B. Miniscalco

Comparison of three Tfr2 -deficient murine models suggests distinct functions for TFR2 alpha and beta isoforms in different tissues

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in hemochromatosis type 3. The TFR2 gene is transcribed in 2 main isoforms: the full-length (alpha) and a shorter form (beta). alpha-Tfr2 is the sensor of diferric transferrin, implicated in the modulation of hepcidin, the main regulator of iron homeostasis. The function of the putative beta-Tfr2 protein is unknown. We have developed a new mouse model (KI) lacking beta-Tfr2 compared with Tfr2 knockout mice (KO). Adult Tfr2 KO mice show liver iron overload and inadequate hepcidin levels relative to body iron stores, even though they increase Bmp6 production. KI mice have normal transferrin saturation, liver iron concentration, hepcidin and Bmp6 levels but show a transient anemia at young age and severe spleen iron accumulation in adult animals. Fpn1 is strikingly decreased in the spleen of these animals. These findings and the expression of beta-Tfr2 in wild-type mice spleen suggest a role for beta-Tfr2 in Fpn1 transcriptional control. Selective inactivation of liver alpha-Tfr2 in KI mice (LCKO-KI) returned the phenotype to liver iron overload. Our results strengthen the function of hepatic alpha-Tfr2 in hepcidin activation, suggest a role for extrahepatic Tfr2 and indicate that beta-Tfr2 may specifically control spleen iron efflux.

The heme exporter Flvcr1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation.

Feline leukemia virus subgroup C receptor 1 (Flvcr1) encodes two heme exporters: FLVCR1a, which localizes to the plasma membrane, and FLVCR1b, which localizes to mitochondria. Here, we investigated the role of the two Flvcr1 isoforms during erythropoiesis. We showed that, in mice and zebrafish, Flvcr1a is required for the expansion of committed erythroid progenitors but cannot drive their terminal differentiation, while Flvcr1b contributes to the expansion phase and is required for differentiation. FLVCR1a-down-regulated K562 cells have defective proliferation, enhanced differentiation, and heme loading in the cytosol, while FLVCR1a/1b-deficient K562 cells show impairment in both proliferation and differentiation, and accumulate heme in mitochondria. These data support a model in which the coordinated expression of Flvcr1a and Flvcr1b contributes to control the size of the cytosolic heme pool required to sustain metabolic activity during the expansion of erythroid progenitors and to allow hemoglobinization during their terminal maturation. Consistently, reduction or increase of the cytosolic heme rescued the erythroid defects in zebrafish deficient in Flvcr1a or Flvcr1b, respectively. Thus, heme export represents a tightly regulated process that controls erythropoiesis.

Morgana acts as an oncosuppressor in chronic myeloid leukemia

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.

Corynebacterium urealyticum urinary tract infection in a cat with urethral obstruction

Corynebacterium urealyticum is an uncommon cause of urinary tract infections in cats. However, it is difficult to diagnose and if left untreated it may result in irreversible bladder lesions. C urealyticum is a multiantibiotic-resistant bacterium whose culture requires special care. Risk factors for the occurrence of this infection include urological procedures, foreign bodies, bladder mucosa abnormalities, immuno-suppressed states and antibiotic treatment. This report describes an unusual case of C urealyticum urinary infection in a young cat with pre-existing urethral obstruction. C urealyticum was isolated in pure cultures from two urine samples. Clinical and ultrasound features, results of the urinalysis and urine culture are described as well as therapeutic treatment and eventual favourable outcome to treatment with amoxycillin–clavulanic acid.

Flow cytometric evaluation of ki67 for the determination of malignancy grade in canine lymphoma

Ki67 is a nuclear antigen significantly correlated with degree of malignancy in human non-Hodgkin lymphomas. We wanted to assess the ability of flow cytometric evaluation of Ki67 index (Ki67I) in differentiating the grade of malignancy in canine lymphomas. Ki67I was determined on lymph node aspirates of 90 immunophenotyped lymphomas classified according to the updated Kiel classification: 80 high grade (HG, 62 B cell and 18 T cell) and 10 low grade (LG, 3 B cell and 7 T cell) lymphomas. HG lymphomas showed significantly higher Ki67I compared with LG lymphomas (P < 0.0001). A significant difference in HG lymphomas was detected between B- and T-immunophenotypes. Receiver operating characteristic (ROC) curve highlighted a high accuracy of Ki67I in recognizing HG lymphomas [area under the curve (AUC) = 99.4] and a cut-off value of 12.2% was established (sensitivity = 96.3% and specificity = 100%). Thus, we suggest the combination of Ki67I flow cytometric determination and immunophenotype as a reliable tool to classify canine lymphomas.

Canine leishmaniosis: in vitro efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum

Despite the availability of different therapeutic options, canine visceral leishmaniosis (CVL) remains a challenging disease to treat. Recently miltefosine has been registered for use in dogs, and different studies have demonstrated its leishmanicidal effect. Moreover, it has been suggested that fluoroquinolones, compared to standard chemotherapeutic agents, could be an effective and pragmatic alternative to treat CVL. The efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum was assessed in vitro by incubating increasing concentrations of the drugs with a standard parasite inoculum. Miltefosine was significantly more efficacious than marbofloxacin (P < 0.05) against the two strains of L. infantum either alone or in combination with allopurinol. Both drugs were significantly (P < 0.05) more efficacious when associated with allopurinol than alone.

Analysis of cerebrospinal fluid from 20 calves after storage for 24 hours

Samples of CSF collected from 20 normal healthy calves were analysed either immediately or after having been stored for 24 hours at 4°C in the presence of 11 per cent autologous serum. There were no significant differences between the total and differential cells counts of the fresh and stored samples, but there was a positive linear correlation between them. There were some morphological changes to the nuclei of the mononuclear cells in the stored samples.

Clinical Usefulness of Peripheral Blood Lymphocyte Subsets in Canine Lymphoma

The classification of canine lymphoid malignancies into groups based on cell of origin (Bor T-cell types) is possible with immunophenotypic analysis. A high frequency of B-cell lymphomas has been described; about 30% of lymphomas are of T-cell origin (Teske et al., 1994a; Fournel-Fleury et al., 1997; Grindem et al., 1998). In human medicine the major diagnostic role of blood lymphocyte subset typing is the screening for non-Hodgkin’s lymphomas with peripheral blood involvement and their differentation from reactive lymphoproliferative conditions (Thalhammer-Scherrer et al., 2000). The aim of this project is to evaluate the diagnostic efficacy of blood immunophenotyping in dogs with lymphoma in order to detect the blood involvement and the prognostic benefits.

Prognostic significance of Ki67 evaluated by flow cytometry in dogs with high-grade B-cell lymphoma.

Ki67 can discriminate between high- and low-grade canine lymphomas, but its prognostic role in specific subtypes of the neoplasm is unknown. We assessed the prognostic significance of Ki67% (percentage of Ki67-positive cells), evaluated by flow cytometry, in 40 dogs with high-grade B-cell lymphoma, treated with a modified Wisconsin-Madison protocol (UW-25). The following variables were investigated for association with lymphoma specific survival (LSS) and relapse free interval (RFI): Ki67%, breed, sex, age, stage, substage, complete remission (CR). By multivariate analysis, Ki67% (P = 0.009) and achievement of CR (P = 0.001) were independent prognostic factors for LSS. Dogs with intermediate Ki67% (20.1-40%) presented longer LSS and RFI (median = 866 and 428 days, respectively) than dogs with low (median = 42 days, P < 0.001; median = 159 days, P = 0.014) or high (median = 173 days, P = 0.038; median = 100 days, P = 0.126) values. Determination of Ki67 is a prognostic tool that improves the clinical usefulness of flow cytometric analysis in canine high-grade B-cell lymphoma.

Analytical and diagnostic validation of a flow cytometric strategy to quantify blood and marrow infiltration in dogs with large b‐cell lymphoma

Lymph node (LN), peripheral blood (PB), and bone marrow (BM) samples are commonly analyzed by flow cytometry (FC) for the immunophenotyping and staging of canine lymphomas. A prognostic value for FC BM infiltration in dogs with large B‐cell lymphoma (LBCL) was demonstrated. Aim of this study was to define the analytical performances of this technique, and to establish a cutoff suitable to safely discriminate between infiltrated and noninfiltrated PB and BM samples.