Anna Maria Cantoni

Targeting MEK/MAPK signal transduction module potentiates ATO-induced apoptosis in multiple myeloma cells through multiple signaling pathways

We demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in human myeloma cell lines (HMCLs) and in tumor cells from patients with multiple myeloma (MM) through a caspase-dependent mechanism. In HMCLs retaining a functional p53, PD treatment greatly enhances the ATO-induced p53 accumulation and p73, a p53 paralog, cooperates with p53 in caspase activation and apoptosis induction. In HMCLs carrying a nonfunctional p53, cotreatment with PD strikingly elevates the (DR4 + DR5)/(DcR1 + DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated cells. In MM cells, irrespective of p53 status, the combined PD/ATO treatment increases the level of the proapoptotic protein Bim (PD-mediated) and decreases antiapoptotic protein Mcl-1 (ATO-mediated). Moreover, Bim physically interacts with both DR4 and DR5 TRAIL receptors in PD/ATO-treated cells, and loss of Bim interferes with the activation of both extrinsic and intrinsic apoptotic pathways in response to PD/ATO. Finally, PD/ATO treatment induces tumor regression, prolongs survival, and is well tolerated in vivo in a human plasmacytoma xenograft model. These preclinical studies provide the framework for testing PD325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two‐step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA‐1)‐coated beads, followed by purification with monoclonal antibody D2–40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8–10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR‐3 molecules. In fact, stimulation with VEGFR‐3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube‐like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro‐ and macrovessel‐derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil‐ and ILV‐derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia. J. Cell. Physiol. 207: 107–113, 2006.

Elucidating the gut microbiome of ulcerative colitis: bifidobacteria as novel microbial biomarkers

Ulcerative colitis (UC) is associated with a substantial alteration of specific gut commensals, some of which may be involved in microbiota-mediated protection. In this study, microbiota cataloging of UC patients by 16S rRNA microbial profiling revealed a marked reduction of bifidobacteria, in particular the Bifidobacterium bifidum species, thus suggesting that this taxon plays a biological role in the aetiology of UC. We investigated this further through an in vivo trial by testing the effects of oral treatment with B. bifidum PRL2010 in a wild-type murine colitis model. TNBS-treated mice receiving 10(9) cells of B. bifidum PRL2010 showed a marked reduction of all colitis-associated histological indices as well as maintenance of mucosal integrity as it was shown by the increase in the expression of many tight junction-encoding genes. The protective role of B. bifidum PRL2010, as well as its sortase-dependent pili, appears to be established through the induction of an innate immune response of the host. These results highlight the importance of B. bifidum as a microbial biomarker for UC, revealing its role in protection against experimentally induced colitis.

Experimental Tracheal Transplantation Using a Cryopreserved Aortic Allograft

Background: The tracheal reconstruction after wide resections remains a critical surgical problem. Our aim was to replace trachea with a tissue easy to vascularize, which allows a simple reconstruction and does not require an immunosuppressive regimen. Materials and Methods: A segment of cryopreserved aorta was used in order to verify its adequacy as tracheal substitute. In phase 1, the thoracic aorta of 10 rabbits was excised, obtaining 20 segments that were cryopreserved. Ten segments were implanted in the omentum of 10 rabbits that were sacrificed on postoperative days 7, 14 and 21, and the grafts were examined histologically. In phase 2, a segment of cryopreserved aorta arranged with a silicone prosthesis was transplanted in 10 rabbits and wrapped with omentum. The animals were sacrificed on postoperative days 7, 14 and 21. Results: In phase 1, the neovascularization of the grafts was present after 7 days, and after 14 days the fibroblasts invaded the lumen of the aorta. In phase 2, 8 rabbits survived and the histologic examination after 7, 14 and 21 days showed neovascularization, the absence of rejection and the proliferation of fibroblasts inside the lumen of the aorta; this growth has been restrained by an endoluminal prosthesis. Conclusions: Our study demonstrated that replacing the trachea with cryopreserved aorta is technically feasible and does not evoke immunologic reactions. It requires, however, a silicone tube inside the allograft to limit the colonization of fibroblasts.

Canine bladderworm (Capillaria plica) infection associated with glomerular amyloidosis.

Capillaria plica (Trichuroidea: Capillariidae), commonly known as bladderworm, is a nematode rarely associated with clinical disease that resides in the lower urinary tract of wild and domestic canids. In the present paper a case of canine urinary capillariosis associated with glomerular amyloidosis is described. The dog, an 8-year-old, male, hunting Jagd terrier had a history of weight loss and diarrhoea and was referred to the University of Parma Teaching Veterinary Hospital (UPTVH). Clinical and laboratory tests shown here suggest that C. plica may be a contributing factor to glomerular amyloidosis.

Clinical, genetic, and pathological features of male pseudohermaphroditism in dog

Male pseudohermaphroditism is a sex differentiation disorder in which the gonads are testes and the genital ducts are incompletely masculinized. An 8 years old dog with normal male karyotype was referred for examination of external genitalia abnormalities. Adjacent to the vulva subcutaneous undescended testes were observed. The histology of the gonads revealed a Leydig and Sertoli cell neoplasia. The contemporaneous presence of testicular tissue, vulva, male karyotype were compatible with a male pseudohermaphrodite (MPH) condition.

Intrapleural polymeric films containing cisplatin for malignant pleural mesothelioma in a rat tumour model: a preliminary study

OBJECTIVE This study aims to investigate the effect of intrapleural polymeric films containing cisplatin on the local recurrence of malignant pleural mesothelioma in a rat tumour model. METHODS An orthotopic rat recurrence model of malignant pleural mesothelioma was used. Five animals per group were evaluated. Polymeric films (4.5 cm diameter) for the local delivery of anticancer drug were constructed: hyaluronate, chitosan and the combined dual-layer polymers were loaded with cisplatin at a concentration of 100 mgm(-2). Animals without any adjuvant therapy were used as control. Mesothelioma cells were injected subpleurally in the anaesthetised rats. Six days later, a pleural tumour of 5.5mm was resected and a left pneumonectomy and pleural abrasion were performed. Thereafter, the cisplatin-loaded and unloaded films or cisplatin solution were intrapleurally applied, according to randomisation. After 6 days, animals were euthanised and organs harvested for morphological and histological evaluations. The primary endpoint was the volume of tumour recurrence. The secondary endpoints were treatment-related toxicity; cisplatin serum concentration evaluated at different time points; and cisplatin concentration in the pleura measured at autopsy. Analysis of variance (ANOVA) was used for statistical analysis. Bonferroni correction was applied for comparison between all groups. RESULTS Tumour volume was significantly reduced in the hyaluronate cisplatin and hyaluronate-chitosan cisplatin groups in comparison to control groups (p=0.001 and p<0.0001, respectively). Animals treated with hyaluronate-chitosan cisplatin had a tumour recurrence significantly lesser than animals treated with cisplatin solution (p=0.003) and hyaluronate cisplatin (p=0.032). No toxicity related to the different treatments was observed. On postoperative days 1 and 2, cisplatin was detected in the serum at a concentration six- and sevenfold significantly higher in the hyaluronate cisplatin and hyaluronate-chitosan cisplatin groups, in comparison to cisplatin solution, and was maintained over time. Cisplatin levels in the pleura were higher in the hyaluronate-chitosan cisplatin group than in all others. CONCLUSIONS Hyaluronate-chitosan cisplatin was significantly effective in reducing tumour recurrence compared with cisplatin solution. Hyaluronate and hyaluronate-chitosan loaded with cisplatin assured significantly higher and more prolonged plasmatic drug concentrations than cisplatin solution without increasing toxicity.

Myocardial damage in dogs affected by heartworm disease (Dirofilaria immitis): Immunohistochemical study of cardiac myoglobin and troponin I in naturally infected dogs

It has recently been reported that dogs affected by canine heartworm disease (Dirofilaria immitis) can show an increase in plasma levels of myoglobin and cardiac troponin I, two markers of muscle/myocardial injury. In order to determine if this increase is due to myocardial damage, the right ventricle of 24 naturally infected dogs was examined by routine histology and immunohistochemistry with anti-myoglobin and anti-cardiac troponin I antibodies. Microscopic lesions included necrosis and myocyte vacuolization, and were associated with loss of staining for one or both proteins. Results confirm that increased levels of myoglobin and cardiac troponin I are indicative of myocardial damage in dogs affected by heartworm disease.

Intoxication with Fumonisin B1 (FB1) in Piglets and Supplementation with Granulated Activated Carbon: Cellular-Mediated Immunoresponse

Fumonisin B1 (FB1), a secondary compound produced by toxigenic strains of Fusarium verticilloides and Fusarium proliferatum, is an important contaminant of cereals, especially maize (Pietri and Piva, 2000). Fumonisins, produced by Fusarium moniliforme, represent one of the five most important mycotoxin groups causing human disease. Although the toxic effect of mycotoxins in humans is very difficult to prove, based upon the toxicological results obtained so far, fumonisins are regarded as potential carcinogens. Farm animals are also susceptible to fumonisin, which causes equine leucoencephalomalacia. In poultry, chickens and turkeys can be considered relatively resistant, however, in young birds, a high dose of FB1 has been reported to cause growth retardation and liver damage. Ruminants are relatively resistant to fumonisin, but in young animals the toxin may cause reduced weight gain and hepatopathy (Zomborszky-Kovacs et al., 2002). It is well known that pigs are very susceptible to fumonisin and depending on its concentration, route of uptake and the duration of exposure, FB1 can cause rather diverse pathological lesions such as pulmonary edema, cardiovascular lesions, pancreatic necrosis (Harrison et al., 1990) and hepatic intoxication (Bucci et al., 1998; Gumprecht et al., 2001; He et al., 2001; Theumer et al., 2002). Unfortunately, until now, the effects of fumonisin on the cellular immune response of the pig and of its defence mechanisms are only partially understood. The aim of this study was to evaluate the behaviour and the susceptibility of T lymphocyte (CD4, CD8) and monocyte (CD14) subpopulations in piglets fed daily with a diet containing 30 ppm of Fumonisin B1 supplemented with 1% granulated activated carbon, as compared to piglets fed daily with a supplementation of only 1% granulated activated carbon, as detoxificant (control group) (Minervini, 2000).

Suppression of inflammatory events associated to intestinal ischemia–reperfusion by 5-HT1A blockade in mice

Intestinal ischemia and reperfusion (I/R) is a potentially life-threatening disease, ensuing from various clinical conditions. Experimentally, either protective or detrimental roles have been attributed to 5-HT in the functional and morphological injury caused by mesenteric I/R. Recently, we proved the involvement of 5-HT2A receptors in the intestinal dysmotility and leukocyte recruitment induced by 45min occlusion of the superior mesenteric artery (SMA) followed by 24h reperfusion in mice. Starting from these premises, the aim of our present work was to investigate the role played by endogenous 5-HT in the same experimental model where 45min SMA clamping was followed by 5h reflow. To this end, we first observed that ischemic preconditioning before I/R injury (IPC+I/R) reverted the increase in 5-HT tissue content and in inflammatory parameters induced by I/R in mice. Second, the effects produced by intravenous administration of 5-HT1A ligands (partial agonist buspirone 10mgkg(-1), antagonist WAY100135 0.5-5mgkg(-1)), 5-HT2A antagonist sarpogrelate (10mgkg(-1)), 5-HT3 antagonist alosetron (0.1mgkg(-1)), 5-HT4 antagonist GR125487 (5mgkg(-1)) and 5-HT re-uptake inhibitor fluoxetine (10mgkg(-1)) on I/R-induced inflammatory response were investigated in I/R mice and compared to those obtained in sham-operated animals (S). Our results confirmed the significant role played by 5-HT2A receptors not only in the late but also in the early I/R-induced microcirculatory dysfunction and showed that blockade of 5-HT1A receptors protected against the intestinal leukocyte recruitment, plasma extravasation and reactive oxygen species formation triggered by SMA occlusion and reflow. The ability of α7 nicotinic receptor (α7nAchR) antagonist methyllycaconitine (5mgkg(-1)) to counteract the beneficial action provided by buspirone on I/R-induced neutrophil infiltration suggests that the anti-inflammatory effect produced by 5-HT1A receptor antagonism could be partly ascribed to the indirect activation of α7nAch receptors.