RNA aptamers specific for transmembrane p24 trafficking protein 6 and Clusterin for the targeted delivery of imaging reagents and RNA therapeutics to human β cells

The ability to detect and target β cells in vivo can substantially refine how diabetes is studied and treated. However, the lack of specific probes still hampers a precise characterization of human β cell mass and the delivery of therapeutics in clinical settings. Here, we report the identification of two RNA aptamers that specifically and selectively recognize mouse and human β cells. The putative targets of the two aptamers are transmembrane p24 trafficking protein 6 (TMED6) and clusterin (CLUS). When given systemically in immune deficient mice, these aptamers recognize the human islet graft producing a fluorescent signal proportional to the number of human islets transplanted. These aptamers cross-react with endogenous mouse β cells and allow monitoring the rejection of mouse islet allografts. Finally, once conjugated to saRNA specific for X-linked inhibitor of apoptosis (XIAP), they can efficiently transfect non-dissociated human islets, prevent early graft loss, and improve the efficacy of human islet transplantation in immunodeficient in mice

In vivo induction of myeloid suppressor cells and CD4+Foxp3+ T regulatory cells prolongs skin allograft survival in mice.

Natural CD4+Foxp3+ T regulatory (Treg) cells can promote transplantation acceptance across MHC barriers, while Myeloid-derived suppressor cells (MDSCs) inhibit effector T cell responses in tumor-bearing mice. One outstanding issue is whether combining the potent suppressive function of MDSCs with that of Treg cells might synergistically favor graft tolerance. In the present study, we evaluated the therapeutic potential of MDSCs and natural Treg cells in promoting allograft tolerance in mice by utilizing immunomodulatory agents to expand these cells in vivo. Upon administration of recombinant human G-CSF (Granulocyte-Colony Stimulating Factor; Neupogen), or interleukin-2 complex (IL-2C), Gr-1+CD11b+ MDSCs or CD4+Foxp3+ Treg cells were respectively induced at a high frequency in the peripheral lymphoid compartments of treated mice. Interestingly, induced MDSCs exhibited a more potent suppressive function in vitro when compared to MDSCs from na\uefve mice. Furthermore, in vivo co-administration of Neupogen and IL-2C induced MDSCs at percentages that were higher than those seen when either agent was administered alone, suggesting an additive effect of the two drugs. Although treatment with either IL-2C or Neupogen led to a significant delay of major histocompatibility complex (MHC) class II disparate allogeneic donor skin rejection, the combinatorial treatment was superior to either alone. Importantly, histological assessment of surviving grafts revealed intact morphology and minimal infiltrates at 60 days post transplant. Collectively, our findings demonstrate that concurrent induction of MDSCs and Tregs is efficacious in downmodulating alloreactive T cell responses in a synergistic manner and highlight the therapeutic potential of these naturally occurring suppressive leukocytes to promote transplantation tolerance

Antitumor DNA vaccines

The inoculation of plasmid DNA coding for a protein antigen by means of a simple intramuscolar (i.m.) or intradermal injection, or the more sophisticated "gene gun", currently represents an easily performed vaccine approach that is safe for host and relatively inexpensive. DNA vaccines have been described to generate humoral as well as cellular immune responses in various preclinical models of infectious and noninfectious diseases, such as autoimmune deseases and tumors, and also induced immunological memory. As this vaccine modality is still quite young, further study is needed to assure that when transferred to a clinical setting it will obtain results similar to those observed in animals. Our research group has studied the potentialities of this approach in the field of tumor immunotherapy. We utilized three different experimental tumor models to study the immune response induced by some DNA vaccines encoding proteins with characteristics similar to tumor-specific or differentiation antigens identified in human neoplasias. We observed that i.m. injection of a plasmid coding for a tumor antigen generated a specific immune response that was measurable in vitro as cytotoxic activity against antigenically correlated tumor cells. This response was efficacious in protecting a high percentage of animals from a subsequent tumor challenge, but was therapeutic only in very few tumor-bearing animals. Moreover, preventive immunity was even observed when animals were immunized with a differentiation antigen also expressed by cells of the tissue from which the tumor originates, thus indicating that the vaccination with DNA was able to break the state of central or peripheral tolerance that normally is established against a self antigen. Finally, we could demonstrate the immunogenic capacity of a plasmid that encodes a subdominant epitope of the tumor specific response, and compared it with that generated by a plasmid encoding the immunodominant epitope. This finding constitutes the premise for the potential use of subdominat epitopes in constructing DNA vaccines that would be particularly useful against tumor variants that have lost the expression of the immunodominant antigen or of the MHC molecule required for its recognition by T lymphocytes

Age is a risk factor for maladaptive changes in rats exposed to increased pressure loading of the right ventricular myocardium

Objective: To study the adaptive potential of the right ventricular myocardium after 30 days of mechanical-induced overload in rats from two different age groups. Materials and methods: We banded the pul- monary trunk, so as to increase the systolic work load of the right ventricle, in 19 adult Sprague-Dawley rats at the age of 10 weeks, and 16 weanlings when they were 3 weeks-old, using 10 adults and 10 weanlings as con- trols. We analysed the functional adaptation and structural changes of the right ventricular myocardium, blood vessels and interstitial tissue after 30 days of increased afterload. Results: The increased workload induced an increase of the right ventricular weight and free wall thickness in animals from both age groups when compared to controls. These changes were mostly related to cardiomyocytic hypertrophy, as confirmed by the expression of myocardial hypertrophic markers, without any apparent increase of their number, a \u201creactive\u201d fibrosis espe- cially evident in the adult rats, with p-value less than 0.0001, and a more extensive neocapillary network in the weanlings compared to the adults aubsequent to banding, the p-value being less than 0.0001. Conclusion: In response to right ventricular afterload, weanlings showed a higher adaptive capillary growth, which hampered the development of fibrosis as seen in the adult rats. Age seems to be a risk factor for adverse structural-functional changes of right ventricle subjected to increased workload

In vivo administration of artificial antigen-presenting cells activates low-avidity T cells for treatment of cancer

The development of effective antitumor immune responses is normally constrained by low-avidity, tumor-specific CTLs that are unable to eradicate the tumor. Strategies to rescue antitumor activity of low-avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low-avidity CTLs, we immunized mice bearing lung melanoma metastases with artificial antigen-presenting cells (aAPC), made by covalently coupling pepMHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system, and complete tumor eradication in a mouse TRP-2 antigen system, when low-avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis, and TCR downregulation in low-avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherap