Loss of CD45 cell surface expression in canine T-zone lymphoma results from reduced gene expression.

Canine T-zone lymphoma (TZL) is a peculiar lymphoma subtype characterized by an indolent clinical course and aberrant CD45-negative phenotype, commonly recognized by flow cytometry (FC). Recent studies have described clinical presentation and behavior, but to date the mechanisms behind the loss of CD45 protein expression have never been investigated. The aims of this study were: 1) to confirm the absence of CD45 in canine TZL via the concomitant use of FC and immunohistochemistry with two different sources of antibody; and 2) to investigate the amount of CD45 transcript and the presence of CD45 gene in the neoplastic cells of dogs affected by TZL. 57 lymph node aspirates were included in the present study: 40 (70.2%) TZLs, 7 (12.3%) high grade T-cell lymphomas and 10 (17.5%) reactive lymph nodes. Neoplastic cells and normal T-cells were isolated from TZL and reactive lymph nodes, respectively, via cell sorting. Immunohistochemistry was performed on 2 TZL, 2 reactive lymph nodes and 2 Peripheral T-cell Lymphomas. Total RNA and genomic DNA were extracted from lymph-nodes aspirates. Two different quantitative real-time PCR experiments were designed, to determine the amount of the CD45 transcript and of the corresponding gene fragment. All TZL cases were negative for CD45 at immunohistochemistry. CD45 transcript amount was significantly lower in TZL compared to controls (p<0.001). This difference was not significant (p=0.584) for CD45 DNA load, that was similar between TZL and controls. Moreover, CD45 transcript amount was inversely correlated with the percentage of neoplastic cells in each TZL sample (p=0.010). These results confirm that CD45 protein is lacking on cell surface irrespective of the technique and antibody source adopted. This phenotypic aberrancy is apparently due to the absence of gene transcription, as CD45 DNA was present, whereas CD45 transcript was virtually absent in the neoplastic cells. The data here reported support further studies investigating possible factors impairing CD45 gene transcription.

X-Linked Hereditary Nephropathy in Navasota Dogs: Clinical Pathology, Morphology, and Gene Expression During Disease Progression.

X-linked hereditary nephropathy (XLHN) in Navasota dogs is a spontaneously occurring disease caused by a mutation resulting in defective production of type IV collagen and juvenile-onset renal failure. The study was aimed at examining the evolution of renal damage and the expression of selected molecules potentially involved in the pathogenesis of XLHN. Clinical data and renal samples were obtained in 10 XLHN male dogs and 5 controls at 4 (T0), 6 (T1), and 9 (T2) months of age. Glomerular and tubulointerstitial lesions were scored by light microscopy, and the expression of 21 molecules was investigated by quantitative real-time polymerase chain reaction with selected proteins evaluated by immunohistochemistry. No significant histologic lesions or clinicopathologic abnormalities were identified in controls at any time-point. XLHN dogs had progressive proteinuria starting at T0. At T1, XLHN dogs had a mesangioproliferative glomerulopathy with glomerular loss, tubular necrosis, and interstitial fibrosis. At T2, glomerular and tubulointerstitial lesions were more severe, particularly glomerular loss, interstitial fibrosis, and inflammation. At T0, transforming growth factor ß, connective tissue growth factor, and platelet-derived growth factor α mRNA were overexpressed in XLHN dogs compared with controls. Clusterin and TIMP1 transcripts were upregulated in later stages of the disease. Transforming growth factor ß, connective tissue growth factor, and platelet-derived growth factor α should be considered as key players in the initial events of XHLN. Clusterin and TIMP1 appear to be more associated with the progression rather than initiation of tubulointerstitial damage in chronic renal disease.

Canine indolent and aggressive lymphoma: clinical spectrum with histologic correlation.

Sixty-three dogs with newly diagnosed lymphoma underwent complete staging and received the same chemotherapy. Diffuse large B-cell lymphoma was the leading histotype (44.4%), followed by peripheral T-cell lymphoma (20.6%). Indolent lymphomas accounted for 30.2% of cases. Most dogs with aggressive B-cell lymphoma had stage IV disease. Dogs with indolent and aggressive T-cell lymphoma had more often stage V disease and were symptomatic. Liver and bone marrow were predominantly involved in B-cell and T-cell lymphoma, respectively. The clinical stage was significantly related to substage, sex and total lactic dehydrogenase (LDH) levels. Aggressive B-cell lymphomas were more likely to achieve remission. Median survival was 55 days for aggressive and indolent T-cell lymphoma, 200 and 256 days for indolent and aggressive B-cell lymphoma, respectively. The prognosis of advanced indolent lymphoma does not appear to be appreciably different from that of aggressive disease. Familiarity with the various histotypes is critical to make the correct diagnosis and drive therapy.

Platelet-derived growth factors and receptors in Canine Lymphoma.

Platelet-derived growth factors (PDGFs) belong to a family of polypeptide growth factors that signal through cell surface tyrosine kinase receptors to stimulate growth, proliferation and differentiation. Platelet-derived growth factor receptors (PDGFRs) are also considered important targets for specific kinase inhibitors in the treatment of several human tumours. The aim of this study was to investigate the role of PDGF-A, PDGF-B, PDGFR-α and PDGFR-ß in canine lymphoma by determining gene and protein expression in lymph nodes of dogs with diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL), T-lymphoblastic lymphoma (T-LBL) and in healthy control dogs. One lymph node was also studied at the end of therapy in a subset of dogs in remission for DLBCL. In controls, PDGF-A, PDGFR-α and PDGFR-ß mRNA levels were significantly higher than in DLBCLs, PTCLs and T-LBLs. However, PDGFR-α and PDGFR-ß were minimally expressed by lymphocytes and plasma cells in normal lymph nodes as determined by immunohistochemistry, while neoplastic B and T cells showed the highest score (P <0.05). This discordant result may be compatible with the constitutive expression of these molecules by endothelial cells and fibroblasts in normal lymph nodes, thereby influencing gene expression results. Furthermore, these cells were not included in the immunohistochemical analysis. Similarly, dogs with DLBCL that were in remission at the end of therapy showed significantly higher gene expression of PDGFs and receptors than at the time of diagnosis and with an opposite trend to the protein assay. PDGF-B protein and mRNA were overexpressed in PTCLs and T-LBLs when compared with DLBCLs and controls (P <0.05). Additionally, there was a correlation between protein expression of PDGF-B and both PDGFRs in PTCLs and T-LBLs, suggesting an autocrine or paracrine loop in the aetiology of aggressive canine T-cell lymphomas. These data provide a rationale for the use of PDGFR antagonists in the therapy of aggressive T-cell lymphomas, but not in DLBCLs.

IrO2 as a promoter of Pt-Ru for methanol electro-oxidation.

A multifunctional catalyst may represent a valid route to enhance methanol electro-oxidation. Ternary catalysts based on Pt modified with both Ru and Ir oxides show better performance for methanol electro-oxidation than bi-metallic Pt-Ru catalysts.

CD44 in canine leukemia: analysis of mRNA and protein expression in peripheral blood.

Hyaluronan receptor CD44 mediates interaction between cells and extracellular matrix. The expression of standard form and its variants is dysregulated in human leukemias and is associated with metastasis and prognosis. The aim of this work is the evaluation of CD44 mRNA and protein expression in canine leukemia. Peripheral blood from 20 acute leukemias (AL) (10 acute lymphoblastic, 6 acute myeloid and 4 acute undifferentiated leukemias), 21 chronic lymphocytic leukemias (CLL) and thirteen healthy dogs were collected. The mRNA expression of all CD44 variants presenting exons 1-5 and/or 16-20 (CD44_ex1-5 and CD44_ex16-20) and CD44 protein were determined by real-time RT-PCR and flow cytometry, using the mean fluorescent index (MFI), respectively. CD44 MFI was significantly higher in leukemic samples compared to controls and a higher expression was found in AL in respect with CLL. No significant differences were found when considering different phenotypic subtypes of AL and CLL. CD44_ex1-5 mRNA expression was significantly higher in AL compared to controls, whereas there was no difference in CLL compared to controls and AL. CD44_es16-20 showed the same trend, but without differences among groups. The high CD44 expression found in canine leukemias could be considered a step toward the definition of their molecular features.

VEGF and MMP-9: biomarkers for canine lymphoma.

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF-ß) stimulates VEGF and MMPs production. VEGF and TGF-ß plasma levels were tested by ELISA, MMP-2 and MMP-9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act-MMP-9 (P < 0.01) and VEGF (P < 0.05), and lower TGF-ß than controls, and a positive correlation between act-MMP-9 and VEGF (P < 0.001). Act-MMP-9 and VEGF were significantly higher in T-cell lymphomas, and in stage V compared with stages III-IV disease, regardless of immunophenotype. VEGF was higher in high-grade compared with low-grade T-cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act-MMP-9 and VEGF decreased in B-cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.

Evaluation of tyrosine-kinase receptor c-kit mutations, mRNA and protein expression in canine lymphoma: might c-kit represent a therapeutic target?

c-kit plays an important role in proliferation, survival and differentiation of hematopoietic progenitor cells. In human hematopoietic malignancies, c-kit is mostly expressed by progenitor cell neoplasms and seldom by mature cell neoplasms. Aim of this study was to evaluate c-kit expression in canine lymphoma. Twenty-five B-cell lymphomas and 21 T-cell lymphomas were enrolled in the study. c-kit mRNA and protein expression was measured in lymph node fine needle aspirates by quantitative real-time RT-PCR, flow cytometry and immunocytochemistry, while the occurrence of KIT mutations on exons 8-11 and 17 was investigated by direct cDNA sequencing. KIT mRNA was amplifiable but below the limit of quantification in 76% of B-cell lymphomas and 33% of T-cell lymphomas. Remaining samples showed a very low expression of KIT, except for some high grade (HG) T-cell lymphomas where a comparatively higher mRNA amount was observed. Transcriptional data were confirmed at the protein level. No gain-of-function mutations were observed. Among canine lymphomas, T-cell lymphoma typically shows an aggressive biological behavior, partly being attributable to the lack of efficacious treatment options, and the evidence of c-kit expression in HG T-cell lymphomas might represent the rationale for its routinely diagnostic evaluation and the use of tyrosine kinase inhibitors in future clinical trials.

Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: might c-KIT represent a therapeutic target?

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8-11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials.

Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases and vascular endothelial growth factor in canine mast cell tumours.

Degradation of the extracellular matrix and angiogenesis are associated with tumour invasion and metastasis in human and canine neoplasia. Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and vascular endothelial growth factor-A (VEGF-A) are key mediators of these respective processes. Mast cell tumour (MCT) is the most common malignant cutaneous tumour in dogs. MCTs are always considered potentially malignant, but their true metastatic potential is unknown. In the present study, samples from seven grade 1, 22 grade 2 and six grade 3 MCTs were subjected to quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) to evaluate MMP-2, MMP-9, membrane-type 1 MMP (MT1-MMP), TIMP-2 and VEGF-A mRNA and protein expression. Gelatin zymography (GZ) was also performed to evaluate MMP-2 and MMP-9 activity. MMP-9 and VEGF-A mRNA increased with histological grade, while TIMP-2 decreased with increasing grade. Gene expression data obtained for MMP-9, VEGF-A and TIMP-2 were confirmed by IHC for evaluation of the respective proteins. In contrast, MMP-2 and MT1-MMP had variable, but similar, expression for both mRNA and protein. Despite the high variability observed, there was correlation between MMP-2 and MT1-MMP mRNA expression (r=+0.91, P<0.0001). The MMP-2:TIMP-2 and MMP-9:TIMP-1 mRNA ratios showed an imbalance between MMPs and their specific inhibitors in MCTs, which increased with the histological grade. Finally, the activities of both latent and active forms of MMP-2 and MMP-9 were evaluated by GZ and there were significant increases in their activities with increasing histological grade and immunohistochemical expression. This study demonstrates that MMP-9, TIMP-2 and VEGF-A expression is related to histological grade and suggests that these markers are possible indicators of malignancy and targets for therapeutic strategies.

Design of efficient methanol impermeable membranes for fuel cell applications.

In this paper, the design of efficient composite membranes based on sulfonated polysulfone and acidic silica material with characteristics and properties such as methanol barrier, high proton conductivity and suitable fuel cells performance is presented. A positive influence of nanosized acidic silica powders, used as an additive filler in the preparation of composite membranes, due to an efficient hydrophilic inter-distribution inside the membrane when compared to pure silica, is found. A series of different techniques such as XRF, FT-IR, TGA, DSC, IEC and conductivity measurements are used to highlight the properties of acidic silica material and composite membranes. The composite membrane based on acidic silica (SPSf-SiO(2)-S) shows the lowest crossover current (only 8 mA cm(-2)), which is 43% lower than that of a pure SPSf membrane and 33% lower compared to a composite membrane based on bare silica (SPSf-SiO(2)). These significant differences are attributed to the increasing diffusion path length of MeOH/H(2)O clusters in the composite membranes. The maximum DMFC performance at 30 °C is achieved with the SPSf-SiO(2)-S membrane (23 mW cm(-2)), whereas the MEAs based on SPSf-SiO(2) and pure SPSf membranes reached 21 and 16 mW cm(-2), respectively. These significant results of the composite SPSf-SiO(2)-S membrane are ascribed at a good compromise among high proton conductivity, low swelling and low methanol crossover compared to pure SPSf and (unmodified silica)-SPSf membranes. A preliminary short durability test of 100 h performed in a cell with the composite SPSf-SiO(2)-S membrane shows remarkable performance stability during chrono-voltammetric measurements (60 mA cm(-2)) at 30 °C.

[Carcinomas of the right colon. A review of 60 consecutive cases]. / I carcinomi del colon destro. Revisione di 60 casi consecutivi.

Sixty consecutive cases of tumor of the right colon operated upon between 1981 and 1991 are reviewed. Age and sex distribution, clinical findings, preoperative diagnostics and system used staging, TNM classification, surgical management, operative mortality, follow up and survival were analyzed. Results are discussed and compared with published data. In the second of the two 5-year period considered an increased incidence of right-sided carcinoma compared with tumors of the left colon and rectum was observed. According to the TNM classification, more than 50% of patients were in stage III. Operative mortality was 5%. The overall 5-year survival rate was 58%, while the survival rate of patients treated with radical intent was 70%.

[Porcelain gallbladder. Case reports]. / La colecisti a porcellana. Contributo casistico.

The Authors report some observations on gallbladder calcification with the presentation of two clinical cases. The pathologic anatomy and the clinical aspects of this infrequent affection are described. The Authors underline the importance of surgical treatment, considering the frequency of carcinoma in porcelain gallbladder.

[Stoma complications. Experience of a care center for enterostomal patients]. / Complicanze delle stomie. Esperienza di un centro di assistenza per enterostomizzati.

Personal experience in an enterostomy centre over a 5-year period is report. The problems deriving from the presence of a stoma are examined with particular emphasis on surgical complications which are grouped into early and late forms.

[Celiac-mesenteric insufficiency. Apropos of a case of aorto-hepatic bypass]. / L'insufficienza celiaco-mesenterica. A proposito di un caso di by-pass aorto-epatico.

The Authors report a case of coeliac axis atheromatous obstruction, with superior mesenteric artery steal. The coeliac axis, stopped up the origin was indirectly revascularized by hepatic artery. The early diagnosis of chronic intestinal ischaemia should avoid the intestinal infarction. The development of intravenous digital angiography will certainly help an early diagnosis.

[Acute cholecystitis. Early diagnostic study using cholescintigraphy with Tc99m-IDA]. / La colecistite acuta. Indagine diagnostica precoce con la colescintigrafia con IDA-99mTc.

34 patients admitted for suspected acute cholecystitis were evaluated using 99mTc IDA cholescintigraphy. The results of these studies are reviewed and compared with other diagnostic tests and the subsequent clinical diagnosis. Cholescintigraphy proved to be a safe, simple, highly accurate and sensitive technique. Therefore, 99mTc-IDA cholescintigraphy is proposed as the initial procedure of choice in the evaluation of patients with suspected acute cholecystitis.