DnIKK2-transfected dendritic cells induce a novel population of inducible nitric oxide synthase-expressing CD4+CD25- cells with tolerogenic properties

BACKGROUND: We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4 T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. METHODS: Brown Norway (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4 T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4 T cells pre-exposed to dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. RESULTS: CD4 T cells pre-exposed to dnIKK2-DCs were CD4CD25 and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:10 ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). CONCLUSIONS: DnIKK2-Treg is a unique population of CD4CD25 T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.     

A cytoprotective role for the heme oxygenase-1/CO pathway during neural differentiation of human mesenchymal stem cells

The inducible protein heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to carbon monoxide (CO) and biliverdin, which play a concerted action in cytoprotection against oxidative stress and in the modulation of cell proliferation and differentiation. Here we report that both HO-1 expression and activity can be highly increased in undifferentiated human mesenchymal stem cells (MSCs) treated with hemin, a known HO-1 inducer. However, HO-1 mRNA and protein expression gradually decrease when MSCs undergo neural differentiation in vitro, making them extremely susceptible to glutamate-mediated cytotoxicity. A time course for HO-1 revealed that this protein is markedly down-regulated after 2 days and returns to control levels 6 days after differentiation. Treatment with glutamate (250 microM) after 2 days of neural differentiation resulted in a more pronounced lactate dehydrogenase release, a marker of cell injury, compared with undifferentiated cells. Notably, cells pretreated with hemin (50 microM) or compounds that release small amounts of CO (10 microM CORM-3 and CORM-A1) rendered cells more resistant to glutamate-induced toxicity; this effect was evident in both undifferentiated and differentiated MSCs. Our findings indicate that MSCs become more vulnerable to oxidative injury during the early stages of differentiation via mechanisms that involve a temporary inhibition of HO-1 expression. Thus, overexpression of HO-1 and CO-releasing molecules could provide a possible therapeutic strategy to improve cell viability during neural differentiation in applications that use stem cell technology.     

Human osteoblast-like cell adhesion on titanium substrates covalently functionalized with synthetic peptides

Biomaterials to be used for the production of endosseous devices in dental, orthopedic and maxillo-facial applications, might be designed to support, guide and enhance osteoblast adhesion. Cell recruitment onto biomaterial surface is a fundamental step within the complex process responsible for implant osseointegration; this process involves several proteins from the extra cellular matrix (ECM), cytoskeleton and cell membrane. A new strategy to improve endosseous implant integration is based on preparing biomimetic surfaces able to present adhesive factors to cells. Osteoblast adhesion takes place by at least two different mechanisms: the most investigated one implies the interaction with RGD sequences via cell-membrane integrin receptors; a further mechanism concerns the interaction between cell-membrane heparan sulfate proteoglycans and heparin-binding sites of ECM proteins. In the present study two different biomimetic surfaces were obtained by covalently grafting two adhesive peptides on oxidized titanium substrates after silanization: an RGD-containing peptide and a peptide mapped on human vitronectin. The two sequences are known to act via different adhesive mechanisms. The amount of human osteoblasts adhered onto peptide-enriched or not enriched titanium oxidized surfaces and the strength of cell binding were estimated, thus comparing the capacity of the bioactive substrates in promoting cell adhesion.     

Thrombectomy and surgical reconstruction for extensive iliocaval thrombosis in a patient with agenesis of the retrohepatic vena cava and atresia of the left renal vein

In 80% of the patients presenting with deep-venous thrombosis (DVT), a risk factor can be identified. An absent or hypoplastic infrarenal vena cava is a rare risk factor for DVT in young adults. In these cases, the prevalence of congenital anomalies of the inferior vena cava (IVC) is estimated at 0.5% of the general population, up to 5% in young people. The association with coagulopathy increases the risk of DVT. We report a case of a young man who presented with a massive caval and iliofemoral-popliteal thrombosis in presence of the agenesis of retrohepatic inferior vena cava and atresia of the left renal vein. Open thrombectomy and caval reconstruction with a polytetrafluoroethylene graft were performed. Surgical option with vein reconstruction was preferred to prevent new episodes of thrombosis and the risk of acute renal failure.     

Complex anal fistula with a recess above the levator ani muscles: report of a case complicated by haemorrhagic colitis

Anal fistula surgery offers very little in the way of new developments. As early as the 14th century John of Arden defined the rules for surgical therapy. The numerous classifications adopted are often contradictory and unclear, and no clearly defined pathogenesis of the condition has been established. Complex anal fistula with a recess above the elevator ani muscles requires very careful and meticulous therapeutic treatment because of the risk of damaging the sphincter. In the case reported here the patient presented a very complex fistula which was followed by onset of severe haemorrhagic colitis without any clinical, endoscopic, radiological, or histological evidence of inflammatory bowel disease prior to surgical treatment.     

Allogeneic Blood Transfusion Does Not Affect Outcome After Curative Resection for Advanced Cholangiocarcinoma

Purpose

To assess the impact of perioperative blood transfusion on overall and disease-free survival in patients undergoing curative resection for cholangiocarcinoma.

Methods

In a single-center study, 128 patients undergoing curative resection for cholangiocarcinoma between 2001 and 2010 were assessed. The median follow-up period was 19 months. Transfused and nontransfused patients were compared by Cox regression and propensity score analyses.

Results

Overall, 38 patients (29.7 %) received blood transfusions. The patient characteristics were highly biased with respect to receiving transfusions (propensity score 0.69 ± 0.22 vs. 0.11 ± 0.16, p < 0.001). In the unadjusted analysis, blood transfusion was associated with a 105 % increased risk of mortality [hazard ratio (HR) 2.05, 95 % CI 1.19–3.51, p = 0.010]. In the multivariate (HR 1.14, 95 % CI 0.52–2.48, p = 0.745) and the propensity score-adjusted Cox regression (HR 1.02, 95 % CI 0.39–2.62, p = 0.974), blood transfusion had no influence on overall survival. Similarly, in the propensity score-adjusted Cox regression (HR 0.62, 95 % CI 0.24–1.58, p = 0.295), no relevant effect of blood transfusion on disease-free survival was observed.

Conclusions

To our knowledge, this is the first propensity score-based analysis providing compelling evidence that the worse oncological outcome after curative resection for advanced cholangiocarcinoma in patients receiving perioperative blood transfusions is caused by the clinical circumstances requiring the transfusions, not by the blood transfusions themselves.     

The role of the renin-angiotensin system in aortic aneurysmal diseases

The renin-angiotensin system has been invoked in the development of both abdominal and thoracic aortic aneurysms. This has been demonstrated experimentally by the chronic subcutaneous infusion of angiotensin II, which consistently leads to development of abdominal aortic aneurysms (AAAs) in mice. Angiotensin II-induced AAAs have highly heterogenous cellular and extracellular matrix characteristics throughout the aorta that change markedly with infusion duration. The mechanistic basis for the reproducible location of AAA development has not been elucidated, but many insights have been provided, especially regarding receptor and inflammatory mechanisms. A recent clinical study provided limited evidence for extrapolating these results to mechanisms of human AAAs. Experimental evidence has also demonstrated that antagonism of angiotensin II type 1 (AT1) receptors prevents ascending aortic aneurysms in a murine model of Marfan’s syndrome. A clinical study is currently ongoing to demonstrate the efficacy of AT1 receptor antagonism in humans.     

Vascular endothelial growth factor gene polymorphisms in Behçet's disease

OBJECTIVE: To evaluate potential associations of vascular endothelial growth factor (VEGF) gene polymorphisms with Beh?et's disease (BD) and disease expression. METHODS: Case patients were 122 consecutive Italian patients with BD followed at the Rheumatology, Ophthalmology, and Neurology Units in Bologna, Ferrara, Milano, Palermo, Potenza, Prato, Reggio Emilia, and Trento over a 3-year period (1997-99) and who satisfied the International Study Group criteria for BD. Also selected as a control group were 200 healthy age and sex matched blood donors. All patients with BD and controls were genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for +936 C/T (rs3025039) and -634 C/G (rs2010963) mutations and for an 18 base pair (bp) insertion/deletion (I/D) polymorphism at -2549 of the the VEGF promoter region. In vitro release of VEGF by peripheral blood mononuclear cells (PBMC) was investigated by ELISA in healthy controls homozygous for the polymorphisms studied. RESULTS: The carriage rates of the alleles I and -634C were significantly more frequent in patients with BD than in healthy controls [p corr = 0.036, OR 1.8 (95% CI 1.1-2.9) and p corr = 0.05, OR 1.8 (95% CI 1.1-3.0), respectively]. While the distribution of allele +936T was similar in patients with BD and healthy controls, its frequency was significantly higher in BD patients with posterior uveitis/retinal vasculitis than in those without (p = 0.022, OR 2.4, 95% CI 1.1-5.0). Lipopolysaccharide-stimulated VEGF production from PBMC of healthy subjects was higher in II homozygous than in DD homozygous. CONCLUSION: Our data indicate that carriers of -634C and I alleles are associated with susceptibility to developing BD.