Type 2 Transglutaminase in Coeliac Disease: A Key Player in Pathogenesis,Diagnosis and Therapy

Type 2 transglutaminase (TG2) is the main autoantigen in coeliac disease (CD), a widespread inflammatory enteropathy caused by the ingestion of gluten-containing cereals in genetically predisposed individuals. As a consequence, serum antibodies to TG2 represent a very useful marker in CD diagnosis. However, TG2 is also an important player in CD pathogenesis, for its ability to deamidate some Gln residues of gluten peptides, which become more immunogenic in CD intestinal mucosa. Given the importance of TG2 enzymatic activities in CD, several studies have sought to discover specific and potent inhibitors that could be employed in new therapeutical approaches for CD, as alternatives to a lifelong gluten-free diet. In this review, we summarise all the aspects regarding TG2 involvement in CD, including its enzymatic reactions in pathogenesis, the role of anti-TG2 antibodies in disease management, and the exploration of recent strategies to reduce deamidation or to use transamidation to detoxify gluten.     

Inclusion of Methoxybutropate in β-and Hydroxypropyl β-Cyclodextrins: Comparison of Preparation Methods

Interactions between methoxybutropate and β-cyclodextrin or hydroxypropyl β-cyclodextrin and the possibility of obtaining inclusion complexes have been evaluated by phase solubility diagram, HPLC, DSC, and x-ray diffractometry. Solid inclusion complexes were prepared by spray drying, kneading, and solid dispersion. The dissolution profiles of the obtained powders were studied in order to define the most appropriate cyclodextrin preparation method and molar ratio to use in the production of methoxybutropate inclusion complexes     

A single-pinhole confocal laser scanning microscope for 3-D imagingof biostructures

Confocal laser scanning microscopy (CLSM) is a powerful three-dimensional (3-D) tool at the microscopic level, both for functional and for structural studies. The confocal microscope provides a practical and well-established method for obtaining microscopic volume imaging. It has become more and more evident that it permits one to obtain a large amount of information on biological systems in terms of their (slow) dynamics and 3-D structure. Moreover, when linked to bioimage-oriented interfaces, CLSM adds the ability to examine the spatial distribution and the temporal behaviour of specific cellular components to the already intriguing properties of confocal imaging. In this article, we discuss a new ultracompact confocal microscope based on a single-pinhole optical architecture     

Liquid phase reactions catalyzed by Fe- and Mn-sulphated ZrO2

Fe- and Mn-promoted ZrO₂–SO₄ (ZS) powders were prepared both by a single step sol–gel reaction and by impregnation of the zirconia hydrous precursor. The samples were calcined at 890 K and characterized for the structural (XRD) and morphological features (BET method). Surface functionalities were investigated by XPS, ¹H MAS NMR and FTIR analyses. The liquid medium catalytic activity was tested with respect to both the esterification of benzoic acid to methylbenzoate and the benzylation of toluene. The acidity features appeared not to be significantly different among the various samples while all surface characterizations showed a lower affinity to retain water by the metal-promoted samples with respect to ZS, the more so in the case of iron-containing samples. The presence of Mn reduced the surface area and depressed the catalytic activity. Iron-doped catalysts appeared, instead, to be more efficient than ZS especially for the benzylation of toluene.     

High levels of cerebrospinal fluid IgM binding to myelin basic protein are associated with early benign course in multiple sclerosis

We assessed human myelin basic protein (MBP) binding IgM levels in CSF. MBP is the most studied putative antigen in multiple sclerosis (MS) and immune responses against it may be involved in the demyelination process. We also correlated these levels with EDSS score and other parameters of disease progression and prognosis, both at the time of CSF analysis and during follow-up. CSF IgM anti-MBP levels were assayed by measuring total IgM levels with solid-phase ELISA in CSF samples from 66 patients with relapsing-remitting MS, 11 subjects without neurological diseases, 20 patients with non-inflammatory neurological diseases and 7 patients with lymphocytic meningitis, before and after immunoabsorption with human MBP. Confirmation of IgM binding specificity was performed by immunoblotting of positive CSF samples onto MBP coated-nitrocellulose sheets. Clinical evaluation (disability score, number and time of attacks) was performed during a mean follow-up of 2.7 +/- 1.1 years. 23 of the 66 relapsing-remitting MS patients (33.8%) had elevated IgM anti-MBP levels. In this patient subgroup, IgM anti-MBP levels correlated with the IgM index (r = 0.71; P = 0.0001), but not with CSF/serum albumin (r = 0.08; P = 0.72). In the first year of follow-up, patients with low IgM anti-MBP suffered from more numerous attacks than those with elevated levels (0.86 +/- 0.63 versus 0.43 +/- 0.58; P = 0.017). Patients with high IgM binding to MBP had a first attack during follow-up in a significantly higher time than those with low binding (28.87 +/- 4.7 versus 17 +/- 2.6 months, respectively; P = 0.005) and reached a decrease of 0.5 EDSS point significantly faster than those with low IgM (16.17 +/- 1.2 versus 29.7 +/- 2.6 months, respectively; P = 0.0002). A similar significant finding was observed when the time to reach low disability score (EDSS < or = 2.0) was analyzed (10.7 +/- 2.57 +/- 3.3 months, respectively; P = 0.014). These findings demonstrate that in a subgroup of MS patients, elevated CSF levels of IgM anti-MBP are associated with early favorable course and therefore suggest that IgM binding to MBP could be a possible prognostic marker in relapsing-remitting MS to select early MS patients for future trials.     

Evaluation of advanced glycation end products and carbonyl compounds in patients with different conditions of oxidative stress

Advanced glycation end products (AGE) and dicarbonyl compounds accumulate in serum and tissues of patients with diabetes and chronic renal failure. Pentosidine, free pentosidine, glyoxal and methylglyoxal have been evaluated in plasma of diabetic patients with poor metabolic control at baseline and after the improvement of glycemic levels, and in plasma and peritoneal dialysate of patients with renal failure before and after 12 h of peritoneal dialysis. In diabetic patients, acceptable metabolic control was unable to normalize levels of pentosidine (after 2 and 10 months), glyoxal and methylglyoxal (after 2 months). In patients with end-stage renal disease, mean values of pentosidine, free pentosidine, glyoxal and methylglyoxal decreased in plasma after dialysis. No pentosidine or free pentosidine were present in the peritoneal dialysate at time 0, but were found after 12 h of peritoneal dialysis; glyoxal and methylglyoxal decreased after 12 h of dialysis. So, glyoxal and methylglyoxal, already present in the dialysis fluid, can react with the peritoneal matrix protein, giving a reason for the gradual loss of peritoneal membrane function often observed in patients undergoing long-term peritoneal dialysis.     

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.     

Temporal occurrence of Cryptosporidium in the Manila clam Ruditapes philippinarum in northern Adriatic Italian lagoons

In order to evaluate the temporal occurrence of Cryptosporidium oocysts in Ruditapes philippinarum clams bred along the northeastern Italian Adriatic coast and molecularly characterize the isolates, 2,160 specimens (180 clams per month) were collected from three clam farms from January to December 2004. Two farms (sites A and B) were located in Venice (Chioggia, Veneto region) and one (site C) in the Marano Lagoons (Friuli Venezia Giulia region). Clams from 36 pools (i.e., one pool of 60 clams per month per site) were subjected to a high-sensitivity seminested PCR assay specific for a 360-bp diagnostic region internal to the Cryptosporidium spp. outer wall protein gene. Positive amplicons were sequenced and analyzed. Cryptosporidium DNA was found in clams from seven pools (sites A and B) during 1 month of sampling at site A and 6 months of sampling at site B, with Cryptosporidium hominis and Cryptosporidium parvum being detected. The expected infection rate of the clams was 0.36%. Site B showed a significantly higher expected infection rate (1.15%) than did the other sites (A = 0.14% and C = 0%). Given its high sensitivity and specificity, this seminested PCR assay can be considered a reliable tool for detecting and distinguishing species within the Cryptosporidium genus. The seasonal pattern of contamination and the related public health risks are of particular concern.