Dana C. Baiu

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1α increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1α-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.

Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies

Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

Linker-free conjugation and specific cell targeting of antibody functionalized iron-oxide nanoparticles

Specific targeting is a key step to realize the full potential of iron oxide nanoparticles in biomedical applications, especially tumor-associated diagnosis and therapy. Here, we developed anti-GD2 antibody conjugated iron oxide nanoparticles for highly efficient neuroblastoma cell targeting. The antibody conjugation was achieved through an easy, linker-free method based on catechol reactions. The targeting efficiency and specificity of the antibody-conjugated nanoparticles to GD2-positive neuroblastoma cells were confirmed by flow cytometry, fluorescence microscopy, Prussian blue staining and transmission electron microscopy. These detailed studies indicated that the receptor-recognition capability of the antibody was fully retained after conjugation and the conjugated nanoparticles quickly attached to GD2-positive cells within four hours. Interestingly, longer treatment (12 h) led the cell membrane-bound nanoparticles to be internalized into cytosol, either by directly penetrating the cell membrane or escaping from the endosomes. Last but importantly, the uniquely designed functional surfaces of the nanoparticles allow easy conjugation of other bioactive molecules.

Potential Pathways to Restore β-Cell Mass: Pluripotent Stem Cells, Reprogramming, and Endogenous Regeneration

Currently available β-cell replacement therapies for patients with diabetes, including islet and pancreas transplantation, are largely successful in restoring normal glucose metabolism, but the scarcity of organ donors restricts their more widespread use. To solve this supply problem, several different strategies for achieving β-cell mass restoration are being pursued. These include the generation of β cells from stem cells and their subsequent transplantation, or regeneration-type approaches, such as stimulating endogenous regenerative mechanisms or inducing reprogramming of non-β cells into β cells. Because these strategies would ultimately generate allogeneic or syngeneic β cells in humans, the control of alloimmunity and/or autoimmunity in addition to replacing lost β cells will be of utmost importance. We briefly review the recent literature on these three promising strategies toward β-cell replacement or restoration and point out the major issues impacting their translation to treating human diabetes.

Interactions of Iron Oxide Nanoparticles with the Immune System: Challenges and Opportunities for their Use in Nano-oncology

Iron oxide (IO) nanoparticles hold great promise as diagnostic and therapeutic agents in oncology. Their intrinsic physical properties make IO nanoparticles particularly interesting for simultaneous drug delivery, molecular imaging, and applications such as localized hyperthermia. Multiple non-targeted IO nanoparticle preparations have entered clinical trials, but more exciting, new tumortargeted IO nanoparticle preparations are currently being tested in preclinical settings. This paper will analyze the challenges faced by this new theranostic modality, with a specific focus on the interactions of IO nanoparticles with the innate and adaptive immune systems, and their effect on nanoparticle biodistribution and tumor targeting. Next, we will review the critical need for innovative surface chemistry solutions and strategies to overcome the immune interactions that prevent existing tumor-targeted IO preparations from entering clinical trials. Finally, we will provide an outlook for the future role of IO nanoparticles in oncology, which have the promise of becoming significant contributors to improved diagnosis and treatment of cancer patients.

Autoantibodies to vascular smooth muscle are pathogenic for vasculitis.

We have previously shown that microvascular smooth muscle activates CD4+ T lymphocytes in sterile co-culture, presents antigen, and produces inflammatory cytokines. Adoptive transfer of lymphocytes co-cultured with syngeneic smooth muscle cells to healthy recipient mice results in vasculitic lesions predominantly in postcapillary venules. The present study assessed the pathogenic role of immunoglobulin and B cells in a murine model of vasculitis. Here, we show that transferred B cells, including plasmablast cells, accumulated, persisted, and proliferated in lung and secondary lymphoid organs of recipient mice. The induction of vasculitis was accompanied by production of IgM and IgG2a autoantibodies specific for vascular smooth muscle intracellular antigens. Circulating immunoglobulin had a pathogenic role in this vasculitis model, because the disease could be induced by transfer of serum from vasculitic mice to untreated animals but not by transfer of serum depleted of anti-smooth muscle autoantibodies. Additionally, the pathogenic mechanisms triggered by the transfer of vasculitogenic serum were dependent on T lymphocytes because both wild-type and B cell-deficient mice developed the disease after serum transfer, whereas RAG2-deficient mice did not. Thus, immunoglobulin and cell-mediated pathways work in concert to produce vasculitis in this model.

High specificity targeting and detection of human neuroblastoma using multifunctional anti-GD2 iron-oxide nanoparticles

AIM To develop biocompatible, tumor-specific multifunctional iron-oxide nanoconstructs targeting neuroblastoma, an aggressive pediatric malignancy. MATERIALS & METHODS Clinical-grade humanized monoclonal antibody (hu14.18K322A), designed to target GD2 antigen on neuroblastoma with reduced nonspecific immune interactions, was conjugated to hydroxyethyl starch-coated iron-oxide nanoparticles. Targeting capability in vitro and in vivo was assessed by immunofluorescence, electron microscopy, analytical spectrophotometry, histochemistry and magnetic resonance R2* relaxometry. RESULTS The biocompatible nanoconstructs demonstrated high tumor specificity in vitro and in vivo, and low background uptake in a mouse flank xenograft model. Specific accumulation in tumors enabled particle visualization and quantification by magnetic resonance R2* mapping. CONCLUSION Our findings support the further development toward clinical application of this anti-GD2 iron-oxide nanoconstruct as diagnostic and therapeutic scaffold for neuroblastoma and potentially other GD2-positive malignancies.

A small population of vasculitogenic T cells expands and has skewed T cell receptor usage after culture with syngeneic smooth muscle cells

Adoptive transfer of lymphocytes co-cultured with syngeneic smooth muscle (SM) cells to healthy recipient mice results in vasculitic lesions predominantly in post-capillary venules. The present study focuses on the mechanisms by which the disease-inducing CD4(+) T cells are generated in co-culture of lymphocytes with SM cells. Microvascular SM cells provide survival signals to both CD4(+) and CD8(+) naïve syngeneic T cells and can activate only a limited range of CD4(+) T lymphocytes in culture. Additionally, approximately 0.4% of the original CD4(+) T cells divide at least twice in co-culture with SM cells. Survival of CD4(+) T cells in co-culture is dependent on a TCR mediated process, since transgenic CD4 (+)cells with a unique specificity for a non-murine peptide do not survive in culture with SM. Analysis of TCR Vbeta shows no superantigen activation of T cells following co-culture with SM cells. Spectratype analysis of TCR Vbeta Jbeta segment usage reveals a skewage in the TCR repertoire of T cells co-cultured with SM, and also of T cells from vasculitic lung. These results are consistent with a specific immune response of pathogenic T cells against one or more activating antigenic determinants of the microvascular SM cells, in contrast to non-specific cytokine activation.

CD4+ T Cells Sensitized by Vascular Smooth Muscle Induce Vasculitis, and Interferon Gamma Is Critical for the Initiation of Vascular Pathology

Primary vasculitis is the result of idiopathic inflammation in blood vessel walls. T cells are believed to play a critical role, but the nature of the pathological T-cell response remains obscure. In this study, we provide evidence that CD4(+) T lymphocytes, activated in the presence of syngeneic vascular smooth muscle cells, were sufficient to induce vasculitic lesions after adoptive transfer to recipient mice. Additionally, the disease is triggered in the absence of antibodies in experiments in which both the donors of stimulated lymphocytes and the transfer recipients were mice that were deficient in B cells. Tracking and proliferation of the transferred cells and their cytokine profiles were assessed by fluorescence tagging and flow cytometry. Proliferating CD4(+) T cells were evident 3 days after transfer, corresponding to the occurrence of vasculitic lesions in mouse lungs. The transferred T lymphocytes exhibited Th1 and Th17 cytokine profiles and minimal Th2. However, 1 week after vasculitis induction, effector functions could be successfully recalled in Th1 cells, but not in Th17 cells. Additionally, in the absence of constitutive interferon-γ expression, T cells sensitized by vascular smooth muscle cells failed to induce vasculitis. In conclusion, our results show that Th1 cells play a key role in eliciting vasculitis in this murine model and that induction of the disease is possible in the absence of pathogenic antibodies.

Targeted Molecular Radiotherapy of Pediatric Solid Tumors Using a Radioiodinated Alkyl-Phospholipid Ether Analog

External-beam radiotherapy plays a critical role in the treatment of most pediatric solid tumors. Particularly in children, achieving an optimal therapeutic index to avoid damage to normal tissue is extremely important. Consequently, in metastatic disease, the utility of external-beam radiotherapy is limited. Molecular radiotherapy with tumor-targeted radionuclides may overcome some of these challenges, but to date there exists no single cancer-selective agent capable of treating various pediatric malignancies independently of their histopathologic origin. We tested the therapeutic potential of the clinical-grade alkyl-phospholipid ether analog CLR1404, 18-(p-iodophenyl)octadecyl phosphocholine, as a scaffold for tumor-targeted radiotherapy of pediatric malignancies. Methods: Uptake of CLR1404 by pediatric solid tumor cells was tested in vitro by flow cytometry and in vivo by PET/CT imaging and dosimetry. The therapeutic potential of 131I-CLR1404 was evaluated in xenograft models. Results: In vitro, fluorescent CLR1404-BODIPY showed significant selective uptake in a variety of pediatric cancer lines compared with normal controls. In vivo tumor-targeted uptake in mouse xenograft models using 124I-CLR1404 was confirmed by imaging. Single-dose intravenous injection of 131I-CLR1404 significantly delayed tumor growth in all rodent pediatric xenograft models and extended animal survival while demonstrating a favorable side effect profile. Conclusion: 131I-CLR1404 has the potential to become a tumor-targeted radiotherapeutic drug with broad applicability in pediatric oncology. Because 131I-CLR1404 has entered clinical trials in adults, our data warrant the development of pediatric clinical trials for this particularly vulnerable patient population.