We formerly demonstrated that vaccination with Wilms' tumor 1 (WT1)-loaded autologous monocyte-derived dendritic cells (mo-DCs) can be a well-tolerated effective treatment in acute myeloid leukemia (AML) patients. Here, we investigated whether we could introduce the receptor for hyaluronic acid-mediated motility (RHAMM/HMMR/CD168), another clinically relevant tumor-associated antigen, into these mo-DCs through mRNA electroporation and elicit RHAMM-specific immune responses. While RHAMM mRNA electroporation significantly increased RHAMM protein expression by mo-DCs, our data indicate that classical mo-DCs already express and present RHAMM at sufficient levels to activate RHAMM-specific T cells, regardless of electroporation. Moreover, we found that RHAMM-specific T cells are present at vaccination sites in AML patients. Our findings implicate that we and others who are using classical mo-DCs for cancer immunotherapy are already vaccinating against RHAMM.

Glioblastoma (GB) is a devastating disease for which new treatment modalities are needed. Efficacious therapy requires the removal of stem-cell like cells, these cells drive tumor progression because of their ability to self-renew and differentiate. In glioblastoma, the GB stem-like cells (GSC) form a small population of tumor cells and possess high resistance to chemo and radiation therapies. To assess the sensitivity of GSC to reovirus-mediated cytolysis, a panel of GSC cultures was exposed to wild-type reovirus Type 3 Dearing (T3D) and its junction adhesion molecule-A (JAM-A)-independent mutant, jin-1. Several parameters were evaluated, including the fraction of cells expressing the JAM-A reovirus receptor, the fraction of cells synthesizing reovirus proteins, the number of infectious reovirus particles required to reduce cell viability, the amount of infectious progeny reovirus produced and the capacity of the reoviruses to infect the GSC in 3-dimensional (3D) tumor cell spheroids. Our data demonstrate a marked heterogeneity in the susceptibility of the cultures to reovirus-induced cytolysis. While in monolayer cultures the jin-1 reovirus was generally more cytolytic than the wild-type reovirus T3D, in the 3D GSC spheroids, these viruses were equally effective. Despite the variation in reovirus sensitivity between the different GSC cultures, our data support the use of reovirus as an oncolytic agent. It remains to be established whether the variation in the reovirus sensitivity correlates with a patient's response to reovirus therapy. Moreover, our data show that the expression of the JAM-A receptor is not a major determinant of reovirus sensitivity in 3D GSC cultures.

Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be a leading cause of mortality among bacterial diseases, and the bacillus Calmette-Guérin (BCG) is the only licensed vaccine for human use against this disease. TB prevention and control would benefit from an improved method of BCG vaccination that simplifies logistics and eliminates dangers posed by hypodermic needles without compromising immunogenicity. Here, we report the design and engineering of a BCG-coated microneedle vaccine patch for a simple and improved intradermal delivery of the vaccine. The microneedle vaccine patch induced a robust cell-mediated immune response in both the lungs and the spleen of guinea pigs. The response was comparable to the traditional hypodermic needle based intradermal BCG vaccination and was characterized by a strong antigen specific lymphocyte proliferation and IFN-γ levels with high frequencies of CD4(+)IFN-γ(+), CD4(+)TNF-α(+) and CD4(+)IFN-γ(+)TNF-α(+) T cells. The BCG-coated microneedle vaccine patch was highly immunogenic in guinea pigs and supports further exploration of this new technology as a simpler, safer, and compliant vaccination that could facilitate increased coverage, especially in developing countries that lack adequate healthcare infrastructure.
Copyright © 2011 Elsevier Ltd. All rights reserved.

The innate immune response is a double-edged sword in systemic inflammation and sepsis. Uncontrolled or inappropriate activation can damage and be lethal to the host. Several studies have investigated inhibition of downstream mediators, including tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Emerging evidence indicates that upstream inhibition is a better therapeutic approach for attenuating damaging immune activation. Therefore, we investigated inhibition of two central innate immune pathways, those of complement and CD14/Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), in a porcine in vitro model of Escherichia coli-induced inflammation. Porcine whole blood anticoagulated with lepuridin, which did not interfere with the complement system, was incubated with E. coli lipopolysaccharide (LPS) or whole bacteria. Inhibitors of complement and CD14 and thus the LPS CD14/TLR4/MD-2 receptor complex were tested to investigate the effect on the inflammatory response. A broad range of inflammatory readouts were used to monitor the effect. Anti-CD14 was found to saturate the CD14 molecule on granulocytes and completely inhibited LPS-induced proinflammatory cytokines in a dose-dependent manner. Anti-CD14 significantly reduced the levels of the E. coli-induced proinflammatory cytokines TNF-alpha and IL-1beta, but not IL-8, in a dose-dependent manner. No effect on bacterial clearance was seen. Vaccinia complement control protein and smallpox inhibitor of complement enzymes, two Orthopoxvirus-encoded complement inhibitors, completely inhibited complement activation. Furthermore, these agents almost completely inhibited the expression of wCD11R3, which is associated with CD18 as a beta2 integrin, on porcine granulocytes and decreased IL-8 levels significantly in a dose-dependent manner. As expected, complement inhibition reduced bacterial clearance. We conclude that inhibition of complement and CD14 attenuates E. coli-induced inflammation and might be used as a therapeutic regimen in gram-negative sepsis along with appropriate treatment with antibiotics.

The pathophysiology of meconium aspiration syndrome (MAS) is complex. We recently showed that meconium is a potent activator of complement. In the present study, we investigated whether the complement activation occurring in experimental MAS is associated with a systemic inflammatory response as judged by granulocyte activation and cytokine and chemokine release. MAS was induced by the instillation of meconium into the lungs of newborn piglets (n = 8). Control animals (n = 5) received saline under otherwise identical conditions. Haemodynamic and lung dynamic data were recorded. Complement activation, revealed by the terminal sC5b-9 complex (TCC), and cytokines [interleukin (IL)-6 and IL-8] were measured in plasma samples by enzyme immunoassays. The expression of CD18, CD11b and oxidative burst in granulocytes was measured in whole blood by flow cytometry. Plasma TCC increased rapidly in the MAS animals in contrast with controls (P < 0.0005). The TCC concentration correlated closely with oxygenation index (r = 0.48, P < 0.0005) and ventilation index (r = 0.57, P < 0.0005) and inversely with lung compliance (r = -0.63, P < 0.0005). IL-6 and IL-8 increased in MAS animals compared with the controls (P = 0.002 and P < 0.001, respectively). Granulocyte oxidative burst declined significantly in the MAS animals compared with the controls (P < 0.02). TCC correlated significantly with IL-6 (r = 0.64, P < 0.0005) and IL-8 (r = 0.32; P = 0.03) and inversely with oxidative burst (r = -0.37; P = 0.02). A systemic inflammatory response associated with complement activation is seen in experimental MAS. This reaction may contribute to the pathogenesis of MAS.