Oncogenic Ras inhibits IRF1 to promote viral oncolysis.

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. Previously, we demonstrated that oncogenic RAS/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. Here, we investigated how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, namely guanylate-binding protein 2 and IFN gamma inducible protein 47 (Ifi47), we identified the IFN regulatory factor 1 (IRF1) binding site as the promoter region responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild type mouse embryonic fibroblasts (MEFs), but not in IRF1(-/-) MEFs, showing that IRF1 is involved in MEK-mediated downregulation of IFN-inducible genes. Furthermore, IRF1 protein expression was lower in RasV12 cells compared with vector control NIH3T3 cells, but was restored to equivalent levels by inhibition of MEK. Similarly, the restoration of IRF1 expression by MEK inhibition was observed in human cancer cells. IRF1 re-expression in human cancer cells caused cells to become resistant to infection by the oncolytic vesicular stomatitis virus strain. Together, this work demonstrates that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis.

Reduced toxicity, myeloablative conditioning with BU, fludarabine, alemtuzumab and SCT from sibling donors in children with sickle cell disease.

BU and CY (BU/CY; 200 mg/kg) before HLA-matched sibling allo-SCT in children with sickle cell disease (SCD) is associated with ~85% EFS but is limited by the acute and late effects of BU/CY myeloablative conditioning. Alternatives include reduced toxicity but more immunosuppressive conditioning. We investigated in a prospective single institutional study, the safety and efficacy of a reduced-toxicity conditioning (RTC) regimen of BU 12.8-16 mg/kg, fludarabine 180 mg/m(2), alemtuzumab 54 mg/m(2) (BFA) before HLA-matched sibling donor transplantation in pediatric recipients with symptomatic SCD. Eighteen patients, median age 8.9 years (2.3-20.2), M/F 15/3, 15 sibling BM and 3 sibling cord blood (CB) were transplanted. Mean whole blood and erythroid donor chimerism was 91% and 88%, at days +100 and +365, respectively. Probability of grade II-IV acute GVHD was 17%. Two-year EFS and OS were both 100%. Neurological, pulmonary and cardiovascular function were stable or improved at 2 years. BFA RTC and HLA-matched sibling BM and CB allo-SCT in pediatric recipients result in excellent EFS, long-term donor chimerism, low incidence of GVHD and stable/improved organ function.

Short-term toxicity of hexavalent-chromium to epipsammic diatoms of a microtidal estuary (Río de la Plata): responses from the individual cell to the community structure.

Diatoms are an integral and often dominant component of the benthic microalgal assemblage in estuarine and shallow coastal environments. Different toxic substances discharged into these ecosystems persist in the water, sediments, and biota for long periods. Among these pernicious agents, the toxicity in diatoms by metal is linked to different steps in the transmembrane and internal movements of the toxicant, causing perturbations in the normal structural and functional cellular components. These changes constitute an early, nontaxonomic warning signal that could potentially serve as an indicator of this type of pollution. The aim of this work was to study the environment-reflecting short-term responses at different levels of organization of epipsammic diatoms from the Río de la Plata estuary, Argentina that had been exposed to hexavalent chromium within experimental microcosms. To this end we monitored: (i) changes in the proportion of the diatoms in relation to other algal groups at the biofilm community level; (ii) shifts in species composition at the diatom-assemblage level; (iii) projected changes in the densities of the most representative species at the population level through comparison of relative growth rates and generation times; and (iv) the cytological changes at the cellular and subcellular levels as indicated by the appearance of teratological effects on individuals and nuclear alterations. The epipsammic biofilms were exposed for 96 h to chromium at a concentration similar to that measured in highly impacted sites along the coast (80 µg L⁻¹). Chromium pollution, at this concentration and short exposure time did not affect the algal biomass and density of these mature biofilms. The biofilm composition, however, did change, as reflected in a decline in cyanophytes and an increment in the proportions of diatoms and chlorophytes; with Hippodonta hungarica, Navicula novaesiberica, Nitzschia palea, and Sellaphora pupula being the most frequent and abundant species. The most notable shifts related to chromium exposure were a decrease in the relative abundance of H. hungarica and a significant increase in the proportion of N. palea. Moreover, the species analyzed in the treatment microcosms showed higher growth rates than in the controls--N. palea grew faster, while H. hungarica replicated more slowly. The total nuclear abnormalities--as recorded in Fallacia pygmaea and N. novaesiberica--were significantly higher in the treatment microcosms; whereas in N. palea, the dominant species in treatment microcosms, neither nuclear alterations nor abnormal frustules were observed.

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression.

Bicistronic vectors are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. Internal ribosome entry site (IRES) elements have been utilized to initiate expression of an additional protein from a bicistronic vector. The IRES element commonly used in current bicistronic vectors originates from the encephalomyocarditis virus (EMCV). As IRES-mediated translation is dependent on availability of IRES trans-acting factors, which vary between cell types and species, adequate gene expression from the EMCV IRES element is not always achieved. To identify a novel IRES element that mediates gene expression consistently with a higher efficiency than the EMCV IRES, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the vascular endothelial growth factor and type 1 collagen-inducible protein (VCIP) IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results suggest that VCIP-IRES containing vectors improve gene expression compared with those harboring an EMCV-IRES. This could increase the potential benefits of bicistronic vectors for experimental and therapeutic purposes.

[Occult rupture of the spleen in a patient with infectious mononucleosis]. / Rottura splenica "occulta" in paziente con mononucleosi infettiva.

INTRODUCTION: Rupture of the spleen can be secondary to abdominal traumas (usually closed trauma) or spontaneous, can interest an organ normal or with morphological alterations secondary to various pathologies. Among the diseases responsible of occult rupture, infectious diseases are the most frequent and, among these, infectious mononucleosis, that is complicated with splenic rupture in 0.5% of the cases, with 30% of mortality. CASE REPORT: P.M., 16 years old female, admitted with acute abdomen, progressive anaemia and incipient cardiovascular instability, associated with suggestive clinical diagnosis of infectious mononucleosis, confirmed by serological findings and histological examination. Because of the imaging of subcapsular splenic haematoma, probably ruptured and with peritoneal bleeding we opt for emergency laparotomy intraoperative findings allows to splenectomy. DISCUSSION: Splenic rupture in infectious mononucleosis often presents as left hypochondrial pain, rare in uncomplicated cases; its occurrence in a patient with a recent diagnosis of infectious mononucleosis or with clinical or laboratory features suggestive of acute EBV infection, should always be investigated with an urgent abdominal ultrasound scan or CT. This approach is mandatory when hypochondrial pain is associated with pain referred to the left shoulder (Kehr's sign), peritoneal irritation and haemodynamic instability. Patients with splenic rupture in infectious mononucleosis generally undergo emergency splenectomy.

[Retained rectal foreign bodies by trans-anal introduction. Case report]. / Corpi estranei endorettali introdotti per via anale. Case report.

The Authors refer about two cases of retained rectal foreign bodies by trans-anal introduction as consequence of anal eroticism: a deodorant aerosol-can cap and a sizeable phallic object. These reports represent an occasion to talk about the etiology (the wide variety of foreign bodies) and the motivations (eroticism or sadism, clumsy diagnostic and therapeutic procedures, true or presume accidents) responsible for this pathological condition and to consider every therapeutic options employed during the past years, without forgetting that, despite difficulties, non-surgical extraction is to prefer, if possible, because of the negative prognostic implications often related to the surgical treatment. The Authors finally confirm, because of the severity of this pathological condition - with negative outcomes especially in that cases with complete or incomplete perforative complications (produced during introduction through the anus or during several attempts of extraction of the object or caused by its long staying in the rectum because of the patient's denial of medical care) - the surgeons can't put aside possible indication for surgical treatment.

[Acute abdomen due to jejunal perforation secondary to metastatic lung carcinoma]. / Addome acuto da perforazione di metastasi digiunale di carcinoma polmonare operato.

INTRODUCTION: Lung cancer metastases of small bowel are rare (1,1%), often with few or not symptoms. This aspecific onset and the difficult physical-instrumental approach to small bowel, led often to diagnosis at autopsy. This is not true for intestinal metastases that cause complications (haemorrhage, obstruction, perforation); in this cases emergency surgery leds to the diagnosis. CASE REPORT: We describe a case of a male 56 years old patient with acute abdomen due to perforation (X-ray and CT). He refers, about 6 months before, an upper right lobectomy for lung cancer, followed by adjuvant chemo-radiotherapy, because the presence of brain and bone metastases. During the emergency surgery we found out a perforation of the Treitz tract, treated with intestinal resection and immediate end-to-end anastomosis with manual suture. Histological examination shows the perforation of the intestinal wall tract with lung cancer metastases. CONCLUSIONS: Our case shows that any acute abdomen in patient with lung cancer can be considered as expression of intestinal metastases. Negative prognosis of this complication imposes to surgeons only a local treatment.

[Gastrointestinal stromal tumors (GISTs). Personal experience on three cases of the tumors of the small intestine complicated and emergency surgically treated]. / I tumori stromali gastrointestinali (GIST). Esperienza personale su tre casi a localizzazione nel tenue mesenteriale trattati in urgenza.

INTRODUCTION: GISTs, a new nosological entity recently described, represent a peculiar model of solid tumor: the identification of the molecular mechanism responsible for the oncogenesis led to the development of a new drug (imatinib) active on the specific molecular target, represented by the product of the mutated proto-oncogene c-kit which is a tyrosine kinase receptor that becomes constitutively active by mutation. Surgical resection, nevertheless, is still the primary treatment and it has to be as complete as possible. These two treatments can be integrated. GISTs are not uniformly kit-positive, and they can be alternatively due to mutations of the PDGFRA gene or, in patients with neurofibromatosis type 1 (NF-1), to generally isolated mutations of the NF-1 gene. PATIENTS AND METHODS: We describe 3 cases of kit-positive GISTs of the small intestine (SISTs), complicated and emergency surgically treated: case 1--53 years, female, with small bowel obstruction and concomitant acute intestinal bleeding; case 2--71 years, male, with NF-1 and acute intestinal bleeding; case 3--47 years, male, with perforation of the Treitz tract. The first two cases have been treated with intestinal resection and immediate mechanical anastomosis; the third one with resection of the pedunculated tumor at its base, where is situated the perforation too. CONCLUSIONS: SISTs (20-30%), with little or no symptoms in the initial phases, show notable diagnostic difficulties. Their aspecific and late clinical presentation--typical of this site and of the pathology that we are talking about--and the difficult physical-instrumental approach to small bowel limit the possibility of an accurate diagnosis and expose the patient to potentially fatal acute complications and to risks related to emergency surgery treatment that decreases the possibility of a radical resection.

Seasonal and spatial distribution of the microbenthic communities of the Rio de la Plata estuary (Argentina) and possible environmental controls.

The relationship between microbenthic communities, the habitat characteristics, and physical and chemical gradients was studied in the Rio de la Plata estuary. Five replicates of the surface layer were collected seasonally, in 10 sampling sites influenced by different land uses. The distribution of microbenthic communities was governed by two gradients, the first one determined by anthropic factors, related to pollution, and the second one to conductivity and turbidity. The higher densities of producers were observed in sites characterized by fine sediments. During winter, spring, and summer cyanophytes were abundant, while in autumn the diatoms, particularly birraphideans, dominated. The ciliates were the most abundant group among consumers, particularly in winter, and their spatial distribution was influenced by the turbidity. The whole study area has an eutrophic condition. Turbidity and the enrichment with nutrients and organic matter explained 50% of the variability in the species' distribution.

[Management of perforations as adverse events of ERCP plus ES. Personal experience]. / Trattamento degli eventi perforativi post-colangiopancreatografia retrograda con sfinterotomia endoscopica. Esperienza personale.

UNLABELLED: INTRODUCTION. ERCP has brought real progress in the study and treatment of pancreatic and biliary diseases, because of its ambivalence as diagnostic and therapeutic procedure. Among its complications, perforations occur in fewer than 1% of patients, but are associated with a mortality rate of 16% -18%. CASE REPORTS: CASE 1- F, 89 years old with obstructive jaundice by choledocholithiasis submitted to ERCP plus ES, during which occurs type II lesion; the partial removing of stones from choledochus during the procedure allow us to opt for a conservative treatment, with resolution on post-ERCP day 12. CASE 2- F, 53 years old with recurring cholangitis and post-cholecystectomy stenosis of choledochus already treated by stenting; for the occurrence of type I lesion during ERCP, the patient undergoes surgery in emergency with healing in postoperative day 23. CASE 3- M, 84 years old with lithiasic cholecystitis, obstructive jaundice, lung emphysema and ischemic heart disease; after percutaneous cholecystostomy in emergency, we attempt to ERCP with evidence of type I lesion. Because of comorbility, we opt for a conservative treatment, not resolving, and then proceed to surgery. Exitus for cardio-respiratory complications. CASE 4- M, 89 years old with obstructive jaundice; ERCP is suspended for respiratory complications and then a PTC is perform; during it we note a type IV lesion, which is treated conservatively with resignation in day 12. CASE 5- F, 68 years old with cholecystitis and choledocholithiasis; during ERCP plus SE a type II lesion occurs with worsening signs of acute abdomen. Because of clinical conditions and the impossibility of carrying out stones from choledochus by endoscopy, we opt for a surgical treatment in emergency. Exitus for respiratory complications. DISCUSSION: Because of the controversy exists on what should be the management of perforations as adverse events of ERCP plus ES (immediate surgery or conservative therapy), we can only hope an eclectic approach based on the anatomical and clinical peculiarity of each case.

Evidence of bovine immunodeficiency virus (BIV) infection: serological survey in Argentina.

This is the first report of serological evidence for bovine immunodeficiency virus (BIV) infection in Argentina. The analysis was performed in 589 dairy bovine sera samples, applying indirect enzyme-linked immunosorbent assay (I-ELISA) using a synthetic antigen (transmembrane peptide, TM) and Immunofluorescent assay (IFA). In this study, 9 dairy herds from 4 Argentinian provinces were evaluated and 12% of the animals tested positive for BIV. Seven of the 9 herds tested were BIV seropositive and the percentage of BIV seropositive animals in the herds ranged from 2% to 42%. Direct detection of BIV provirus applying nested PCR was not conclusive. Antibody detection against bovine leukemia virus (BLV) in all sera was also performed applying immunodiffusion (ID) assay and 59% resulted seropositive. Statistical analysis of the results was carried out and possible evidence of association between BIV and BLV infection was considered. Future studies should be performed including local field isolates strains of BIV.

An experimental pilot study on controlled portal vein arterialization with an extracorporeal device in the swine model of partial liver resection and ischemia.

AIM: To determine whether the physiologically oxygenated arterial blood reversed in the portal system by means of portal vein arterialization (PVA) through an extracorporeal device which we have called L.E.O2.NARDO (Liver Extracorporeal Oxygen. NARDO) is effective in treating swine with subtotal hepatectomy leading to acute liver failure (ALF). METHODS: Ten swine with ALF induced by 85-90% liver resection and five minutes of ischemia-reperfusion injury were randomly divided into two groups: five animals received PVA extracorporeal treatment and five swine were not-treated (control group). Blood was withdrawn from the iliac artery and reversed in the portal venous system. An extracorporeal device was interposed between the outflow and the inflow in order to monitoring the hemodynamic parameters. Each treatment lasted 6 hours. Serum and liver samples were collected in both groups. The survival was assessed at 1 week. RESULTS: The PVA-extracorporeal treatment yielded beneficial effects for subtotal hepatectomy-induced ALF swine with decreased serum ammonia, transaminases and total bilirubin as compared with the untreated group. INR recovered rapidly in the PVA-extracorporeal group remaining significantly lower than in untreated animals. The 7-day survival of PVA-extracorporeal group swine was significantly higher than that of untreated animals, with a statistically significant difference (p<0.05). Four swine in the PVA-extracorporeal group survived at 1 week while none of the swine in the control group were alive at that time; an average time of 144h+/-13h and 24.4h+/-5h was observed in the PVA-extracorporeal and control groups, respectively. CONCLUSIONS: Arterial blood supply in the portal system through the extracorporeal device is easily applicable, efficacious, safe and may represent a novel approach for ALF swine induced by subtotal liver resection.

The first case of ureteral duplication in a combined liver-kidney transplantation.

AIM: Kidney transplantation with ureteral duplication may represent a doubled risk factor in terms of ureteral stenosis or necrosis with urinary leakage usually from the site of ureteroneocystostomy. The incidence of complete duplication is very low at 0.19%. We report a kidney with ureteral duplication in the specific setting of multiorgan transplantation since it could be considered an adjunctive risk factor for urological complications. METHODS: The recipient was a 67-year old man, suffering from terminal renal insufficiency. He was also affected by HCV-related cirrhosis. The patient had been waiting for the combined transplantation for 27 months and in the last two months his hepatic function dramatically worsened. The donor was a 53-year old man who died of non-traumatic subarachnoid hemorrhage. Good HLA compatibility was observed between donor and recipient. During harvest both kidneys presented a complete ureteral duplication. So the ureters were freed together with a wide cuff of periureteral tissue and dissected distally. No vascular abnormalities were noted during the removal of either kidney. The grafts were flushed with University of Wisconsin solution and stored in the same solution. RESULTS: The liver was reperfused after 9 hours of cold ischemia. Subsequently the kidney was vascularized after 15 hours of cold ischemia. Urine production occurred immediately after revascularization. Two separated ureteroneocystostomies with a single antireflux technique were performed. Cyclosporine and steroids were given. Post-operative course was uneventful and liver and kidney function were normal. The 7-day cystography was normal. The 6, 12, 24 month ultrasonographies showed no signs of hydronephrosis or hydroureter. After 28 months renal cancer was diagnosed and the patient underwent a right nephrectomy. The liver-kidney recipient had excellent hepatic and renal function for 84.7 months. He died of malignancy from de novo tumor. CONCLUSIONS: On the basis of this experience, a kidney with an ureteral duplication, while rare, can be satisfactorily used also in combined liver-kidney transplantation.

Portal vein oxygen supply through a liver extracorporeal device to treat acute liver failure in Swine induced by subtotal hepatectomy: preliminary data.

AIM: To determine whether the increase of oxygen supply in the portal system by a liver extracorporeal (L.E.O.NARDO) device is effective in treating swine with subtotal hepatectomy leading to acute liver failure (ALF). METHODS: Eight swine with ALF induced by 85% to 90% liver resection and 5 minutes of ischemia-reperfusion injury were randomly divided into two groups: four animals received L.E.O.NARDO treatment and four swine were not treated (control group). Blood was withdrawn from the iliac artery and reversed in the portal venous system. An extracorporeal device was interposed between the outflow and the inflow in order to monitor the hemodynamic parameters. Each treatment lasted 6 hours. Serum and liver samples were collected in both groups. The survival was assessed at 1 week. RESULTS: L.E.O.NARDO treatment yielded beneficial effects for subtotal hepatectomy-induced ALF in swine with decreased serum transaminases as compared with the untreated group. International normalized ratio recovered rapidly in the L.E.O.NARDO group, remaining significantly lower than in untreated animals. The 7-day survival of L.E.O.NARDO group swine was significantly higher than that of untreated animals, with a significant difference. Three swine in the L.E.O.NARDO group survived 1 week while none of the swine in the control group were alive at that time. CONCLUSIONS: Oxygen supply in the portal vein through the L.E.O.NARDO device is easily applicable, efficacious, and safe and may represent a novel approach for ALF in swine induced by subtotal liver resection.