Long-Term Follow-Up, Association between CARD15/NOD2 Polymorphisms, and Clinical Disease Behavior in Crohn's Disease Surgical Patients.

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.

Preliminary study of short- and long-term outcome and quality of life after minimally invasive surgery for Crohn's disease: Comparison between single incision, robotic-assisted and conventional laparoscopy.

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 <30 g/L (P = 0.031) resulted to be a risk factor for post-operative complications. HEQ had a better result for RALS (P = 0.019), while no differences resulted for SF-36, BIQ and GIQLI. CONCLUSIONS: Minimally invasive technique for CD is feasible, even for complicated and recurrent disease. Our study demonstrated low rates of post-operative complications. However, it is a preliminary study with a small sample size. Further studies should be performed to assess the best surgical technique.

Crohn's disease recurrence updates: first surgery vs. surgical relapse patients display different profiles of ileal microbiota and systemic microbial-associated inflammatory factors.

Background and aims: Crohn's disease (CD) pathogenesis is still unclear. Remodeling in mucosal microbiota and systemic immunoregulation may represent an important component in tissue injury. Here, we aim to characterize the ileal microbiota in both pathological and healthy settings and to evaluate the correlated systemic microbial-associated inflammatory markers comparing first-time surgery and relapse clinical conditions. Methods: We enrolled 28 CD patients at surgery; we collected inflamed and non-inflamed mucosa tissues and blood samples from each patient. Bacterial wall adherence was observed histologically, while its composition was assessed through amplicon sequencing of the 16S rRNA gene. In addition, we evaluated the systemic microRNA (miRNA) using quantitative real-time PCR amplification and free fatty acids (FFAs) using gas chromatography-mass spectroscopy. Results: The total number of mucosal adherent microbiota was enriched in healthy compared to inflamed mucosa. In contrast, the phylum Tenericutes, the family Ruminococcaceae, and the genera Mesoplasma and Mycoplasma were significantly enriched in the pathological setting. Significant microbiota differences were observed between the relapse and first surgery patients regarding the families Bacillaceae 2 and Brucellaceae and the genera Escherichia/Shigella, Finegoldia, Antrobacter, Gemmatimonas, Moraxella, Anoxibacillus, and Proteus. At the systemic level, we observed a significant downregulation of circulating miR-155 and miR-223, as well as 2-methyl butyric, isobutyric, and hexanoic (caproic) acids in recurrence compared to the first surgery patients. In addition, the level of hexanoic acid seems to act as a predictor of recurrence risk in CD patients (OR 18; 95% confidence interval 1.24-261.81; p = 0.006). Conclusions: We describe a dissimilarity of ileal microbiota composition comparing CD and healthy settings, as well as systemic microbial-associated inflammatory factors between first surgery and surgical relapse. We suggest that patterns of microbiota, associated with healthy ileal tissue, could be involved in triggering CD recurrence. Our findings may provide insight into the dynamics of the gut microbiota-immunity axis in CD surgical recurrence, paving the way for new diagnostics and therapeutics aimed not only at reducing inflammation but also at maintaining a general state of eubiosis in healthy tissue.

Analysis of Oxidative Stress-Related Markers in Crohn's Disease Patients at Surgery and Correlations with Clinical Findings.

Crohn' 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, 5.02-15.15, vs. 1.36, 0.75-2.70, median, interquartile range; p < 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 < 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.

Multiplex gene expression profile in inflamed mucosa of patients with Crohn's disease ileal localization: A pilot study.

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.

Bombesin promotes vasculogenesis and angiogenesis in chick chorio-allantoic membrane: A morphometric, structural, and ultrastructural study.

Experiments were performed on the chorio-allantoic membrane (CAM) of the chick to evaluate the effects of bombesin (BN) on vascular neoformation. In morphometrical assays, 10(-13)-10(-4) M BN promoted dose-dependent vascular development. Newly formed vessels converged toward the BN release site in a spoked wheel arrangement, suggesting a diffusion gradient mechanism. Structural and ultrastructural analysis of CAM specimens collected near the BN release site showed that both vasculogenetic and angiogenetic processes cooperated in vascular neoformation that involved committed cells from the mesenchyme (angioblasts) as well as endothelial cells. No pattern of vascular development was detected away from the BN release site. Findings from the present study emphasize the role of BN in vascular net development of respiratory organs.

Crohn's Colitis: Development of a multiplex gene expression assay comparing mRNA levels of susceptibility genes.

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.

Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes.

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.

Ultrastructural Evidence of Serous Gland Polymorphism in the Skin of the Tungara Frog Engystomops pustulosus (Anura Leptodactylidae).

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.

Transforming growth factor-beta isoform and receptor expression in chondrosarcoma of bone.

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.

Larval epidermis of the red eye tree frog Agalychnis callidryas (Anura, Hylidae): ultrastructural investigation on the Kugelzellen, specialized forms of the constitutive skein cell line.

An ultrastructural study was carried out on the epidermis of Agalychnis callidryas tadpoles during limb development. Larval epidermis consisted of four cell layers: basal, lower intermediate, upper intermediate, and surface or apical layers. Basal cells represented the stem compartment of intermediate cells: both belong to the skein cell (SC) lineage, described in several anuran species, on account of the conspicuous intracytoplasmic tonofilament bundles. Apical cells were secretory in nature and released mucus on the body surface. Intermediate SCs exhibited a hydrated central cytoplasm and peripheral tonofilament bundles. They closely resembled the epidermal ball-like cells, Kugelzellen (KZn) of Xenopus laevis tadpoles, and possibly shared their turgor-stiffness properties. In A. callidryas, the stratification of intermediated SCs on their stem cell layer provided the chance to study their cytodifferentiation in a suitable sequence, until basal cell differentiation shifted toward the keratinocyte lineage in premetamorphic stages. Present data assign A. callidryas to the anuran species with a constitutive SC population in larval epidermis, and demonstrate that KZn express the ultimate specialization of such cell line. SCs were arranged in the fashion of a random-rubble stone groundwork, and possessed long processes. These cytoplasmic outgrowths contained a tonofilament axial rod and held together contiguous cells. Ultrastructural findings suggest that this complex structure may impart compressive as well as sliding strengths to the larval epidermis, representing a possible adaption to the fresh water environment.

Association of low bone mass with vitamin d receptor gene and calcitonin receptor gene polymorphisms in juvenile idiopathic arthritis.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our data suggest that patients with particular VDR and CTR genotypes may be at higher risk to lose bone mass.

Influence of calcium-sensing receptor gene on urinary calcium excretion in stone-forming patients.

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.