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BACKGROUND Crohn’s disease (CD) is a complex disorder resulting from the interaction of genetic, environmental, and microbial factors. The pathogenic process may potentially affect any segment of the gastrointestinal tract, but a selective location in the terminal ileum was reported in 50% of patients. AIM To characterize clinical sub-phenotypes (colonic and/or ileal) within the same disease, in order to identify new therapeutic targets. METHODS 14 consecutive patients undergoing surgery for ileal CD were recruited for this study. Peripheral blood samples from each patient were collected and the main polymorphisms of the gene Card15/Nod2 (R702W, G908R, and 1007fs) were analyzed in each sample. In addition, tissue samples were taken from both the tract affected by CD and from the apparently healthy and disease-free margins (internal controls). We used a multiplex gene assay in specimens obtained from patients with ileal localization of CD to evaluate the simultaneous expression of 24 genes involved in the pathogenesis of the disease. We also processed surgery gut samples with routine light microscopy (LM) and transmission electron microscopy (TEM) techniques to evaluate their structural and ultrastructural features. RESULTS We found a significant increase of Th17 (IL17A and IL17F, IL 23R and CCR6) and Th1 (IFN-γ) gene expression in inflamed mucosa compared to non-inflamed sites of 14 CD patients. DEFB4 and HAMP, two genes coding for antimicrobial peptides, were also strongly activated in inflamed ileal mucosa, suggesting the overwhelming stimulation of epithelial cells by commensal microbiota. IFN-γ and CCR6 were more expressed in inflamed mucosa of CD patients with ileal localization compared with patients with colonic localization suggesting a more aggressive inflammation process in this site. Morphological analysis of the epithelial lining of Lieberkün crypts disclosed enhanced release activity from goblet mucocytes, whereas the lamina propria contained numerous cells pertaining to various lines. CONCLUSION We observed that the expression of ileal genes related to Th1 and Th17 activity is strongly activated as well as the expression of genes involved in microbiota regulation.

Background. CARD15/NOD2 is the most significant genetic susceptibility in Crohn’s disease (CD) even though a relationship between the different polymorphisms and clinical phenotype has not been described yet. The study is aimed at analyzing, in a group of CD patients undergoing surgery, the relationship between CARD15/NOD2 polymorphisms and the clinical CD behavior after a long-term follow-up, in order to identify potential clinical biomarkers of prognosis. Methods. 191 surgical CD patients were prospectively characterized both for the main single nucleotide polymorphisms of CARD15/NOD2 and for many other environmental risk factors connected with the severe disease form. After a mean follow-up of 7.3 years, the correlations between clinical features and CD natural history were analyzed. Results. CARD15/NOD2 polymorphisms were significantly associated with younger age at diagnosis compared to wild type cases ( p < 0.05 ). Moreover, patients carrying a 3020insC polymorphism presented a larger Δ between diagnosis and surgery ( p = 0.0344 ). Patients carrying an hz881 and a 3020insC exhibited, respectively, a lower rate of responsiveness to azathioprine ( p = 0.012 ), but no difference was found in biologic therapy. Finally, the risk of surgical recurrence was significantly associated, respectively, to age at diagnosis, to familial CD history, to diagnostic delay, to arthritis, and to the presence of perioperative complications. Conclusions. 3020insC CARD15 polymorphism is associated with an earlier CD onset, and age at CD diagnosis < 27 years was confirmed to have a detrimental effect on its clinical course. In addition, the familiarity seems to be connected with a more aggressive postoperative course. Finally, for the first time, we have observed a lower rate of responsiveness to azathioprine in patients carrying an hz881 and a 3020insC.

Summary Crohn's disease (CD) is a multifactorial immunologically mediated disease. In this study we explored, for the first time, the efficacy of the Multiplex Gene Assay technology for detecting mRNA expression profile of 24 selected CD related genes in endoscopic biopsies and surgical specimens from CD patients with colonic localization of the disease. The polymorphisms of genes most frequently associated with CD were also analysed in DNA samples from the same patients. The analysis of endoscopic samples showed increased expression of 7 genes in inflamed mucosa compared to non-inflamed mucosa and suggests the activation of the autophagy process and of a Th17 adaptive response. The analysis of surgical specimens showed increased expression of 16 genes in inflamed tissue compared to non-inflamed internal controls and revealed the activation of immune-adaptive Th17 response in association with a Th1 response. Furthermore, an increased expression of genes involved in ionic transport and signal transduction was found in inflamed mucosa compared to non-inflamed internal controls. This study confirms the activation of Th17 and Th1 adaptive-immune response also in colonic CD. It should be stressed that these responses have been disclosed in biopsy tissue, while only Th17 differentiation is revealed in endoscopic tissue. Interestingly, the polymorphisms analysis revealed that a homozygous genotype is associated to a more complicated clinical course.

Crohn&rsquo, disease (CD) patients are at high risk of postoperative recurrence and new tools for the assessment of disease activity are needed to prevent long-term complications. In these patients, the over-production of ROS generated by inflamed bowel tissue and inflammatory cells activates a pathogenic cascade that further exacerbates inflammation and leads to increased oxidative damage to DNA, proteins, and lipids. We measured the products of protein/lipid oxidation and the total antioxidant capacity (ferric reducing ability of plasma, FRAP) in the serum of CD patients with severe disease activity requiring surgery with the aim to characterize their redox status and identify associations between oxidative stress-related markers and their clinical characteristics. At the systemic level, CD was associated with increased levels of protein and lipid oxidation products when compared to healthy volunteers, even though the FRAP values were similar. Advanced oxidation protein product (AOPP) levels showed the highest difference between patients and the controls (11.25, 5.02&ndash, 15.15, vs. 1.36, 0.75&ndash, 2.70, median, interquartile range, p &lt, 0.0001) and the analysis of receiver operating characteristic (ROC) curves, indicated for AOPP, the best area under the curve (AUC) value for CD prediction. Advanced glycated end-products (AGEs) were also significantly higher in CD patients (p &lt, 0.01), which is of interest since AOPP and AGEs are both able to activate the membrane receptor for advanced glycation end products (RAGE) involved in inflammatory diseases. Thiobarbituric acid reactive substance (TBARS) levels were significantly higher in CD patients with ileal localization and aggressive disease behavior, in smokers, and in patients suffering from allergies. In conclusion, our data indicate that circulating oxidative stress biomarkers may be attractive candidates as disease predictors as well as for clinical or therapeutic monitoring of CD. Our results also suggest that AOPP/AGEs and RAGE signaling may represent a pathogenic factor and a potential therapeutic target in CD.

Background: The feasibility of minimally invasive approach for Crohn's disease (CD) is still controversial. However, several meta-analysis and retrospective studies demonstrated the safety and benefits of laparoscopy for CD patients. Laparoscopic surgery can also be considered for complex disease and recurrent disease. The aim of this study was to investigate retrospectively the effect of three minimally invasive techniques on short- and long-term post-operative outcome. Patients and Methods: We analysed CD patients underwent minimally invasive surgery in the Digestive Surgery Unit at Careggi University Hospital (from January 2012 to March 2017). Short-term outcome was evaluated with Clavien–Dindo classification and visual analogue scale for post-operative pain. Long-term outcome was evaluated through four questionnaires: Short Form Health Survey (SF-36), Gastrointestinal Quality Of Life Index (GIQLI), Body Image Questionnaire (BIQ) and Hospital Experience Questionnaire (HEQ). Results: There were 89 patients: 63 conventional laparoscopy, 16 single-incision laparoscopic surgery and 10 robotic-assisted laparoscopy (RALS). Serum albumin

Three types of serous products were detected in the syncytial cutaneous glands of the leptodactylid tungara frog, Engystomops pustulosus: type Ia, granules with wide halos and variable density cores; type Ib, high density granules without halos; and type II, vesicles containing a finely dispersed product. Ultrastructural evidence revealed that these products were manufactured by different serous gland types and excluded that they represented different steps in the secretory cycle of a single gland type. Indeed, secretory maturation affecting the products released by the Golgi apparatus proceeded through different mechanisms: confluence (vesicles), interactions between syncytium and secretory product (type Ib granules), and a combination of both processes (type Ia granules). In conclusion, this investigation of secretory maturation was shown to be a suitable approach for the identification of serous gland polymorphism and demonstrated that the tungara frog belongs to the minority of anuran species characterized by this peculiar morpho-functional trait. Anat Rec, 298:1659–1667, 2015. © 2015 Wiley Periodicals, Inc.

Secretory activity of serous (poison) cutaneous glands was studied with light and transmission electron microscopy over the ontogenetic cycle of the Black-legged Dart Frog Phyllobates bicolor. Serous gland activity includes two steps: biosynthesis and maturation. Biosynthesis is performed by the rough endoplasmic reticulum, smooth endoplasmic reticulum, and Golgi apparatus, and follows the usual pathways of exocrine secretory processes. Maturation is typical of anuran serous glands, which accumulate their products in syncytial secretory units. Maturation involves merging processes between secretory deposits (vesicles and/or granules) distally from the Golgi stacks, as well as an increase in the surface area between the syncytial cytoplasm and the secretory vesicles/granules. In P. bicolor, this interface area increases through the formation of peculiar cytoplasmic outgrowths. These outgrowths allow transport processes involved in maturational changes of secretory product. During maturational storage, the secretory products of P. bicolor acquire a repeating substructure of spherical to short tubular subunits. This recurrent pattern is seen in cutaneous poisons of several anuran species, regardless of their phylogenetic relationships. Among anurans, condensing serous cutaneous products into repeating aggregations of subunits appears to be a successful strategy, which allows intracytoplasmic accumulation of pharmacologically active and/or toxic molecules through various post-Golgian processes. The present findings provide evidence of a novel repertory of cytological activities that stress the large diversity of the maturational pathways leading to the peculiar repeating arrangement of secretory subunits.

Three types of serous products were detected in the syncytial cutaneous glands of the leptodactylid tungara frog, Engystomops pustulosus: type Ia, granules with wide halos and variable density cores; type Ib, high density granules without halos; and type II, vesicles containing a finely dispersed product. Ultrastructural evidence revealed that these products were manufactured by different serous gland types and excluded that they represented different steps in the secretory cycle of a single gland type. Indeed, secretory maturation affecting the products released by the Golgi apparatus proceeded through different mechanisms: confluence (vesicles), interactions between syncytium and secretory product (type Ib granules), and a combination of both processes (type Ia granules). In conclusion, this investigation of secretory maturation was shown to be a suitable approach for the identification of serous gland polymorphism and demonstrated that the tungara frog belongs to the minority of anuran species characterized by this peculiar morpho-functional trait.

The embryonic horny layer of vertebrates contains distinctive cell lines that account for the remarkable, diverse secretory performances of their cutaneous apparatuses. Aquatic classes (jawless, cartilaginous and bony fishes) possess single gland cells, scattered among ordinary epidermal cells, that manufacture proteinaceous or mucous substances. This dichotomy is retained in adult amphibians, although the secretory cells in anamnionic tetrapode begin to be arranged in complex glands located in the loose dermis. In the Amniota, intensive keratinisation involving the epidermis results in the loss of the ancestral secretory lines, accompanied by the onset of a novel, lipid- producing gland type, which in mammals is flanked by the exclusive sweat gland apparatus.In this concise up-to-date review, we attempt to phylogenetically narrow the large variety of secretory structures in vertebrate skin into a few basic schemes, in the light of the close relationships between environmental challenges and ecolo...

It has been shown that transforming growth factor-beta (TGF-beta) has a potent stimulatory effect on the growth of chondrosarcoma cells in vitro. In order to examine the production of this family of growth factors and their receptors in vivo, we studied the expression of TGF-beta isoforms 1, 2, and 3 and of TGF-beta receptor types I and II (TGF-betaRI and TGF-betaRII) in a series of 24 chondrosarcomas of bone using immunohistochemistry and reverse-transcription polymerase chain reaction analysis. For comparison, five enchondromas and five osteochondromas were also analyzed. TGF-beta1 was expressed in 3 benign lesions (30%) and 18 chondrosarcomas (75%), with a significantly higher expression in grade-2 and -3 tumors than in grade-1 tumors ( P=0.002). TGF-beta2 was identified in 8 benign lesions (89%) and 21 chondrosarcomas (87.5%), with increased expression in grade-2 and -3 chondrosarcomas in comparison with grade-1 tumors ( P=0.05). TGF-beta3 was detected in 6 benign lesions (60%) and 17 chondrosarcomas (70.8%), with no significant differences between chondrosarcomas of different histologic grade ( P=0.6). Twenty-three chondrosarcomas (95.8%) expressed both TGF-beta receptor types I and II. Reverse-transcription polymerase chain reaction analysis performed on ten chondrosarcomas confirmed the presence of low or absent levels of TGF-beta1 and -beta2 mRNA in grade-1 chondrosarcomas, while grade-2 chondrosarcomas presented high levels of transcript of both cytokines. High levels of TGF-betaRI and RII mRNA were also detected. Chondrosarcomas with TGF-beta1 and TGF-beta2 overexpression (20% of tumor cells) had a significantly higher expression of the cell proliferation marker MIB-1 ( P=0.006 and P=0.0003, respectively), while no significant correlation was found between TGF-beta3 expression and proliferative activity ( P=0.5). When TGF-beta isoform and receptor expression were examined with respect to disease-free survival, TGF-beta1 overexpression was significantly associated with a shorter disease-free survival ( P=0.004, log-rank test). Our data indicate that TGF-beta isoforms are produced by neoplastic cells of chondrosarcomas and could have a potential role as autocrine growth stimulators in these neoplasms.

We described guest nematodes in serous and lipid skin glands in two anuran species from the Old and New Worlds: Rana camerani and Phyllomedusa hypochondrialis, respectively. These glands are involved in three types of response: a) invaginations from the periluminal plasma membrane which accommodate the “round worms”; b) secretory release into such hosting chambers; c) migration of macrophages from the periglandular stroma to the secretory unit. There are no obvious signs suggesting the glands are harmed by the nematodes, nor are the latter affected by defence reactions, either secretory or cell-mediated, of their hosts. Nematodes are natural tracers which allow analysis of the inner space system (lumina as well as interstitia) in the glands. Furthermore, they provide the opportunity for functional characterisation of anuran serous glands under intrusion stress, revealing peculiar reactive traits, namely a pliable periluminal plasma membrane and inducible processes of secretory release.

To compare bone density with polymorphisms in the calcitonin receptor (CTR) and vitamin D receptor (VDR) genes in 50 patients with juvenile idiopathic arthritis and 80 matched controls.Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry at the lumbar spine. Genomic DNA was isolated from EDTA blood samples by standard procedures. Polymerase chain reaction was performed using genomic DNA and 100 pmol of each oligonucleotide primer for VDR and CTR genes. Products from genomic PCR were digested by Alu I enzyme for CTR polymorphism and Fok I enzyme for VDR polymorphism.In the total population, higher prevalence of CC genotype (41.5%) for the CTR gene and FF genotype (59.8%) for the VDR gene was found, in agreement with data for Caucasian populations. No significant differences in distribution of CTR and VDR genotypes were observed between patients and controls. However, patients with TT genotype had lumbar BMD (L-BMD) that was lower in comparison to those with CC genotype (p = 0.04). For VDR gene polymorphism, we observed that patients with ff genotype had lower L-BMD in comparison with FF genotype (p = 0.02). Patients with heterozygosity for the 2 genotypes showed intermediate L-BMD. The differences in L-BMD among these groups did not seem to be related to corticosteroid therapy.Our data suggest that patients with particular VDR and CTR genotypes may be at higher risk to lose bone mass.

Calcium-sensing receptor (CaSR) is a plasma membrane protein that regulates tubular reabsorption of Ca. To establish its role in idiopathic hypercalciuria, the association of urinary Ca excretion with the polymorphisms of CASR gene has been studied in healthy subjects and in hypercalciuric and normocalciuric Ca stone formers. CASR exon 7 single nucleotide polymorphisms (SNP), G/T at codon 986, G/A at codon 990, and C/G at codon 1011, were evaluated by PCR amplification and direct sequencing in 97 normocalciuric stone formers, 134 hypercalciuric stone formers, and 101 normocalciuric healthy controls. Four haplotypes were defined on the basis of CASR gene SNP: haplotype 1 was characterized by the most frequent sequence; haplotypes 2, 3, or 4 by the presence of a single polymorphic variant at codon 986, 990, or 1011, respectively. The relative risk of hypercalciuria was calculated with multinomial logistic regression and was significantly increased only in individuals carrying haplotype 3 (Odds ratio, 13.0 [95% confidence interval, 1.7 to 99.4]). Accordingly, Ca excretion was higher in subjects bearing haplotype 3, whereas those bearing haplotype 2 showed a slight increase of plasma Ca concentration. Multiple regression analysis showed that haplotype 3 explained 4.1% of the total variance of Ca excretion and 12.6% of the variance explained by the variables considered in the study. In conclusion, CASR gene could be a component of the complex genetic background regulating Ca excretion. Arg990Gly polymorphism could facilitate activation of CaSR and increase Ca excretion and susceptibility to idiopathic hypercalciuria.

The authors examined the distribution of tumor growth factor-beta (TGF-beta) isoforms and receptors in 35 giant cell tumor (GCT) of bone in comparison with a group of benign giant cell-containing lesions of bone, including 5 aneurysmal bone cysts, 2 cases of brown tumor of hyperparathyroidism, 3 nonossifying fibromas, and 7 cases of giant cell reparative granuloma. The results of immunohistochemical analysis of GCT showed a complete absence of TGF-beta1 expression in both mononuclear tumor cells and giant cells. Only reactive bone present within the tumor showed an intense immunoreactivity. Transforming growth factor-beta2 and TGF-beta3 were detected in the majority of cases (97.1% and 82.8%, respectively), whereas TGF-beta receptor type I (TGF-beta RI) and type II (TGF-beta RII) were diffusely expressed in all cases. Reverse transcription-polymerase chain reaction (RT-PCR) analysis performed on 10 GCTs with specific oligonucleotide primers demonstrated the presence of mRNA transcripts for TGF-beta1, 2, 3, and for TGF-beta RI and RII. Quantitative measurements of TGF-beta1 in conditioned media from primary cultures of GCT showed undetectable or very low amounts of the cytokine (0-23 pg/mL). The results of immunohistochemical analysis showed that all giant cell-containing lesions of bone were at least focally positive for the 3 isoform of TGF-beta, with positivity present both in osteoclast-like giant cells and mononuclear cells, and diffusely positive for TGF-beta RI and RII. Reverse transcription-polymerase chain reaction analysis conducted on samples from 3 nonossifying fibromas and 1 giant cell reparative granuloma confirmed the expression of the corresponding mRNA. In conclusion, according to the current data, GCT of bone can be distinguished from other giant cell-containing lesions of bone on the basis of the absence of TGF-beta1 expression at the protein level, which appears to be the result of posttranslational regulation processes.

In vitro studies have demonstrated that the extracellular matrix modulates the cell phenotype. In the present study we have investigated in vitro proalpha1(I) collagen mRNA expression in a human pre-osteoclastic cell line (FLG 29.1 cells) in basal condition and after various stimuli. In addition, in order to evaluate the effect of cell-cell interactions on collagen type I mRNA expression, we have cultured the human pre-osteoclastic cells FLG 29.1 with either the human osteoblast-like cell line Saos-2 or the bovine bone endothelial cell line BBE. We showed that the FLG 29.1 cells express proal (I) collagen mRNA, whose expression is modulated by phorbol esters (TPA). Co-culturing FLG 29.1 cells with either Saos-2 or BBE cells induced decrease of proalpha1(I) collagen mRNA expression.

Background The minor histocompatibility antigens (mHags) are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD) in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed. Methods We used sequence specific primer (SSP) PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm) assay that may help identification of HA-1 alleles without oligonucleotide probes. Results We report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO) during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed. Conclusion The possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp) amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability.