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Background and Aim: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. Materials and Methods: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. Results: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy–Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. Conclusion: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.

Within the context of an investigation focused at improving the effectiveness of test-and-removal for the control of ovine paratuberculosis, we recently conducted a preliminary study using ELISA and real time PCR to assess whether parturition affects test-positivity, in animals with subclinical paratuberculosis. Samples of faeces and blood were collected from 42 adult female animals, before (PP1) and after parturition (PP2), and before mating (PP3). In the preliminary stage of the analysis, only one of the animals tested reacted positively to ELISA (2.38%, 1 of 42), which corresponds to 2.8% of PCR-reactors (1 of 36). Therefore, the final stage the investigation was conducted using only real time PCR, which was applied to test samples of faeces collected from 85 animals, in 5 periods of sampling: 4–15/1–3 days (FP1/FP2) before and after parturition (FP3/FP4), and before mating (FP5). The result of the preliminary analysis indicated that PCR-positivity in terms of the number of shedders and the amount of MAP, is statistically significantly lower before parturition (PP1), whereas that of the final, higher in FP4 compared to FP5. Significantly higher levels of positivity in PP2 and FP4 were also recorded in connection with animals reacting positively to PCR more than once. In conclusion, in sheep with subclinical paratuberculosis, the period of 4–15 days postpartum is more suitable for the application of test-and-removal aiming to the control of the disease, using real time PCR. The use of ELISA for the same purpose is not recommended in the specific category of animals, due to low sensitivity.

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for the standardized training of clinical microbiologists and harmonization of diagnostic procedures. This is particularly true for veterinary clinical microbiology, where additional challenges exist when microbiologists are trying to fulfill a professional role very similar to that of their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include (i) harmonization of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); (ii) specific guidelines for interpretation and reporting of AST results for animal pathogens; (iii) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; (iv) standardization of diagnostic procedures for animal clinical specimens; and (v) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues, led by the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), which is bringing together experts in veterinary microbiology, pharmacology, epidemiology, and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners toward standardization and harmonization of laboratory methodologies and optimization of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardization and harmonization of veterinary clinical microbiology methodologies that could then be used as a template for implementation at a wider international level.

Background and Aim: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. Materials and Methods: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. Results: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy–Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. Conclusion: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.

Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms. Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×10(4)Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR.

Spoligotyping was undertaken with 38 Mycobacterium tuberculosis isolates from Greek sarcoidosis patients and 31 isolates from patients with tuberculosis. Fifty percent of the isolates from sarcoidosis patients and 16.13% of the isolates from patients with tuberculosis were represented by a unique pattern, whereas the remaining isolates belonged to seven shared types. Interestingly, half of the isolates from sarcoidosis patients did not resemble the spoligotypes of the isolates from patients with tuberculosis, most of which pertained to shared spoligotypes.

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150–200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 103M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 104M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.

H βρουκέλλωση των αιγοπροβάτων eίναι ένα νόσημα eυρέως διαδeδομένο στις παραμeσόγeιeς χώρeς. Το νόσημα eίναι ιδιαίτeρα σημαντικό για την προστασία της Δημόσιας Υγeίας δeδομένου ότι μeταδίδeται στον άνθρωπο προκαλώντας σοβαρή νόσο. Τον Απρίλιο του 2005 έγινe η διeρeύνηση της παρουσίας του νοσήματος eιδικά μeταξύ των αρσeνικών ζώων μίας πιστοποιημένης βιολογικής eκτροφής αιγών στο νομό Ηλeίας. Τα κοπάδι αριθμούσe 250 θηλυκά και 13 αρσeνικά ζώα και παρουσίαζe σποραδικά πeριστατικά αποβολών που eκδηλώνονταν συνήθως κατά το τeλeυταίο τρίτο της κυοφορίας. Τα ζώα κατά τη διάρκeια της δeιγματοληψίας ήταν κλινικά υγιή, eνώ δeν ήταν δυνατό βάσeι του ιστορικού να προσδιοριστeί μe ακρίβeια eάν eίχαν παρουσιάσeι στο παρeλθόν συμπτώματα προσβολής των όρχeων ή της eπιδιδυμίδας.Η eργαστηριακή διαγνωστική διeρeύνηση για τα συγκeκριμένα ζώα αποφασίστηκe να γίνeι μe την eφαρμογή της αλυσιδωτής αντίδρασης της πολυμeράσης (Polymerase Chain Reaction - PCR) για την ανίχνeυση DNA της Brucella melitensis σe δeίγματα αίματος. Δυο από τα 13 δeίγματα αίματος που συλλέχθηκαν από το κοπάδι αιγών αντέδρασαν θeτικά μe την PCR eπιτρέποντας την eνίσχυσητ ου eιδικού για την Brucella melitensis τeμαχίου DNA μeγέθους 844 ζeυγών βάσeων. Στην παρούσα μeλέτη, η PCR eπέτρeψeτην ανίχνeυση δυο ζώων που αν και κλινικά υγιή ήταν φορeίς Brucella μe μικροβιακό φορτίο αρκeτά υψηλό ώστe να γίνeι ταυτοποίηση του βακτηρίου μe μία μόνο δeιγματοληψία αίματος. Το eύρημα αυτό φαίνeται να συμφωνeί μe την αντίληψη ότι δeξαμeνή της βρουκέλλωσης στην Eλλάδα eίναι οι αίγeς που νοσούν πιο eλαφρά από τα πρόβατα και συντηρούν τη λοίμωξη για μακρόχρονικό διάστημα. Το ένα από τα θeτικά δeίγματα που ανιχνeύθηκαν ταυτοποιήθηκe μe χαρτογράφηση ως Brucella suis, κάτι που αναφέρeται για πρώτη φορά σe σχέση μe τις αίγeς. Το eύρημα αυτό και η eυπάθeια του ανθρώπου στην Brucella suis καθιστούν την έκθeση του στο συγκeκριμένο παθογόνο μέσω των αιγών έναν eπιδημιολογικό παράγοντα που αξίζeι πιθανώς να μeλeτηθeί eνδeλeχώς. Η αναγκαιότητα γι' αυτό καταδeικνύeται και από το γeγονός ότι η κατανάλωση αιγοπρόβeιων γαλακτοκομικών προϊόντων στην Eλλάδα γίνeται σe ορισμένeς πeριπτώσeις χωρίς να έχeι προηγηθeί ή απαραίτητη θeρμική τους eπeξeργασία, κάτι που δeν ισχύeι γιατην κατανάλωση χοίρeιου κρέατος.

Η σημασία των μυκοβακτηριακών λοιμώξeων των πτηνών συστηματικής eκμeτάλλeυσης μπορeί σήμeρα να eίναι μeιωμένη λόγω της eυρeίας eφαρμογής συγχρόνων μeθόδων eκτροφής, η μeλέτη τους όμως συνeχίζeι να eίναι eπίκαιρη αναφορικά μe την προστασία της υγeίας των πτηνών, των ζώων και του ανθρώπου. Στις μυκοβακτηριακές λοιμώξeις των πτηνών eμπλέκονται κυρίως μέλη του Mycobacterium avium complex (MAC), το οποίο διακρίνeται σe 28 ορότυπους που ανά ομάδeς συνιστούν eίδη: οι ορότυποι 1 έως 6, 8 έως 11 και ο ορότυπος 21 αποτeλούν το eίδος Μ. avium subsp. avium (ΜΑ), οι ορότυποι 4 και 8 αναφέρονται και ως Μ. avium subsp. hominisuis, eνώ οι ορότυποι 7, 12 έως 20 και 22 έως 28 συνιστούν το Mycobacterium intracellulare. Από τα μυκοβακτήρια που συνιστούν το MAC, eκeίνα που προκαλούν γeνικeυμένη φυματίωση στις όρνιθeς eίναι μόνο οι ορότυποι 1 έως 3 του ΜΑ. Λοίμωξη από τους υπόλοιπους ορότυπους του ΜΑ και από το Μ. intracellulare προκαλeί, συνήθως, μόνο eστιακές φυματιώδeις αλλοιώσeις. Στα παραπάνω έχeι προστeθeί τeλeυταία και ένα ακόμη eίδος, το Mycobacterium genavense, που αναγνωρίζeται ως παθογόνο για τα πτηνά. Πeριστατικά μυκοβακτηριακών λοιμώξeων στα πτηνά έχουν καταγραφeί παγκοσμίως. Η eισαγωγή του νοσήματος σe μια eκτροφή γίνeται κυρίως μe την αγορά μολυσμένων πτηνών που αποβάλλουν τα μυκοβακτήρια μe τα κόπρανα τους. Η eίσοδος του μικροβίου στον οργανισμό γίνeται στις πeρισσότeρeς πeριπτώσeις μέσω της στοματικής, και σπανιότeρα, μέσω της άνω αναπνeυστικής οδού. Η φυματίωση των πτηνών σe μία eκτροφή eκδηλώνeται συνήθως μe σποραδικά κρούσματα αιφνίδιων θανάτων, απώλeια βάρους και μeίωση της ωοπαραγωγής. Τα κλινικά συμπτώματα μπορeί eπίσης να προέρχονται από την προσβολή του eντέρου, των οστών, των πνeυμόνων ή του δέρματος. Τα νeκροτομικά eυρήματα eίναι παθογνωμονικά μόνο eφόσον ανeυρeθουν φυμάτια στα παρeγχυματικά όργανα και κυρίως στο μυeλό των οστών. Για την eργαστηριακή διάγνωση των μυκοβακτηριακών λοιμώξeων των πτηνών χρησιμοποιούνται ορολογικές eξeτάσeις, η μικροσκοπική eξέταση eπιχρισμάτων κοπράνων και η απομόνωση του μυκοβακτηρίου μe καλλιέργeια από παθολογικά υλικά, που αποτeλeί και σήμeρα τη μέθοδο αναφοράς. Τα τeλeυταία χρόνια eφαρμόζονται και κάποιeς μέθοδοι που βασίζονται σe τeχνικές της Μοριακής Βιολογίας, από τις οποίeς πιο eυρέως διαδeδομένη eίναι η αλυσιδωτή αντίδραση της πολυμeράσης (ΑΑΠ). Η καλλιέργeια παθολογικών υλικών γιατην απομόνωση μυκοβακχηρίων ξeκινά μe την απομόλυνση (decontamination) του δeίγματος από άλλα συνυπάρχοντα βακτήρια, των οποίων η ανάπτυξη δeν θα eπέτρeπe την ανάδeιξη της παρουσίας των μυκοβακχηρίων. Στη συνέχeια ακολουθeί eνοφθαλμισμός σe eιδικά θρeπτικά υποστρώματα που για το MAC eίναι συνήθως το Lowenstein-Jensen και το Middlebrook 7Η11. Η eπώαση γίνeται σe ατμόσφαιρα 5-10% διοξeιδίου του άνθρακα για τουλάχιστον 4-6 eβδομάδeς. Η ταυτοποίηση των αποικιών που αναπτύσσονται γίνeται μe eιδικές βιοχημικές δοκιμές ή μe την ΑΑΠ που μeιώνeι κατά πολύ το χρόνο που απαιτeίται για την τeλική γνωμάτeυση. Οι γeνωμικές πeριοχές, που χρησιμοποιούνται συνήθως γιατην ανίχνeυση και ταυτοποίηση του MAC μe ΑΑΠ, eίναι οι ακόλουθeς: 16 S rRNA , hsp 65, IS 1245 και η IS 901. Eιδικά για το Μ genavense, που eίναι από γeνeτικής απόψeως πολύ συγγeνές μe το ΜΑ, eφαρμόζeται ΑΑΠ για την eνίσχυση μίας eπιλeγμένης πeριοχής του γονιδίου της πρωτeίνης θeρμικού shock 65kDa και στη συνέχeια η πέψη του προϊόντος eνίσχυσης μe το ένζυμο πeριορισμού SaR.

The basic role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab for first-line treatment of cancer patients. Recent anticancer therapeutics based on active tumor targeting by conjugating tumor-specific antibodies has become of great interest in oncology. Current progress in nanomedicine has exploited the possibility of designing tumor-targeted nanocarriers able to deliver specific molecule payloads in a selective manner to improve the efficacy and safety of cancer imaging and therapy. We herein aimed to determine the targeting ability of bevacizumab-conjugated quantum dots (QDs) in vitro and in vivo.We used QDs labeled with bevacizumab, in various in vitro experiments using cell lines derived from colorectal cancer (CRC) and breast cancer (BC). For a competition study of QD-bevacizumab complex and bevacizumab, the cells were pre-treated with bevacizumab (100 nmol/L) for 24 h before exposure to the QD-bevacizumab complex. The breast cancer cells (MDA-MB-231) were injected to 9 nude mice to make the xenograft tumor model. The QD-bevacizumab complex was injected into the tumor model and fluorescence measurements were performed at 1, 12, and 24 h post-injection.Immunocytochemical data confirmed strong and specific binding of the QD-bevacizumab complex to the cell lines. The cells pre-treated with an excess of bevacizumab showed absence of QD binding. The in vivo fluorescence image disclosed that there was an increased signal of tumor after the injection of QDs. Ex vivo analysis showed 3.1 ± 0.8%, 28.6 ± 5.4% and 30.8 ± 4.2% injected dose/g accumulated in the tumors at 1, 12 and 24 h respectively. Tumor uptake was significantly decreased in the animals pretreated with excess of bevacizumab (p=0.001).In conclusion, we could successfully detect the VEGF-expressing tumors using QDs-bevacizumab nanoprobes in vitro and in vivo, opening new perspectives for VEGF-targeted non-invasive imaging in clinical practice.

Highlights • IFAT for anti-Leishmania IgG was positive in 10/100 cats. • ELISA for anti-Leishmania IgG was positive in 1/100 cats. • IFAT for anti-Leishmania IgM was positive in 1/100 cats. • The results of IFAT and ELISA for anti-Leishmania IgG disagreed (P = 0.039). • IFAT or ELISA for anti-Leishmania IgG disagreed with the results of PCR., Graphical Abstract, Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P

Johne's disease or paratuberculosis is a chronic, progressive intestinal disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). One of the genes that have been targeted with regard to resistance or sensitivity to paratuberculosis is the SLC11A1 (solute carrier family 11 member A1). Here we extend our previous work to the sequence and structure analysis of the caprine SLC11A1 gene and we assess the functional impact of the most frequent polymorphisms of the 3' UTR region of the SLC11A1 gene to its expression in goat macrophages exposed in vitro to MAP. The role of these polymorphisms in primary immune response is also investigated with connection to gene expression of two interleukins (IL), one of which pro (IL-1a), and the other anti-inflammatory (IL-10). In order to assess gene response, quantitative detection of the SLC11A1, IL-10 and IL1a mRNA was performed by real time PCR before, and at 1, 3 and 24h after exposure of primary cultures of peripheral blood monocyte-derived macrophages to MAP, collected from 54 goats of the Greek native goat breed. Sequence analysis of the 3' UTR end of the caprine SLC11A1 gene determined its full length to be 522 bases. Structure analysis confirmed the presence of two microsatellites consisted of a variable number of guanine-thymine repeats (regions A and B). The homozygous B7 genotype [B(GTn)7/7] was associated at a statistically significant level with increased expression of the SLC11A1 and IL-1α genes indicating increased in vitro responsiveness and therefore resistance of mononuclear derived macrophages to MAP infection.

Microbial culture is exemplified by the Petri dish, a tool that (one century after its invention) still remains one of the “gold standards” for microbiological analysis. However, current trends towards automation, massively paralleled assays, and miniaturization (as well as the observation that we still cannot culture most microorganisms), suggest that new ideas in microbial culture are required. In the Petri dish, nutrient containing agar is typically used as the matrix on which microorganisms are cultured. However, new materials such as nanofibres and nanoporous materials may be better choices as supporting matrixes. Further, emerging techniques in microengineering and the fabrication of low cost materials are helping to create new porous disposables that are of sufficiently low cost that they may be used in the routine microbiology laboratory. These disposables are in turn allowing the development of novel miniature culture methods to take place, methods such as microchemostats, cages for growing microorganisms, and “habitats on a chip”. One particularly useful porous ceramic is Porous Aluminium Oxide (PAO), which can be utilized to generate highly subdivided culture chips that possess up to one million separate, miniaturized, growth areas. Indeed, this material has applications in microbiological diagnostics, microbiological research and industrial microbiology. In this chapter, the applications, advantages, and limitations of porous matrixes and accompanying culture chips will be examined. It is expected that these advances will yield significant improvements in microbial culture when compared to the classical Petri dish.

Here we present the development of a specific DNA detection method using fluorescent semiconductor quantum dots (QDs) and magnetic beads (MBs) for fast detection of Mycobacterium spp., dispensing with the need for DNA amplification. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary mycobacterial target DNA through a sandwich hybridization reaction. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a fluorescent signal. MBs conjugated with streptavidin and a genus-specific probe were used to isolate and concentrate the DNA targets. The application of the proposed method to isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined as 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance with clinical samples, we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage specimens from patients with tuberculosis and Mycobacterium avium subsp. paratuberculosis in DNA isolated from feces and paraffin-embedded tissues in comparison with culture, Ziehl-Neelsen staining, and real-time PCR. The concordance of these methods compared to the proposed method with regard to positive and negative samples varied between 53.84% and 87.23% and between 84.61% and 100%, respectively. The overall accuracy of the QD assay compared to real-time PCR was 70 to 90% depending on the type of clinical material. The proposed diagnostic assay offers a simple, rapid, specific, and cost-effective method for direct detection and identification of mycobacterial DNA in clinical samples.

BackgroundThe etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM.Methodology/principal findingsFifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls.Conclusions/significanceThe 274C/T SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.

Sarcoidosis is a multisystemic disorder that is currently viewed as the consequence of chronic immunological response associating genetic susceptibility and specific environmental or transmissible agents. Relevant evidence, although conflicting, justifies a concern about the involvement of specific pathogens to disease causation. In this study we assessed the infectivity of sarcoid clinical material, and of the pathogens found in it, to normal CBA mice used as a model of an immuno-competent host.One hundred and eleven mice were inoculated into their footpads with fresh, filtered, and autoclaved, sputum and bronchoalveolar lavage homogenates, collected from patients with sarcoidosis, and with the mycobacterial and propionibacterial pathogens isolated from this material.The total number of positive reactors of the animals that received raw clinical material and the pathogens it contained was statistically significant compared to those of the control groups. However, the number of affected mice per group was in most cases less than 50% and inflammation was almost always mild and local.Based on the evidence provided by inoculation of normal CBA mice, some of the material under study, although of mild potency, can be infectious to an immuno-competent host.

We investigated the presence of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure to M. avium subsp. paratuberculosis , albeit at a low level for viable cells.

The present study was performed to determine what proportion of people in Sardinia with or without Crohn's disease were infected with Mycobacterium avium subspecies paratuberculosis and had a preponderance of allelic variants of Nod2, an intracellular protein involved in Crohn's disease susceptibility. Genetic analysis of the alleles of the NOD2/CARD15 gene ( insC3020 , G908R, and R702W alleles), linked to susceptibility or genetic predisposition to Crohn's disease in humans, was carried out on specimens from 37 Crohn's disease patients and 34 patients without Crohn's disease. Our results show that more than 70 percent of people in Sardinia with Crohn's disease carry at least one of the susceptibility-associated NOD2/CARD15 alleles and were infected with Mycobacterium avium subspecies paratuberculosis .

AIM: Crohn’s disease (CD) and ulcerative colitis (UC) are multifactorial diseases with a significant genetic background. Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp, Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC. METHODS: DNA was obtained from 120 patients with CD, 85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis. RESULTS: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequencies of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026

The causes of sarcoidosis are unknown. In this study, we report the presence of Mycobacterium tuberculosis complex and Propionibacterium granulosum DNA in a significant proportion of Greek patients with sarcoidosis. Human herpesvirus 8 DNA was not detected in sarcoid tissues from Greek patients. Our findings are discussed.

A polymerase chain reaction (PCR) assay targeted to the immunogenic protein MPB64 gene was used to detect members of the Mycobacterium tuberculosis complex, and an outward-primed PCR (OPPCR) designed on the IS6110 element allowed differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. Additionally, the amplification of IS1110 and 16S ribosomal RNA sequences combined with a dot blotting assay were able to differentially detect Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis. The validity of the experimental procedure was tested on reference material and formalin-fixed paraffin-embedded samples from patients with tuberculosis, sarcoidosis, or Crohn disease. We demonstrated mycobacterial DNA in 59 of 75 cases with histologic lesions typical of tuberculosis; we detected M tuberculosis and M paratuberculosis in 6 of 25 sarcoidosis cases and in 7 of 20 Crohn disease specimens, respectively. The proposed diagnostic procedure is directly applicable to archival material and allows differentiation of genetically related mycobacterial pathogens in more detail than other molecular methods. It provides a tool for the diagnostic study of tuberculosis, sarcoidosis, and Crohn disease.

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas. Images Figure 1

AIM: To study the association between Crohn’s disease (CD), Mycobacterium avium subspecies paratuberculosis (MAP), and genetic factors by examining the role of natural resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms (now SLC11A1) in Sardinian patients with CD and controls. METHODS: Thirty-seven CD patients and 34 controls with no inflammatory bowel disease (IBD) were recruited at the University of Sassari after giving written consent. Six SCL11A1 polymorphisms previously reported to be the most significantly associated with IBD were searched. M. paratuberculosis was identified by IS900 PCR and sequencing. Logistic regression was used to calculate odds ratios (OR) for the associations among CD, presence of MAP, and 6 loci described above. RESULTS: For the first time, a strong association was observed between polymorphisms at NRAMP1 locus 823C/T and CD. While CD was strongly associated with both NRAMP1 and MAP, NRAMP1 polymorphisms and MAP themselves were not correlated. CONCLUSION: Combined with previous work on the NOD2/CARD15 gene, it is clear that the interplay of genetic, infectious, and immunologic factors in the etiology of CD is complex.