A novel pancreatic tumour and stellate cell 3D co-culture spheroid model.

BACKGROUND: Pancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells. METHOD: A spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting. RESULTS: We found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids. CONCLUSIONS: We developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

Medical management and positive outcome after prolonged recumbency in a case of equine herpesvirus myeloencephalopathy.

A 17-year-old mare presenting with acute fever, weakness and bladder dysfunction was diagnosed with equine herpesvirus myeloencephalopathy (EHM). The mare become transiently recumbent, underwent parenteral fluid therapy, plasma infusion, steroidal/nonsteroidal anti-inflammatory drugs (SAID/NSAIDs) and bladder catheterization. After 10 days the mare was hospitalized. Neurological evaluation revealed ataxia and proprioceptive deficits mainly in the hind limbs. The mare was able to stand but unable to rise from recumbency or walk. Secondary complications included Escherichia coli cystitis, corneal ulcers and pressure sores. A full-body support sling was used for 21 days. Medical treatment included systemic antimicrobials, NSAIDs, gradual discontinuation of SAIDs, parenteral fluid therapy and bladder lavage. The mare tested positive for Varicellovirus equidalpha 1 (EHV-1) DNA in nasal swab and blood samples on day 13 and in urine samples on days 13 and 25 after the onset of fever. Neurological signs improved over a period of 34 days and the mare was discharged with mild hind limb weakness/ataxia. Secondary complications resolved within 2 weeks. At the eight-month follow-up, marked improvement in locomotory function had been achieved.

Spread of Toxoplasma gondii among animals and humans in Northern Italy: A retrospective analysis in a One-Health framework.

Toxoplasmosis occurs worldwide and is considered one of the most important food-borne parasitic zoonoses. The consumption of undercooked meat containing viable tissue cysts and ingestion of environmental oocyst are the most important sources of infection. The aim of this retrospective study was to evaluate the spread of Toxoplasma gondii in the province of Bologna (Emilia-Romagna region) in northern Italy, with a One Health approach, comparing seropositivity rates in different animal species and in humans over the last 19 and 4 years respectively. Analyses were performed on serological data collected over different periods at three separate locations: Istituto Zooprofilattico Sperimentale della Lombardia e della Emilia-Romagna (IZSLER); Veterinary University Hospital Clinical Pathology Service, Department of Veterinary Medical Sciences, University of Bologna; and Unit of Microbiology, St. Orsola Hospital, Bologna. Most relevant seropositivity rates observed in animals were 15.5% (wild boar), 25% (roe deer), 18.7% (goat), 29.9% (sheep), 9.7% (pigs), 42.9% and 21.8% in cat and dog, respectively. A comprehensive screening was conducted on a population of 36,814 individuals, revealing a prevalence of 20.4%. Among pregnant women, a frequence of 0.39% for active toxoplasmosis was observed. Despite certain limitations, this study provided valuable insights into the extensive distribution of this parasitic infection among diverse animal species and human populations in the province of Bologna. These findings underscore the importance of implementing consistent and proactive toxoplasmosis screening protocols during pregnancy, while emphasizing the critical need for adopting a One Health approach for effective control of this parasitic disease.

Detection of a virus related to betacoronaviruses in Italian greater horseshoe bats.

The association between coronaviruses and bats is a worldwide phenomenon and bats belonging to genus Rhinolophus are the reservoir host for several coronaviruses, including a large number of viruses closely related genetically to severe acute respiratory syndrome-coronavirus (SARS-CoV). We carried out a survey in colonies of Italian bats (Rhinolophus ferrumequinum) for the presence of coronaviruses. Two of 52 R. ferrumequinum captured from different Italian areas tested positive by reverse transcription-PCR for a fragment of RNA-dependent RNA polymerase (RdRp) gene of viruses related to Coronavirus. Phylogenetic analysis revealed close correlations between one of the positive samples and SARS-related CoV belonging to the genus Betacoronavirus.

Comparison of clinicopathological patterns of renal tubular damage in dogs with acute kidney injury caused by leptospirosis and other aetiologies.

In humans, leptospiral acute kidney injury (AKI) is characterised by tubulointerstitial involvement and renal electrolyte losses, impacting clinical presentation and case management. The aim of this study was to evaluate urine chemistry findings in dogs with leptospirosis in order to identify characteristic patterns of tubular damage associated with this disease. Dogs with intrinsic AKI caused by leptospirosis and by other aetiologies were prospectively enrolled. Clinical and clinicopathological variables, including serum and urine chemistry, fractional excretion (FE%) of electrolytes, and urinary neutrophil gelatinase-associated lipocalin (NGAL), were evaluated in both groups and compared statistically. Dogs with leptospirosis (n = 38) had significantly higher serum creatinine concentration than dogs with AKI caused by other aetiologies (n = 37). Serum potassium and glucose concentrations were comparable between groups. Dogs with leptospiral AKI had significantly higher FE of potassium (median 100%, range 20-480 vs. median 68%, range 5-300; P = 0.048), as well as higher magnitude of glucosuria (urine glucose to creatinine ratio, median 0.64, range 0-26 vs. median 0.22, range 0-13; P = 0.023) and frequency of positive glucose dipstick reaction (59% vs. 18%; P = 0.002), than dogs with AKI of other aetiologies. Additional markers of tubular damage considered in this study, including FE of other electrolytes and urinary NGAL, did not differ between groups. In conclusion, when compared to other aetiologies of intrinsic AKI, canine leptospirosis was characterised by increased glucosuria and kaliuresis.

Prospective evaluation of rapid point-of-care tests for the diagnosis of acute leptospirosis in dogs.

The early diagnosis of acute leptospirosis is still a major challenge in dogs. The aim of this prospective study was to evaluate the suitability of two in-clinic tests detecting anti-leptospiral IgM and IgG antibodies in diagnosing canine leptospirosis. The performances of the two rapid tests were compared to the microscopic agglutination test (MAT) carried out on acute sera and to diagnostic criteria adopted in this study to confirm leptospirosis infection (MAT upon admission, convalescent MAT and quantitative real-time PCR on blood and/or urine). The dogs were enrolled on the basis of reported exposure to known risk factors and clinical presentation (acute kidney injury and/or systemic inflammatory response syndrome with multi-organ damage). Eighty-nine dogs included in the study were sub-grouped on the basis of the results of the diagnostic criteria adopted: (1) confirmed leptospirosis cases (42/89 dogs); (2) negative leptospirosis cases (36/89 dogs); and (3) unconfirmed leptospirosis cases (11/89 dogs). The results supported the usefulness of the two rapid diagnostic tests as a first in-clinic screening tool for suspected leptospirosis; positive results in the in-clinic tests in dogs with suggestive clinical and laboratory signs strongly indicated acute leptospirosis, while negative results required additional diagnostic investigation to exclude the infection. Confirmatory tests recommended for canine leptospirosis are still necessary in addition to the use of rapid in-clinic tests.

Novel sequence variants of viral hexon and fibre genes in two dogs with canine adenovirus type 1-associated disease.

There is little information on sequence variation of canine adenovirus type 1 (CAdV-1), the aetiological agent of infectious canine hepatitis (ICH). This study reports hexon and fibre gene sequence variants of CAdV-1 in a dog with systemic ICH and a dog with the ocular form of the disease ('blue eye') in Northern Italy in 2013. One of the sequence variants matched a CAdV-1 fox sequence previously detected in Italy.

Altered proliferative kinetics in PHA-activated human T-lymphocytes treated with the anti-HLA class I monoclonal antibody 01.65.

Anti-HLA class I monoclonal antibody (mAb) 01.65 inhibited phytohaemagglutinin (PHA)-induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G1-phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA-induced proliferation of human T-lymphocytes.

Identification of a novel protein kinase C inhibitor in microsomes from phytohaemagglutinin activated human peripheral blood mononuclear cells.

A peptide inhibiting either corpuscolate or purified PKC has been identified from microsomes of PHA-activated human PBMC but it is not detectable in microsomes of resting PBMC. The peptide was obtained from a microsomal preparation in an oligomeric form that could be transformed into a monomeric form by beta-MSH. The active peptide (IN) was retained on a PC-11 chromatographic column and could be eluted with NaCl. IN is ineffective on PKC-dependent protamine phosphorylation of protamine and on Ca2+ and phospholipid-independent activity generated by mild hydrolysis with trypsin of PKC. Ca2+ binding is permissive for IN activity. IN inhibits particulate PKC in PHA-activated PBMC, but is ineffective after TPA activation. All these data indicate that IN acts at the regulatory domain of PKC.

Behcet's disease associated with HLA-B51 and DRw52 antigens in Italians.

Thirty-eight Italian patients with Behcet's disease, all with ocular involvement, (28 complete type and ten incomplete) were typed for HLA A,B,DR, and DQ antigens. A significant increase of HLA-B51 (p less than 0.00001) and DRw52 (p = 0.045) with no significant difference between complete and incomplete syndrome was found. The involvement of B51 antigen as the main immunogenetic factor in the disease is suggested by the high value of relative risk (RR = 16.03). However, the association with the II class antigen DRw52 (RR = 2.77) cannot be easily explained as a secondary association due to linkage disequilibria with B51.

Failure to find a B51 IEF variant associated with Behçet's disease.

HLA B51 antigen from 8 Italian Behçet's disease (BD) patients and 8 healthy controls were analyzed by isoelectric focusing (IEF). No differences were found in the migration patterns.

HLA B51 antigen associated with neutrophil hyper-reactivity.

HLA B51 specificity is strongly associated with Behcet's disease (BD) (for references see Baricordi et al., 1986), a multisystem vasculitis of unknown aetiology, for which an immunological pathogenesis has been proposed (O'Duffy et al., 1983; O'Duffy et al., 1990). Neutrophil abnormalities observed in BD patients even during clinical remission suggest prominent involvement of these phagocytic cells in the pathogenesis of the disease (Niwa et al., 1982). In order to clarify how HLA B51 antigen might confer susceptibility to BD, we have investigated neutrophil function in 13 B51-positive and 13 B51-negative healthy subjects. Lymphocyte spontaneous proliferation and circulating immune complexes were also evaluated. Whereas neutrophils from B51-positive subjects showed an increase in the chemotactic response toward casein (P = 0.003) and LPS (P = 0.033) and also in the PMA-induced superoxide production (P = 0.008) no evidence of enhanced lymphocyte activation emerged. These results suggest that the HLA region can exert a regulatory control on PMN functions.

Investigation of the presence of canine adenovirus (CAdV) in owned dogs in Northern Italy.

The use of a modified live canine adenovirus (CAdV) vaccine has greatly reduced the incidence of infectious canine hepatitis (ICH) in dogs. Nevertheless, cases of CAdV type 1 and 2 (CAdV-1 and CAdV-2) infection have been recently reported posing questions about the epidemiological situation of CAdV in dogs. In order to assess the presence of CAdV, samples from 51 dogs presented at a Veterinary Teaching Hospital in Bologna, Italy, for reasons unrelated with CAdV infection, were tested with a polymerase chain reaction (PCR) assay for CAdV. Thirty dogs (58.8%) were PCR positive for CAdV-2 infection and four of them (7.8%) were positive for CAdV-1. Sequence analysis performed on the obtained PCR products suggests that a genetically stable CAdV-1 strain and different CAdV-2 strains circulate in the canine population examined and that coinfections are relatively frequent.

AGR2 is a SMAD4-suppressible gene that modulates MUC1 levels and promotes the initiation and progression of pancreatic intraepithelial neoplasia.

The mechanisms controlling expression of the putative oncogene Anterior gradient 2 (AGR2) in pancreatic ductal adenocarcinoma (PDAC) are not well understood. We now show that AGR2 is a transforming growth factor-ß (TGF-ß)-responsive gene in human pancreatic cancer cells, whose downregulation is SMAD4 dependent. We also provide evidence supporting a role for AGR2 as an ER-chaperone for the cancer-associated mucin, MUC1. AGR2 is both sufficient and required for MUC1 expression in pancreatic cancer cells. Furthermore, AGR2 is coexpressed with MUC1 in mouse pancreatic intraepithelial neoplasia (mPanIN)-like lesions and in the cancer cells of four distinct genetically engineered mouse models of PDAC. We also show that Pdx1-Cre/LSL-Kras(G12D)/Smad4(lox/lox) mice heterozygous for Agr2 exhibit a delay in mPanIN initiation and progression to PDAC. It is proposed that loss of Smad4 may convert TGF-ß from a tumor suppressor to a tumor promoter by causing the upregulation of AGR2, which then leads to increased MUC1 expression, at which point both AGR2 and MUC1 facilitate mPanIN initiation and progression to PDAC.

MiR203 mediates subversion of stem cell properties during mammary epithelial differentiation via repression of ΔNP63α and promotes mesenchymal-to-epithelial transition.

During reproductive life, the mammary epithelium undergoes consecutive cycles of proliferation, differentiation and apoptosis. Doing so relies on the retained proliferative capacity, prolonged lifespan and developmental potency of mammary stem cells (MaSCs). ΔNp63α, the predominant TP63 isoform in mammary epithelia, is robustly expressed in MaSCs and is required for preservation of self-renewing capacity in diverse epithelial structures. However, the mechanism(s) underlying subversion of this activity during forfeiture of self-renewing capacity are poorly understood. MicroRNAs (miRNAs) govern critical cellular functions including stem cell maintenance, development, cell cycle regulation and differentiation by disrupting translation of target mRNAs. Data presented here indicate that expression of miR203, a miRNA that targets ΔNp63α and ΔNp63ß is activated during luminal epithelial differentiation and that this pattern is observed in the murine mammary hierarchy. In addition, we present evidence that the transcription factor Zeb1 represses miR203 expression, thus enhancing ΔNp63α protein levels. Furthermore, ectopic miR203 suppresses ΔNp63α expression, proliferation and colony formation. The anti-clonogenic effects mediated by miR203 require suppression of ΔNp63α. In addition, ectopic miR203 promotes mesenchymal-to-epithelial transition and disrupts activities associated with epithelial stem cells. These studies support a model in which induction of miR203 mediates forfeiture of self-renewing capacity via suppression of ΔNp63α and may also have anti-tumorigenic activity through its reduction of EMT and cancer stem cell populations.

Molecular analysis of cell cycle-related gene expression in anti-HLA class I monoclonal antibody (01.65) treated PHA-activated human T-lymphocytes.

HLA class I antigens seem to be involved in the proliferative response of PHA-activated human T-lymphocytes. We have previously reported that the treatment of PHA-activated peripheral blood mononuclear cells (PBMC) with an anti-HLA class I monoclonal antibody, 01.65, (i) inhibits the tritiated thymidine incorporation, (ii) inactivates cytosolic protein kinase C (PKC) and (iii) causes an increase in the duration of the cell cycle. Northern Blot kinetic analysis of c-fos, c-myc, cdc2, IL-2R, c-myb, ODC, TK and H3, from 10 minutes to 120 hours, was performed in MAb 01.65 treated cultures. We found that the expression of four genes (c-myc, IL-2R, cdc2 and TK) was depressed 24 hours after PHA stimulation.