5 research outputs found
Exogenous HIV-1 Nef Upsets the IFN-γ-Induced Impairment of Human Intestinal Epithelial Integrity
The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions
CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin
Purpose: The paucity of new drugs for the treatment of Ewing
sarcoma (EWS) limits the cure of these patients. CD99has a strong
membranous expression inEWScells and, being also necessary for
tumor survival, is a suitable target to aim at. In this article, we
described a novel human monospecific bivalent single-chain
fragment variable diabody (dAbd C7) directed against CD99 of
potential clinical application.
Experimental Design: In vitro and in vivo evaluation of cell
death and of the molecular mechanisms triggered by anti-CD99
agents were performed alone or in combination with doxorubicin
to demonstrate efficacy and selectivity of the new dAbd C7.
Results: The dAbd C7 induced rapid and massive EWS cell
death through Mdm2 degradation and p53 reactivation. Mdm2
overexpression as well as silencing of p53 in p53wt EWS cells
decreased CD99-induced EWS cell death, whereas treatment with
nutlin-3 enhanced it. Furthermore, cell death was associated
with induction of p21, bax, and mitochondrial depolarization
together with substantial inhibition of tumor cell proliferation.
Combined treatment of anti-CD99 dAbd C7 with doxorubicin
was additive both in vitro and in vivo against EWS xenografts.
Normal mesenchymal stem cells showed no p53 activation and
were resistant to cell death, unless transformed by EWS-FLI, the
oncogenic driver of EWS.
Conclusions: These results indicate that dAbd C7 is a suitable
candidate tool to target CD99 in patients with EWS able to spare
normal stem cells from death as it needs an aberrant genetic
context for the efficient delivery of CD99-triggered cell death
Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing's sarcoma
Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60Â % soluble fraction, we obtained a yield of 25Â mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75Â % soluble fraction 35Â mg C7 dAbd per L of cell culture grown in a synthetic media containing 3Â g/L of vegetable peptone and 1Â g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells