Development of a droplet digital PCR assay to detect illicit glucocorticoid administration in bovine

Glucocorticoids are often used illegally in food-producing animals for the growth promotion of livestock animals. In accordance to official chemical methods for glucocorticoid detection, an animal is declared as non-compliant when a residue is identified in the sample. Neverthless, growth promoting molecules can often escape identification due to their rapid elimination or due to the use of non-detectable new generation drugs. Therefore, an indirect screening method able to detect the biological effect of long-term administration of low doses of dexamethasone and prednisolone on livestock has been developed to support official methods. As already described, FKBP5 (FKBP prolyl isomerase 5) expression in bovine thymus is regulated by glucocorticoids, and this specific regulation can be exploited in an indirect screening assay. In the present study, male veal calves and young bulls were considered in three different trials in which estradiol, dexamethasone, and prednisolone were administered alone or in combination with Revalor-200 subcutaneous pellets. Thoracic thymus was sampled from all animals and molecular analysis was performed. A duplex droplet digital PCR assay with EvaGreen(®) was employed to detect the target gene expression using absolute quantification. The developed droplet digital PCR assay was precise, showing intra- and inter-assay mean coefficient of variation values of about 6.16% and 3.17%, respectively. It was also highly specific (100%) with Youden’s index of 76.92% and 53.57% applied to veal calves and young bulls, respectively. The lowest detection limit in which the target gene expression level was kept constant, was 0.05 ng/μl of cDNA with 1 copies/μL and 0.5 copies/μL for target and reference gene, respectively. This study establishes the basis for using a digital PCR-based assay as an efficient test to identify animals illegally treated with glucocorticoids

Condizioni per collaborare. Scuole e terzo settore nella costruzione della comunità educante

Il presente contributo parte dalle specifiche esperienze dei progetti OpenSpace e FA.C.E. e dal processo Lab Sperone Children per esplorare i caratteri del rapporto tra scuola e terzo settore a livello territoriale, contestualizzandolo nelle vicende che configurano l’attuale scenario. Procede quindi puntualizzando alcuni apprendimenti e proposte riguardanti, da un lato, la creazione di figure dedicate al lavoro di rete e, dall’altro, alla configurazione istituzionale nella quale le alleanze educative territoriali possono prosperare. Nelle conclusioni si tratteggiano brevemente alcune ulteriori condizioni necessarie affinché ciò possa verificarsi

The involvement of T regulatory lymphocytes in a cohort of lupus nephritis patients: a pilot study

T regulator lymphocytes (Tregs) play a key role in the maintenance of immune tolerance and in the development of autoimmune diseases. Expression of Foxp3 is specific for Tregs, and can be used for the identification of these cells. This study investigated the variations of Tregs Foxp3? in the kidney biopsies inflammatory infiltrate of different lupus nephritis classes compared to that of ANCA glomerulonephritis, acute tubulointerstitial nephritis and nephroangiosclerosis. Sections of paraffin-embedded tissue have been stained by immunohistochemistry with anti-CD3 and anti-FoxP3 antibodies. We find that the ratio of FoxP3?/CD3? cells is significantly lower in patients with lupus nephritis class IV and in patients with vasculitides than in the course of nephroangiosclerosis, tubulointerstitial nephritis and lupus nephritis class V. The data presented herein demonstrate a decrease of FoxP3? Treg cells in the inflammatory infiltrate of lupus nephritis, particularly during the most active phases of lupus nephritis, as observed in the course of a IV class nephritis

POT1 mutations are frequent and associated with Ki-67 index in canine diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) represents one of the most frequent and deadliest neoplasia in dogs worldwide and is characterized by a remarkable degree of clinical heterogeneity, with poor chances to anticipate the outcome. Even if in the last years some recurrently mutated genes have been identified, the genetic origin of canine DLBCL (cDLBCL) is not yet completely understood. The aim of the present study was to assess the prevalence of POT1 mutations in cDLBCL and to elucidate the role of such gene in the pathogenesis of this tumor. Mutations in POT1 were retrieved in 34% of cases, in line with previous reports, but no significant associations with any clinico-pathological variable were identified. Likewise, POT1 mutations are not predictive of worse prognosis. Interestingly, Ki-67 index was significantly higher in dogs harboring POT1 mutations compared to wild-type ones. These results suggest that POT1 mutations may exert their pathogenic role in cDLBCL by promoting cellular proliferation

Phenotypical Characterization and Clinical Outcome of Canine Burkitt-Like Lymphoma

In dogs, Burkitt-like lymphoma (B-LL) is rare tumor and it is classified as a high-grade B-cell malignancy. The diagnosis is challenging because of the similar histologic appearance with other histotypes, no defined phenotypical criteria and poorly described clinical aspects. The aim of the study was to provide a detailed description of clinical and morphological features, as well as immunophenotypical profile of B-LL in comparison with the human counterpart. Thirteen dogs with histologically proven B-LL, for which a complete staging and follow-up were available, were retrospectively selected. Immunohistochemical expression of CD20, PAX5, CD3, CD10, BCL2, BCL6, MYC, and caspase-3 was evaluated. Histologically, all B-LLs showed a diffuse architecture with medium to large-sized cells, high mitotic rate and diffuse starry sky appearance. B-phenotype of neoplastic cells was confirmed both by flow-cytometry and immunohistochemistry. Conversely, B-LLs were negative for BCL2 and MYC, whereas some cases co-expressed BCL6 and CD10, suggesting a germinal center B-cell origin. Disease stage was advanced in the majority of cases. All dogs received CHOP-based chemotherapy with or without immunotherapy. Despite treatment, prognosis was poor, with a median time to progression and survival of 130 and 228 days, respectively. Nevertheless, ~30% of dogs survived more than 1 year. An increased apoptotic index, a high turnover index and caspase-3 index correlated with shorter survival. In conclusion, canine B-LL shows phenotypical differences with the human counterpart along with features that might help to differentiate this entity from diffuse large B-cell lymphoma

Co-occurrence of genomic imbalances on Xp22.1 in the SHOX region and 15q25.2 in a girl with short stature, precocious puberty, urogenital malformations and bone anomalies

BACKGROUND: Mutations of SHOX represent the most frequent monogenic cause of short stature and related syndromes. The genetic alterations include point mutations and deletions/duplications spanning both SHOX and its regulatory regions, although microrearrangements are confined to either the downstream or upstream enhancers in many patients. Mutations in the heterozygous state have been identified in up to 60-80% of Leri-Weill Dyschondrosteosis (LWD; MIM #127300) and approximately 4-5% of Idiopathic Short Stature (ISS; MIM#300582) patients. Homozygous or compound heterozygous mutations as well as biallelic deletions of SHOX and/or the enhancer regions result in a more severe phenotype, which is known as Langer Mesomelic Dysplasia (LMD; MIM #249700). CASE PRESENTATION: A 17 year old girl, presented with severe short stature, growth hormone deficiency (GHD), precocious puberty, dorsal scoliosis, dysmorphisms and urogenital malformations. She was born with agenesis of the right tibia and fibula, as well as with a supernumerary digit on the left foot. Array comparative genomic hybridization (aCGH) analysis detected the presence of two distinct duplications on Xp22.1 flanking the SHOX coding sequence and involving its regulatory regions. An additional duplication of 1.6-2.5 Mb on 15q25.2 that included 13 genes was also identified. The girl was adopted and the parent's DNA was not available to establish the origin of the chromosome imbalances. CONCLUSIONS: The complex phenotype observed in our patient is probably the result of the co-occurrence of rearrangements on chromosomes Xp22.1 and 15q25.2. The duplicated region on 15q25.2 region is likely to contain dosage-sensitive genes responsible for some of the clinical features observed in this patient, whereas the extreme short stature and the skeletal anomalies are likely attributable to the comorbidity of GHD and copy number variants in the SHOX region