-HT) is an important signaling molecule in the gastrointestinal motor and sensory functions. A key regulator of 5-HT levels is the transmembrane serotonin transporter (5-HTT; SLC6A4) that governs the reuptake of 5-HT. Recent studies have indicated 5-HTT expression may be regulated by epigenetic mechanisms. We investigated DNA methylation status of SLC6A4 gene in the gastric mucosa from functional dyspepsia (FD) because of their potential role in dyspeptic symptoms.

Methods

Endoscopic gastric biopsies were obtained from 78 subjects with no upper abdominal symptoms and 79 patients with FD. Bisulfite Pyrosequencing was carried out to determine the methylation status of promoter CpG islands (PCGIs), promoter non-CpG islands (PNCGIs) and gene body non-CpG islands (NPNCGIs) in the SLC6A4 gene. Gene expression was examined by real-time PCR.

Results

In overall, methylation level of PCGIs was significantly lower in FD compared to control subjects (p = 0.04). On the other hand, methylation level of NPNCGIs was significantly higher in FD compared to control subjects (p = 0.03). Lower methylation level in PNCGIs was highlighted in the patients with PDS (p = 0.01), while higher methylation level in NPNCGIs was more prominent in the patients with EPS (p = 0.017). Methylation levels of PCGIs and PNCGIs were inversely correlated, while methylation levels of NPNCGIs was positively correlated with SLC6A4 mRNA levels in FD patients.

Conclusions

Our data suggest that change in DNA methylation pattern of SLC6A4 in the gastric mucosa may have a role for developing FD. A role of epigenetics for developing FD needs to be further evaluated.