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Background: Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolising enzyme that induces immune tolerance by modulating T-cell responses. Carcinomas may create an immunosuppressive state via IDO1 expression. Here we examined a possible contribution of IDO1 on this phenomenon and investigated whether IDO1 has prognostic value in colorectal cancer (CRC).Methods: IDO1 expression was investigated by quantitative PCR and western blotting in three colon cancer cell lines, in basal state and after interferon (IFN)-γ stimulation. Semi-quantitative immunohistochemistry was used to evaluate IDO1 expression in 265 pT1-4N0-2Mx-staged CRCs. Results were related to clinical variables and correlated with amounts of CD3(+) and CD8(+) T lymphocytes, which were quantitatively evaluated using image analysis.Results: In vitro expression of IDO1 depended on IFN-γ stimulation. Higher IDO1 expression at the tumour invasion front was an independent adverse prognostic factor in pT1-4N1Mx-staged CRC. It was associated with overall survival (P=0.001) and with metachronous metastases (P=0.018). IDO1 expression was not associated with the presence of CD3(+) or CD8(+) T lymphocytes.Conclusion: Higher IDO1 expression at the tumour invasion front is involved in CRC progression and correlates with impaired clinical outcome, suggesting that IDO1 is an independent prognostic indicator for CRC. [ABSTRACT FROM AUTHOR]

Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increased IL-8 transcription in ileum of controls (p < 0,001; n = 18-20/group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p <0,001; n = 20/group). In the colon, TRPV1 mRNA levels were decreased (p = 0,046) in smoking healthy controls (n = 20/group). Likewise, healthy mice chronically exposed to cigarette smoke (n = 10/group) showed elevated ileal Cxcl2 (p = 0,0075) and colonic Kc mRNA levels (p = 0,0186), whereas TRPV1 mRNA and protein levels were elevated in the ileum (p = 0,0315). Although cigarette smoke exposure prior to trinitrobenzene sulphonic acid administration did not alter disease activity, increased pro-inflammatory cytokine production was observed in the distal colon (Kc: p = 0,0273; Cxcl2: p = 0,104; Il1-β: p = 0,0796), in parallel with the increase of Trpv1 mRNA (p < 0,001). We infer that CS affects pro-inflammatory cytokine expression in healthy and inflamed gut, and that the simultaneous modulation of TRPV1 may point to a potential involvement of TRPV1 in cigarette smoke-induced production of inflammatory mediators., Competing Interests: The authors have declared that no competing interests exist.

Inflammatory bowel disease (IBD) is characterized by severe gastrointestinal inflammation and results from a complex interplay between genetic and environmental factors. IBD includes two prominent subtypes: Crohn's disease (CD) and ulcerative colitis (UC). One of the main risk factors for the development of CD is cigarette smoking, while UC is rather a disease of ex-smokers. To date, many of the mechanisms underlying the immune imbalance in IBD and the involvement of cigarette smoke (CS) are incompletely understood. Transient receptor potential (TRP) proteins are non-selective cation channels that, upon activation, lead to plasma membrane depolarization and, in general, to Ca2+ influx. TRP channels of the ankyrin and vanilloid family, expressed by sensory neurons in the central and enteric nervous systems, have been extensively studied in the context of intestinal inflammation. Moreover, recent advances made on the role of non-neuronal expressed TRP channels shed light on the involvement of epithelial cells in inflammatory processes. This review focuses on how CS may impact TRP channel function in intestinal inflammation. Firstly, we discuss the current knowledge on neuronal TRP channels, known to be linked to IBD, in health, immune homeostasis and intestinal inflammation. Subsequently, we address how TRP channels are activated by CS and its components in other organ systems and also hypothesize on the potential implications for CS-mediated TRP channel activation in gut inflammation., (© 2016 S. Karger AG, Basel.)

Introduction: Microscopic bowel inflammation is present in up to 50% of patients with spondyloarthritis (SpA) and is associated with more severe disease. Currently no reliable biomarkers exist to identify patients at risk. Calprotectin is a sensitive marker of neutrophilic inflammation, measurable in serum and stool., Objectives: To assess whether serum and faecal calprotectin in addition to C-reactive protein (CRP) can be used to identify patients with SpA at risk of microscopic bowel inflammation., Methods: Serum calprotectin and CRP were measured in 125 patients with SpA. In 44 of these patients, faecal samples were available for calprotectin measurement. All 125 patients underwent an ileocolonoscopy to assess the presence of microscopic bowel inflammation., Results: Microscopic bowel inflammation was present in 53 (42.4%) patients with SpA. Elevated serum calprotectin and CRP were independently associated with microscopic bowel inflammation. Faecal calprotectin was also significantly higher in patients with microscopic bowel inflammation. Patients with CRP and serum calprotectin elevated had a frequency of bowel inflammation of 64% vs 25% in patients with low levels of both. When either CRP or serum calprotectin was elevated, the risk was intermediate (40%) and measuring faecal calprotectin provided further differentiation. Hence we suggest a screening approach where initially serum calprotectin and CRP are assessed and, if necessary, faecal calprotectin. The model using this scenario provided an area under the ROC curve of 74.4% for detection of bowel inflammation., Conclusions: Calprotectin measurements in stool and serum, in addition to CRP, may provide a promising strategy to identify patients with SpA at risk of bowel inflammation and could play a role in overall patient stratification., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Inflammatory bowel diseases (IBD) are complex multifactorial diseases characterized by an inappropriate host response to an altered commensal microbiome and dysfunctional mucus barrier. Cigarette smoking is the best known environmental risk factor in IBD. Here, we studied the influence of chronic smoke exposure on the gut microbiome, mucus layer composition and immune factors in conventional mice. We compared smoke-exposed with air-exposed mice (n = 12) after a smoke exposure of 24 weeks. Both Illumina sequencing (n = 6) and denaturing gradient gel electrophoresis (n = 12) showed that bacterial activity and community structure were significantly altered in the colon due to smoke exposure. Interestingly, an increase of Lachnospiraceae sp. activity in the colon was observed. Also, the mRNA expression of Muc2 and Muc3 increased in the ileum, whereas Muc4 increased in the distal colon of smoke-exposed mice (n = 6). Furthermore, we observed increased Cxcl2 and decreased Ifn-γ in the ileum, and increased Il-6 and decreased Tgf-β in the proximal colon. Tight junction gene expression remained unchanged. We infer that the modulating role of chronic smoke exposure as a latently present risk factor in the gut may be driven by the altered epithelial mucus profiles and changes in microbiome composition and immune factors., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593-0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73-2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83-7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice.

The inflammatory cytokine TNF-α is a central mediator in many immune-mediated diseases, such as Crohn's disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNFΔARE mice; in which a systemic TNF-α overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNFΔARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNFΔARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNFΔARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNFΔARE mice. The lung responses towards CS in TNFΔARE mice however depend on the duration of CS exposure.

Testosterone (T) levels are decreased in obese men, but the underlying causes are incompletely understood. Our objective was to explore the relation between low (free) T levels and male obesity, by evaluating metabolic parameters, subcutaneous adipose tissue (SAT) aromatase expression, and parameters of the hypothalamic-pituitary-gonadal axis. We recruited 57 morbidly obese men [33 had type 2 diabetes (DM2)] and 25 normal-weight men undergoing abdominal surgery. Fourteen obese men also attended a follow-up, 2 years after gastric bypass surgery (GBS). Circulating T levels were quantified by LC-MS/MS, whereas free T levels were measured using serum equilibrium dialysis and sex hormone-binding globulin, luteinizing hormone, and follicle-stimulating hormone by immunoassay. SAT biopsies were used to determine adipocyte cell size and aromatase expression by real-time PCR. Total and free T levels were decreased in obese males versus controls, with a further decrease in obese men with DM2 versus obese men without DM2. There were no differences in aromatase expression among the study groups, and sex steroids did not correlate with aromatase expression. Pearson analysis revealed an inverse association between (free) T and SAT cell size, triglycerides, and HOMA-IR. Multivariate analysis confirmed the inverse association between (free) T and SAT cell size (β = -0.321, P = 0.037 and β = -0.441, P = 0.011, respectively), independent of age, triglycerides, HOMA-IR, obesity, or diabetes. T levels were normalized 2 years after GBS. These data suggest that SAT cell size rather than SAT aromatase expression or parameters of the hypothalamic-pituitary-gonadal axis is related to low T in male obesity, which points to adipose cell size-related metabolic changes as a major trigger in decreased T levels.

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life., (© 2015 The Authors.)

Isolation of human follicles is based on digestion of the tissue by combinations of enzymes. Follicle vitality and morphology are often based on the analysis of pooled follicles of different maturation stages. Information is therefore lacking on the effect of the isolation protocol to individual follicles of different maturation stages. A study was conducted using five protocols combining different enzymes and varying concentrations. Isolated follicles were classified according to their maturation stages, counted and characterized for vitality, morphology, early apoptosis and organization of transzonal projections. No statistical differences were found between the protocols when outcome parameters were analysed on a pool of follicles regardless of their maturation status. Differences were observed in quality when the follicles were analysed separately according to their maturation status. Combining morphologic characteristics and vitality, both Liberase DH and Liberase TM combined with collagenase IV were better at isolating high-quality primordial follicles, compared with collagenase IV. No statistical difference between the isolation protocols was found for primary follicles. If only high-quality isolated secondary follicles are needed, collagenase IV is found to be most advantageous. Follicles of different maturation stages react differently when enzymatic isolation protocols are compared., (Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Neoadjuvant radio(chemo)therapy is increasingly used in rectal cancer and induces a number of morphologic changes that affect prognostication after curative surgery, thereby creating new challenges for surgical pathologists, particularly in evaluating morphologic changes and tumour response to preoperative treatment. Surgical pathologists play an important role in determining the many facets of rectal carcinoma patient care after neoadjuvant treatment. These range from proper handling of macroscopic specimens to accurate microscopic evaluation of pathological features associated with patients' prognosis. This review presents the well-established pathological prognostic indicators and discusses challenging features in order to provide both surgical pathologists and treating physicians with a checklist that is useful in a neoadjuvant setting.

During the past decade, extensive research has undeniably improved the formulation and delivery of oral vaccines. Nevertheless, several factors, such as the harsh gastrointestinal environment together with tolerance induction to exogenous antigens, have thus far impeded the optimal effectiveness and clinical application of oral delivery systems. The current study encompasses an initial evaluation of the stability, biocompatibility, and cellular uptake of two promising candidate systems for oral antigen delivery, that is, calcium carbonate- (CP) and mannitol-templated (MP) porous microspheres. Both spray-dried formulations were efficiently internalized by human intestinal epithelial cells (Caco-2 and HT-29) and degraded into phagolysosomal intracellular compartments. In addition, cellular particle uptake and processing significantly up-regulated the expression of (HLA) class-II and costimulatory molecules on intestinal epithelial cells. Even though the high surface-area-to-volume ratio of the microspheres was expected to favor protease access, antigen release was remarkably limited in simulated intestinal fluid and was even absent under gastric conditions. Finally, neither CP nor MP exerted cytotoxicity upon prolonged in vitro incubation with high antigen concentration. Altogether, these data support the potential of CP and MP for oral antigen delivery and motivate the further development of these promising carrier systems in in vivo studies.

Oral vaccination is the most challenging vaccination method due to the administration route. However, oral vaccination has socio-economic benefits and provides the possibility of stimulating both humoral and cellular immune responses at systemic and mucosal sites. Despite the advantages of oral vaccination, only a limited number of oral vaccines are currently approved for human use. During the last decade, extensive research regarding antigen-based oral vaccination methods have improved immunogenicity and induced desired immunological outcomes. Nevertheless, several factors such as the harsh gastro-intestinal environment and oral tolerance impede the clinical application of oral delivery systems. To date, human clinical trials investigating the efficacy of these systems are still lacking. This review addresses the rationale and key biological and physicochemical aspects of oral vaccine design and highlights the use of yeast-derived β-glucan microparticles as an oral vaccine delivery platform.

Continuously improving the developmental process and the efficacy of oral vaccines is essential in the fight against intestinal pathogens. A promising strategy for vaccination applying safe, biodegradable and non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. The current study evaluates the potential of β-glucan particles (GP) as an oral antigen delivery system and their adjuvant characteristics. GP are efficiently internalized by human intestinal epithelial cell lines (Caco-2 and HT-29 cells), without exerting negative effects on cell viability. GP triggered the expression of pro-inflammatory cytokines IL-23p19, IL-8 and the β-glucan receptors dectin-1 and TLR2 by activated Caco-2 cells, and CCL20 in HT-29 cells. In contrast, the expression level of TGF-β, an important mediator of oral tolerance, was significantly downregulated in HT-29 cells. Additionally, adoptive transfer experiments showed proliferating ovalbumin (OVA)-specific CD4(+) T cells mainly in the spleens of GP-OVA-fed mice. Furthermore, we detected a significantly increased IL-17 and a trend towards increased IFN-γ production in the spleen of GP-OVA-fed mice upon antigen restimulation. Oral administration of GP-OVA induced increased OVA-specific IgA, secretory-IgA (S-IgA) and secretory component (SC) production in intestinal fluids. Our data show that GP vehicles are able to deliver OVA via an oral route allowing efficient antigen presentation alongside adaptive immune activation, resulting in a Th17-biased response and the production of OVA-specific IgA, secretory-IgA and secretory component antibodies., (© 2013.)

Aim: Data on quality control of the pathologic evaluation of total mesorectal excision (TME) specimens are scarce. We aimed to assess differences between evaluation by local pathologists participating in PROject on CAncer of the REctum (PROCARE; a Belgian improvement project on rectal cancer) and by a review panel of experts., Method: Based on photographic material and histopathology slides, a Review Committee of gastrointestinal expert pathologists re-evaluated the mesorectal plane, the tumour differentiation grade, the (y)pT stage and the tumour regression grade in 444 patients previously routinely assessed by local pathologists., Results: The surgical plane was reported in 89% of patients and the circumferential resection margin in 88% of patients by the local pathologist. The median number of lymph nodes harvested in patients undergoing neoadjuvant radiochemotherapy was 11 and 14 in the other patients. The Review Committee downgraded the surgical plane from (intra)mesorectal to intramuscular in 17% of patients, and upgraded it from intramuscular to (intra)mesorectal in 27%. Tumour differentiation grade, T stage and tumour regression grade differed between local pathologists and the Review Committee in 15%, 10% and 38%, respectively, of patients. T stage was upgraded, mainly from T2 to T3, in 8% of patients. Tumour regression was judged by the Review Committee to be less advanced in 15% of patients., Conclusion: Acknowledging some shortcomings, this study gives a realistic view of clinical practice. There are differences in interpretation with regard to both macroscopic and microscopic analysis of TME specimens. These findings indicate a need for more objective and reproducible criteria in histopathology. Being aware of this is a first step for improvement., (Colorectal Disease © 2013 The Association of Coloproctology of Great Britain and Ireland.)

Background: Inflammatory patterns of nasal polyps (NPs) may vary. Changes over time have not been investigated so far. This study was designed to evaluate the inflammatory patterns of NPs in Thailand at two time points 12 years apart, explore differences in Staphylococcus aureus (SA) mucosal carriage rates over time, and the latter's relationship with the inflammatory patterns., Methods: Formalin-fixed nasal tissue was obtained from 89 (47 in 1999 and 42 in 2011) patients suffering from chronic rhinosinusitis with NPs (CRSwNPs). Tissues were evaluated for eosinophils, neutrophils, IgE(+) cells, IgE and macrophage mannose receptors, interleukin (IL)-5 and IL-17 cytokine profile, and the presence of SA, using automated immunohistochemistry and peptide nucleic acid-fluorescence in situ hybridization., Results: We found a significant increase in the absolute values of eosinophils and IgE(+) cells in the 2011 CRSwNP tissue series compared with 1999 and a significant but smaller increase in neutrophils. Semiquantitative evaluation revealed significantly higher mean values of positive cells for all studied inflammatory markers in the 2011 group of patients, except for the high-affinity IgE receptor. This "eosinophilic shift" of inflammation was accompanied by higher SA carriage, as well as higher frequencies of SA invasion (54.8% versus 10.6%; p < 0.001) in the 2011 compared with 1999 subjects. Patients with asthma were more likely to have higher SA carriage rates compared with nonasthmatic patients., Conclusion: There was a shift from predominantly neutrophilic to eosinophilic CRSwNPs in Thai patients within 12 years, with an increase in various inflammatory markers including IgE, which is associated with an increase in intramucosal presence of SA.

Introduction: Regeneration of pulp-like tissue in the pulp chamber after tooth transplantation, replantation, or in regenerative endodontic treatment is only possible if the apical foramen is open. According to the literature, the success of regeneration decreases considerably if the foramen is smaller than 1 mm when measured on radiographs. The aim of this study was to study histologically the relation between the width of the apical foramen and regeneration of tissue in the pulp chamber after autotransplantation., Methods: Fifteen single-rooted mature teeth of 3 adult beagle dogs were used. All experimental teeth were extracted and underwent apicoectomy. The teeth were photographed from the apical side, and the width of the foramen was calculated. The foramen width ranged from 0.24-1.09 mm. All teeth were replanted in infraocclusion. The observation period was 90 days after transplantation., Results: The 10 teeth with the smallest apical diameter, ranging between 0.24 and 0.53 mm, showed vital tissue in at least one third of the pulp chamber. The 6 most successful teeth showing vital tissue in the entire pulp chamber had an apical diameter between 0.32 and 0.65 mm, and 80% of the experimental teeth with a diameter varying between 1.09 and 0.31 mm showed vital tissue in at least one third of the pulp chamber 90 days after transplantation., Conclusions: The size of the apical foramen seems not to be the all decisive factor for successful revascularization and ingrowth of new tissue after transplantation. The minimum width of the apical foramen has not been determined, but a size smaller than 1 mm does not prevent revascularization and ingrowth of vital tissue. In this animal study an apical foramen of 0.32 mm did not prevent ingrowth of new tissue in two-thirds of the pulp chamber 90 days after transplantation., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: Composition changes of extracellular matrix (ECM) can lead to functional disorders of the upper airways (UA). The aim of this study was to systematically measure both the association patterns and the correlation degree between tissue composition parameters in UA inflammatory diseases., Methodology: Nasal samples were obtained from patients with chronic rhinosinusitis with (CRS+NP), without nasal polyps (CRS), with post-operative adhesions (S) and normal nasal mucosa (NM). A reproducible semi-quantitative method, which takes epithelial and lamina propria damages into account was applied for haematoxylin and eosin, alpha-smooth muscle actin, reticulin, elastin, laminin and collagen type IV stainings., Results: The most severe cases of epithelial shedding have been found in a significant higher amount in CRS+NP when compared with NM. The most severe cases of inflammatory reaction were mainly found in CRS+NP. CRS+NP had significantly more severe cases of oedema than NM. Excluding elastin, networks in other ECM proteins were found modified in fibrotic fields but to a lesser extend in oedematous regions in all conditions., Conclusion: Although non specific, oedema in the lamina propria is a key-feature of CRS+NP, while fibrosis, massively present in CRS and S, affects profoundly the distribution of ECM proteins in these areas.

Cigarette smoke (CS) exposure is associated with increased autophagy in several cell types, such as bronchial epithelial cells. Smoking is also an environmental risk factor in Crohn's disease, in which impairment of the autophagy-mediated anti-bacterial pathway has been implicated. So far, it is unknown whether CS induces autophagy in the gut. Here, we examined the effect of chronic CS exposure on autophagy in the follicle-associated epithelium (FAE) of murine Peyer's patches. Transmission electron microscopy revealed that the proportion of cell area occupied by autophagic vesicles significantly increased in the FAE after CS exposure. An increased number of autophagic vesicles was observed in the FAE, whereas the vesicle size remained unaltered. Besides enterocytes, also M-cells contain more autophagic vesicles upon CS exposure. In addition, the mRNA level of the autophagy-related protein Atg7 in the underlying Peyer's patches is increased after CS exposure, which indicates that the autophagy-inducing effect of CS is not limited to the FAE. In conclusion, our results demonstrate that CS exposure induces autophagy in murine FAE and in the underlying immune cells of Peyer's patches, suggesting that CS exposure increases the risk for Crohn's disease by causing epithelial oxidative damage, which needs to be repaired by autophagy.

Epidemiological evidence demonstrates that smoking is the most important environmental risk factor in Crohn's disease while it positively interferes with the disease course of ulcerative colitis. However, the underlying mechanisms through which smoking exerts this divergent effect and affects pathogenesis of inflammatory bowel disease are largely unknown. Animal smoke models are good models to investigate the impact of cigarette smoke on intestinal physiology and inflammation. They enable one to explore the interaction of smoke components and the gut on cellular and molecular level, clarifying how smoking interferes with normal gut function and with disease course in inflammatory conditions. This review describes the currently used animal models for studying the impact of cigarette smoke on the intestinal tract. We first discuss the different methods for simulation of smoking. Furthermore, we focus on the effect of smoke exposure on normal gut physiology and immunology, on experimental (entero)colitis, and on inflammation-induced neoplasia. Based on this current knowledge, a hypothesis is formulated about the mechanisms through which cigarette smoke interferes with the gut in normal and pathological conditions., (Copyright © 2011 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Smokers have a twofold increased risk to develop Crohn's disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Especially Crohn's ileitis is negatively influenced by smoking. Interestingly, the ileum and, more in particular, the Peyer's patches in the terminal ileum are also the sites where the first CD lesions are found. Several chemokines are implicated in the pathogenesis, among which is the CCL20-CCR6 pathway. Here, we studied the gut-associated lymphoid tissue in C57BL/6 wild-type mice and in CCR6-deficient mice after exposure to air or cigarette smoke for 24 weeks. Apoptotic index of the follicle-associated epithelium overlying the Peyer's patches was evaluated. We found that chronic smoke exposure induced apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in Peyer's patches. Important changes in immune cell composition were observed: total dendritic cells, CD4+ T cells (including regulatory T cells) and CD8+ T cells increased significantly after smoke exposure. The CD11b+ dendritic cell subset almost doubled. Interestingly, these changes were accompanied by an upregulated mRNA expression of the chemokines CCL9 and CCL20. However, no differences in the increase of dendritic cells were observed between wild-type and CCR6-deficient mice. Our results show that cigarette smoke exposure increases apoptosis in the follicle-associated epithelium and is associated with immune cell accumulation in Peyer's patches.

BACKGROUND In some couples, not all retrieved oocytes mature, even after prolonged in vitro culture. The underlying mechanisms are not known, although ionophore treatment may alleviate metaphase I (MI) arrest in some mouse strains. We attempted to induce first polar body (PB) extrusion and fertilization using assisted oocyte activation (AOA) after ICSI in maturation-resistant human MI oocytes. METHODS Four ICSI patients are described in this retrospective study. A pilot study tested the calcium ionophore ionomycin (10 µM) on donated MI oocytes from patients with a normal number of metaphase II (MII) oocytes. Subsequently, ionomycin was used to induce first PB extrusion in two patients showing maturation-resistant MI oocytes. AOA, by calcium injection and ionomycin exposure, was applied when mature oocytes were available. Oocytes were analysed by polarized microscopy and immunostaining. RESULTS Ionomycin induced the first PB extrusion in MI oocytes from patients with a normal number of retrieved MII oocytes, while extended in vitro culture failed to achieve the MII stage. Similarly, ionomycin induced first PB extrusion in one of two patients with recurrent maturation-resistant MI oocytes. Use of ICSI combined with AOA on MII oocytes matured in vitro or in vivo resulted in failed or abnormal fertilization with no further embryo cleavage potential. Highly abnormal spindle and chromosome configurations were observed in MI maturation-resistant oocytes, in contrast to control MI oocytes. CONCLUSIONS Ionophore induced first PB extrusion in MI oocytes from patients without maturation arrest but to a lower extent in maturation-resistant MI oocytes. Immunofluorescence staining and confocal analysis revealed, for the first time, highly abnormal spindle/chromosomal structures that may be responsible for this maturation arrest.

In an attempt to extend the indication area for autotransplantation of vital teeth, two possibilities can be proposed: (i) The enlargement of the apical foramen, with the aim to facilitate revascularization and ingrowth of new tissue. The ingrowth of tissue will eliminate the need for endodontic treatment when mature teeth are transplanted and (ii) the cryopreservation of teeth in case they cannot be transplanted immediately to the receptor site. Teeth with an ideal stage of root formation can be cryopreserved to perform transplantation later. Although pulpcell cultures survive crypreservation in vitro, the pulp tissue cannot survive the cryopreservation procedures when it is kept inside the pulpchamber. Therefore, the pulp tissue has to be removed before cryopreservation. It has been demonstrated that revascularization and ingrowth of new tissue can occur in an empty pulp chamber (1). The aim of this study was to find out if revascularization and ingrowth of new pulp tissue is influenced by removal of the original pulp tissue before autotransplantation. Twenty nine single-rooted teeth from three adult beagle dogs were transplanted after resection of the root tip. One group of teeth (n = 14) had the pulp tissue removed before transplantation. The other group (n = 15) had the original pulp left in situ. The transplanted teeth were histologically analysed 90 days post-transplantation. In the group with the tissue left in situ, 12 teeth (80%) showed a pulp chamber totally filled or at least 1/3 to 2/3 filled with viable tissue. In the group with the pulp tissue removed, 11 teeth (79%) had no or little vital tissue in the pulp chamber. The necrotic masses that develop in the original pulp tissue immediately after transplantation are a possible stimulating factor in the repair process of the pulp. As a conclusion, it can be stated that in case of autotransplantation of teeth, it is advisable to leave the pulp tissue in situ to stimulate the revascularization and ingrowth of new tissue after transplantation., (© 2010 John Wiley & Sons A/S.)

Background: Ovarian tissue (OT) cryopreservation and transplantation are options for fertility preservation in young female cancer patients., Methods: We investigated xenotransplantation of human OT into back muscle (B) of severe combined immunodeficiency mice. OT follicle content was evaluated by stereomicroscopy and pre-transplantation. Xenograft survival, follicular development (with/without FSH administration), apoptosis and vascularization were compared in B- versus K-site (under the kidney capsule) several times after grafting using histology, immunohistochemistry and magnetic resonance imaging. In vitro maturation (IVM) was also performed., Results: Anastomoses which developed from existing human and invading murine vessels were seen in OT at both sites, but angiogenesis was more prominent at the B- than K-site (P < 0.001). Vascularization and follicle size were correlated in the B-group (Spearman's coefficient 0.73; P < 0.001). FSH increased early (8 days) micro-vessel formation in B but not in K grafts (P < 0.0001, versus no FSH). B-site grafts showed a better histological morphology and survival (P = 0.0084), formation of larger antral follicles (P = 0.005), more metaphase-II (MII) oocytes, growing follicles (P = 0.028) and slightly fewer apoptotic follicles than K grafts. One MI oocyte from B underwent IVM and reached MII stage next day., Conclusions: To our knowledge, this is the first report of MII and IVM-MII oocytes obtained from B xenografts. We report the largest oval-shaped antral follicles containing an MII oocyte obtained after OT xenotransplantation to date. Xenografting in the mouse B should be further explored as a method for human OT transplantation.

Unlabelled: The aim of this study was to examine systemic and local nasal leptin and leptin receptor expression in patients with nasal polyposis and healthy controls., Methods and Results: Serum leptin and soluble leptin receptor levels were examined by enzyme-linked immunosorbent assay (ELISA). The presence of leptin and leptin receptor mRNA was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), and tissue leptin and leptin receptor protein expression was analysed by immunohistochemistry and ELISA. Serum levels of biologically active leptin were significantly elevated in patients with nasal polyps compared with control subjects. These serum leptin levels were strongly correlated with the levels found in tissue in both study groups, although leptin was not significantly elevated in nasal polyp tissue. Using RT-PCR, we showed that both leptin and its receptors were produced in nasal mucosa. Finally, immunohistochemistry showed that leptin and leptin receptor protein were expressed in several cells of the normal and inflamed nasal mucosa., Conclusions: Leptin receptors and their biological ligand leptin are expressed in the nasal mucosa, suggesting a possible role in upper airway immunology.

Background: Restoring p53 function by antagonizing its interaction with the negative regulator MDM2 is an appealing nongenotoxic approach to treating tumors with wild-type p53. Mutational inactivation of p53 is rare in neuroblastoma tumors at diagnosis and occurs in only a subset of multidrug-resistant neuroblastomas., Methods: The antiproliferative and cytotoxic effect of nutlin-3, a small-molecule MDM2 antagonist, was examined in chemosensitive (UKF-NB-3) and matched chemoresistant neuroblastoma cells with wild-type p53 (UKF-NB-3(r)DOX20) or with mutant p53 (UKF-NB-3(r)VCR10). Activation of the p53 pathway was assessed by expression analysis of p53 target genes, flow cytometric cell cycle analysis, and apoptosis assays. Mice with established chemoresistant tumor xenografts were treated orally with nutlin-3 or vehicle control (n = 5-10 mice per group) and were used to evaluate effects on tumor growth, p53 pathway activity, and metastatic tumor burden. All statistical tests were two-sided., Results: Nutlin-3 induced a similar activation of the p53 pathway in UKF-NB-3 and UKF-NB-3(r)DOX20 cells, as evidenced by increased expression of p53 target genes, G1 cell cycle arrest, and induction of apoptosis. No such response was observed in UKF-NB-3(r)VCR10 cells with mutant p53. Oral administration of nutlin-3 to UKF-NB-3(r)DOX20 xenograft-bearing mice led to inhibition of primary tumor growth (mean tumor volume after 3 weeks of treatment, nutlin-3- vs vehicle-treated mice: 772 vs 1661 mm3, difference = 890 mm3, 95% confidence interval = 469 to 1311 mm3, P < .001), p53 pathway activation, and reduction in the extent of metastatic disease. The growth of UKF-NB-3(r)VCR10 xenografts was unaffected by nutlin-3., Conclusions: Nutlin-3 activates the p53 pathway and suppresses tumor growth in this model system of chemoresistant neuroblastoma, provided that wild-type p53 is present.

Objectives: Xenogenic extracellular heart valve matrices have been suggested as scaffolds for tissue engineering. However, these matrices are immunogenic and stimulate an intense cell-mediated immune response and calcification. Mitigating the immunogenicity was attempted by different doses of gamma irradiation., Methods: Mechanical properties of gamma-irradiated porcine matrices and control matrices (nonirradiated) were examined by tensile strength testing. Irradiated matrices (1, 10, 50, and 100 gray [Gy]) and control matrices were implanted subcutaneously in Wistar rats (n = 20). After 24 h, 1, 2, 3, and 4 weeks the explants were examined by light microscopy and transmission electron microscopy. Calcium (Ca) content was determined using inductively coupled plasma-mass spectrometry. Antibody reaction against porcine tissue in the rat serum was determined., Results: Tensile strength increased in irradiated matrices at the expense of elasticity. Ten gray-irradiated leaflets showed minimal lymphocytic inflammatory infiltration with preservation of ultrastructure. Ca levels after 2 weeks were as follows: control (0 Gy), 388 +/- 264 microg/mg; 1 Gy, 240 +/- 95 microg/mg; 10 Gy, 188 +/- 54 microg/mg; 50 Gy, 289 +/- 94 microg/mg; 100 Gy, 651 +/- 57 microg/mg. All implants still elicit an antibody immunoglobulin G reaction., Conclusions: Exposure to 10 Gy gamma irradiation reduces lymphocytic inflammatory infiltrates and Ca levels in acellular porcine matrices with preservation of structural integrity. This could prolong the durability of these matrices.

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.

In medical care cervical cancer screening is important because it enables the detection of precancer and cancer at an early stage. By adequate treatment after a screening-detected lesion it helps to reduce the mortality related to cervical cancer. Worldwide, many millions of women have smears taken at a more or less regular base and of these, approximately 7% are abnormal, and follow-up is thus required.As this represents an important cost in medical health care and has serious consequences for the affected women, it is important to have uniform and clear guidelines to allow an optimal follow-up and clinical management. A system for the uniform reporting of cervical cytology has been designed by the National Cancer Institute (U.S.A.) and resulted in the Bethesda System 1991. The present paper and the terminology used are based on the Bethesda System revised in 2001. It explains the guidelines, based on the 2001 Bethesda System and the 2004 consensus guidelines for the management of women with cervical cytological abnormalities, as developed by the members of the Board of the Belgian Society of Clinical Cytology, and adapted to the Belgian situation.

Objective: To assess the role of human papillomavirus (HPV) testing and cytology as predictors of residual/recurrent disease after treatment of high-grade cervical intraepithelial lesions., Methods: One hundred and thirty-eight women with cervical intraepithelial neoplasia (CIN) grade 2/3 lesion on biopsy were included in a prospective follow-up study in Belgium and Nicaragua. All women were treated with loop electrosurgical excision procedure (LEEP) and follow-up visits took place at 6 weeks, 6 months, 1 year and 2 years. During these visits, a Papanicolaou (Pap) smear test was taken, colposcopy was performed and specimens were collected for HPV testing. Cytology, high-risk (HR) HPV presence, persistent HR HPV infection and combinations of these tests at different time points during follow-up were correlated with histologically confirmed residual/recurrent disease., Results: Thirteen patients (9%) developed residual/recurrent disease during follow-up. Abnormal cytology at 6 weeks after treatment was significantly correlated with residual/recurrent disease. Nine of thirty-seven patients with abnormal cytology at 6 weeks had recurrent disease versus three of seventy with a normal cytology [odds ratio (OR): 7.2; 95% confidence interval (CI): 1.8-28.5; P = 0.003). Sensitivity of this test was 75.0%, specificity 70.5%. Combining abnormal cytology and the presence of HR HPV within the first 6 months after treatment gave the best correlation with residual/recurrent disease: of the 54 women with abnormal cytology and/or HR HPV presence within the first 6 months, 11 developed residual/recurrent disease (OR 10.2; 95% CI: 2.2-48.3). Sensitivity of this combination was 84.6% and specificity 65.0%., Conclusion: Cytology remains the cornerstone in the early follow-up after LEEP for CIN lesions of the cervix. HPV testing can add value as it increases the sensitivity of cytology in concomitant testing within the first 6 months.

Galectins are increasingly the focus of biomedical research. Although they are involved at different stages in inflammation, data on galectins in colitis remain scarce. The aim of this study was to determine and compare the expression of galectins in acute and chronic experimental colitis in mice. Immunohistochemistry for galectins-1, -3 and -4 was performed on colon tissue from C57BL/6 and BALB/c mice with acute dextran sodium sulphate colitis and from 129 Sv/Ev IL-10 knock-out (IL-10(-/-)) mice. From these three mouse strains, we first detected major differences in galectin expression related to the genetic background in the control animals. With regard to inflammation, chronic colitis in IL-10(-/-) mice was associated with increased galectin-4 expression; in contrast with the two other models, no galectin-1 and -3 alterations were observed in IL-10(-/-) mice. Acute colitis in C57BL/6 and BALB/c mice showed increased galectin-3 expression in the lamina propria and the crypt epithelium, together with a decreased nuclear expression. These results suggest an involvement of galectins in the development and perpetuation of colonic inflammation and illustrate that the choice of the mouse strain for studying galectins might influence the outcome of the experiments.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first step in the degradation of tryptophan, an essential amino acid. During inflammation IDO can be induced in different cell types resulting in local tryptophan depletion. This inhibits T cell proliferation and may induce apoptosis. High expression of IDO was previously found in inflammatory bowel disease and is thought to represent a mechanism for downregulation of the local immune response. Our aim is to investigate the expression pattern of IDO in normal and inflamed murine and human intestinal mucosa. Immunohistochemical staining for IDO was performed on paraffin sections of colon of two mouse models for colitis and their controls and on paraffin sections of human ileum and colon in normal and two different inflammatory conditions, namely inflammatory bowel disease and diverticulitis. IDO immunohistochemistry showed similar results in murine and human tissue. In normal, as well as in inflamed mucosa, some mononuclear cells, fibroblasts and endothelial cells were positive for IDO. In inflamed mucosa a specific expression pattern of epithelial IDO was found where epithelial cells flanking ulcers or bordering crypt abscesses showed high IDO expression. Moreover, in human intestinal inflammation, IDO was expressed in ulcer associated cell lineage. Since bacterial invasion is more pronounced in erosions and in crypt abscesses and since IDO activity and the resulting local tryptophan depletion can cause growth arrest of several tryptophan-dependent microorganisms, IDO expression in the vicinity of interruptions of the epithelial barrier may point to a role for IDO as a local anti-infectious agent. Furthermore, expression of IDO at the margin of ulcerations and in the reparative ulcer-associated cell lineage suggests involvement of IDO in repair processes.

Background: Genetically modified Lactococcus lactis secreting interleukin-10 (IL-10) has been demonstrated to provide localized delivery of a therapeutic agent through active in situ synthesis in murine colitis. At present, many aspects of the exact mechanism by which the beneficial effect of the IL-10-producing L. lactis on the mucosa is mediated remain to be clarified., Methods: Our aim was to determine the interaction of L. lactis with the intestinal mucosa. Therefore, we administered IL-10-producing L. lactis to healthy mice and in 2 mouse models of chronic colitis. Paraffin sections of ileum and colon samples were examined with confocal and transmission electron microscopy. Ileum and colon homogenates were prepared after flushing and after removal of mucus layer and epithelium. These homogenates and homogenates of mesenteric lymph nodes and spleen were plated on agar and immunoblotting for L. lactis and IL-10 was performed., Results: Both confocal and electron microscopy showed the presence of lactococci in inflamed intestinal mucosa of mice with colitis. We recovered viable bacteria that could still produce IL-10 from homogenates of inflamed ileum and colon of which mucous and epithelial layers were removed. We did not find lactococci in mesenteric lymph nodes or in the spleen of mice with colitis., Conclusions: This study demonstrates uptake of IL-10-secreting L. lactis by the paracellular route in inflamed mucosal tissue. We suggest that IL-10 production by L. lactis residing inside the mucosa in the vicinity of responsive cells can improve the local action of interleukin-10 in inflamed tissue and the efficiency of the treatment.

The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer's patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer's ring. Immunohistochemistry for clusterin in human Peyer's patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer's patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.

Aim: To assess the clearance rate of human papillomavirus (HPV) after out-patient treatment of cervical intraepithelial neoplasia (CIN)., Methods and Results: A total of 122 Nicaraguan women with HPV DNA-positive and histologically confirmed CIN lesions were included in the study. Fifty-five patients with CIN1 and 67 with CIN2-3 were treated by cryotherapy and loop electrosurgical excision procedure (LEEP), respectively. Follow-up visits were scheduled at 6 weeks, 6 months, 1 year and 2 years. Investigations included cytology, HPV DNA testing and colposcopy/biopsy if needed. The clearance rate of HPV was calculated by multivariate logistic regression. Immediately after treatment, a pronounced decrease in presence of HPV was observed in both groups, with a significantly higher clearance in the LEEP group than in the cryotherapy group (P = 0.019). Subsequently, clearance continued over time and was similar between the cryotherapy group and the LEEP group (P = 0.73). Approximately the same detection rates were obtained for persistence of all HPV types and for high-risk types separately: 43.9, 37.6, 29.9 and 17.7% in the cryotherapy group and 24.9, 20.3, 15.3 and 8.4% in the LEEP group at 6 weeks, 6 months, 1 year and 2 years, respectively., Conclusions: Out-patient treatment of precancerous lesions of the cervix usually results in clearance of HPV. Both LEEP and cryotherapy are highly effective methods of eradicating HPV. HPV DNA testing may have added value in the follow-up of patients.

Objectives: To determine the prevalence of high risk human papillomavirus (HPV) types in Nicaraguan women with histological proved pre-neoplastic and neoplastic cervical lesions, and to assess its potential impact on preventive strategies., Methods: 206 women with histopathological confirmed cervical lesions (CIN I or worse) were screened for HPV DNA on a liquid based cytology sample, using an HPV short fragment polymerase chain reaction based assay. HPV positive samples were genotyped with a reverse hybridisation line probe assay (Lipa). HPV negative samples were re-analysed using type specific real time polymerase chain reaction., Results: Of all lesions CIN II or worse, 12% tested negative. Prevalence of high risk HPV increased from 48.1% in cervical intraepithelial neoplasia I (CIN I) to 94.7% in invasive squamous cervical carcinoma (SCC). The most prevalent high risk HPV types were, in order of prevalence rate, HPV 16, 58, 31 and 52. HPV 16 and/or HPV 31 were present in 63.2% of SCC cases., Conclusion: Targeting HPV 16 and 31 with prophylactic vaccines could possibly have an important impact on the incidence of invasive cervical carcinoma in Nicaragua. Further research is needed to define the oncogenic potential of other high prevalent HPV genotypes. Meanwhile, primary prevention and cervical cancer screening programmes should be optimised.

Objectives: In this study, we focus on the prevalence and occurrence of different anogenital human papillomavirus (HPV) genotypes in a first abnormal cervical screening test, and correlate HPV genotyping with the cytological diagnosis on thin-layer liquid-based preparations in routine gynaecological screening., Methods: Out of 780 abnormal smears, 513 tested positive for HPV. All 25 different HPV types were identified by Line Probe Assay., Results: The prevalence of high-risk HPV types increased from 72% in atypical squamous cell of undetermined significance to 94.5% in high-grade intra-epithelial lesion (HSIL). Co-infection with multiple HPV types was predominantly found in HSIL (35.8%). In the HSIL group the most common HPV types were 16, 52, 51 and 31; type 18 was rarely present., Conclusion: The role of types 31, 51 and 52 should be considered in future studies on vaccine development.

Background: Crohn's disease is associated with an increased number of macrophages in ileal and colonic mucosa. Data on macrophages in gut mucosa of patients with spondyloarthritis (SpA) are scarce., Objective: To investigate macrophages and other antigen presenting cells in gut mucosa from patients with SpA and Crohn's disease, given the relationship between both entities., Methods: Biopsy specimens from patients with SpA, Crohn's disease, ulcerative colitis, and from controls were immunohistochemically stained with different markers for macrophages and dendritic cells. Slides were scored semiquantitatively on a four point scale., Results: SpA and Crohn's disease were associated with large numbers of CD68+ macrophages. Colon mucosa of both patients with SpA and Crohn's disease, but not ulcerative colitis, showed increased numbers of macrophages expressing the scavenger receptor CD163., Conclusions: Macrophages expressing the scavenger receptor CD163 are increased in colonic mucosa in SpA and in Crohn's disease, highlighting the relationship between these entities. The increased number of CD163+ macrophages in colon mucosa of patients with SpA suggests this is another argument for a role of macrophage scavenger receptors in this group of diseases.

Purpose: Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m- (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis., Patients and Methods: Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices., Results: Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P =.012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered., Conclusion: In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.

Background: University of Wisconsin (UW) solution (Viaspan) is currently used to preserve organs from nonheartbeating donors. Histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) is of proven efficacy in experimental pancreas preservation, but its efficacy in combined warm ischemia (WI) and cold ischemia (CI) is unknown. The viability of HTK-preserved porcine pancreatic grafts was assessed after various periods of WI and compared with grafts flushed and preserved with UW solution., Methods: A total of 14 pigs were used: G1 (n=4, UW) and G2 (n=4, HTK) with 15-min WI and 16-hr cold storage; G3 (n=3, UW) and G4 (n=3, HTK) with 30-min WI and 16-hr cold storage., Results: All animals in G1 and G2 were normoglycemic, whereas only 66% of pancreases were functioning in G3 and G4. HTK perfusion was associated with increased wet weight. Transient hyperinsulinemia was noted in all the groups on postoperative day 1 (mean range: 8.9-12.4 microU/L). Postoperative serum amylase and lipase were more pronounced in G3 and G4. However, HTK-stored grafts exhibited less evidence of biochemical pancreatitis as compared with UW-stored grafts on the first postoperative day in the group with 15-min WI. Mean K values of intravenous glucose tolerance tests on postoperative day 14 were similar in both groups. Vascular congestion was uniformly observed and was considered a typical feature of WI., Conclusions: Porcine pancreatic grafts are viable after 16-hr CI following 15-min WI in this experimental nonheartbeating donor model. HTK solution seems to provide reliable graft function in this setting and to be equivalent to UW.

The objective of this study was to investigate CD163+ macrophages in the synovial membrane of patients with spondyloarthropathy (SpA). Immunohistochemistry was performed on synovium of 17 SpA and 18 rheumatoid arthritis (RA) patients, on colonic biopsies of 16 SpA patients and ten healthy controls, and on paired synovial biopsies of eight SpA patients, before and after anti-TNFalpha therapy. Phenotype and cytokine production were analysed by flow cytometry. CD163+ macrophages were increased in the synovial lining and sublining in SpA versus RA, as well as in colonic lamina propria in SpA versus controls. The number of CD163+ macrophages in the synovial sublining correlated with C-reactive protein levels and erythrocyte sedimentation rate. Paralleling the increase of CD163, HLA-DR was increased in the synovial lining and sublining of SpA. In contrast, the co-stimulatory molecules CD80 and CD86 and the dendritic cell markers CD1a and CD83 were scarce in SpA synovium. Flow cytometry indicated that CD163+ macrophages expressed high levels of HLA-DR and could produce in vitro tumour necrosis factor alpha (TNFalpha) but not interleukin-10 (IL-10). Finally, anti-TNFalpha therapy in vivo induced a decrease of CD163+ macrophages in the synovial lining and sublining. In conclusion, macrophages expressing the scavenger receptor CD163 are increased in synovium and in colonic mucosa in SpA, highlighting the relationship between joint and gut in this disease. The correlation with inflammatory parameters, the expression of HLA-DR, the production of TNFalpha but not IL-10, and the reduction by anti-TNFalpha therapy support a role for CD163+ macrophages in the synovial inflammation in SpA., (Copyright 2002 John Wiley & Sons, Ltd.)

The E-cadherin-catenin complex is important for the maintenance of epithelial architecture. We studied its expression in Crohn disease, ulcerative colitis, acute ileitis, and controls. Immunohistochemical stainings for E-cadherin, alpha-catenin, beta-catenin and gamma-catenin were performed. E-cadherin messenger RNA (mRNA) was detected using riboprobes. In active inflammation, there was up-regulation of the complex. In particular, epithelium adjacent to ulcers showed increased expression of protein and mRNA, but in ulcer-associated cell lineage, the intensity of staining was weak to negative. In focal inflammation, up-regulation was found in affected areas. Reparative epithelium growing over denuded areas showed weaker expression. Since structural or functional perturbation in any of the molecules of the E-cadherin-catenin complex results in loss of intercellular adhesion, the preexistent epithelium may benefit from up-regulation to try to maintain its normal architecture under inflammatory conditions. Reduced expression in reparative epithelium and ulcer-associated cell lineage could facilitate the motility of these cells.

Objective: Previously an upregulation of E-cadherin and its associated molecules alpha-catenin, beta-catenin and plakoglobin has been demonstrated in clinically overt inflammatory bowel disease (IBD). The aim of this study was to investigate the expression of the E-cadherin/catenin complex in subclinically inflamed bowel mucosa from spondyloarthropathy (SpA) patients., Methods: Ileal and colonic biopsy specimens from 19 SpA patients with subclinical inflammatory gut lesions and from seven controls were stained with monoclonal antibodies against E-cadherin, beta-catenin and plakoglobin and a polyclonal antibody against alpha-catenin. E-cadherin mRNA was detected using a riboprobe. Inflammation was histologically classified into acute, chronic active and chronic quiescent forms., Results: In acute and chronic active bowel inflammation of SpA patients, upregulation of the E-cadherin/catenin glycoprotein complex could be observed. Chronic lesions in a quiescent state did not show such an upregulation. Furthermore, chronic inflammation was associated with an increase in E-cadherin mRNA., Conclusions: As some of the SpA patients with subclinical gut inflammation develop IBD, upregulation of the E-cadherin/catenin complex in inflamed bowel mucosa from SpA patients may point to early cellular changes in the development of IBD. However, at present it cannot be excluded that increased E-cadherin/catenin complex expression is a bystander phenomenon of active inflammation.

The idiopathic hypereosinophilic syndrome is empirically defined as the presence of prolonged eosinophilia without identifiable underlying cause, and with evidence of end-organ dysfunction. Virtually any organ system may be involved, most frequently the heart, the central and peripheral nervous system, the lungs and the skin. We report two cases where the diagnosis of hypereosinophilic syndrome was proposed although the classic criteria were not met. In the first case total peripheral eosinophil counts were relatively low, but pathological evidence clearly showed infiltration of eosinophils in the damaged tissues. An hypothesis to explain this discrepancy is formulated. The second case did not fulfil the first feature either, although the clinical presentation and disease course corresponded well with other cases reported in the literature. The delay in diagnosis was caused by early institution of corticosteroids, clearing all evidence of eosinophil involvement in the observed tissue damage.