Dragan Jevremovic

Diverse Origin and Function of Cells With Endothelial Phenotype Obtained From Adult Human Blood

Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14+ subpopulation of PBMCs but OECs developed exclusively from the CD14− fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new cell-therapeutic approaches to ischemic cardiovascular disease.

Autologous Culture-Modified Mononuclear Cells Confer Vascular Protection After Arterial Injury

Background—Bone marrow–derived cells have been shown to contribute to endothelial replacement after vascular injury. In vitro culture of peripheral blood mononuclear cells produces cells with phenotypic characteristics of endothelium. To test the hypothesis that delivery of autologous culture-modified mononuclear cells (CMMCs) to injured arteries could attenuate the vascular response to injury, a rabbit model was studied. Methods and Results—Rabbit peripheral blood mononuclear cells were cultured in endothelial growth media for 7 to 12 days, yielding highly proliferative cells with distinct endothelial phenotype (expressing CD31 and endothelial nitric oxide synthase and capable of acetylated LDL uptake). A rabbit model of balloon carotid injury was used to evaluate the effect of day 7 CMMC delivery on vascular responses. Animals underwent balloon injury and immediate delivery of autologous CMMCs or buffered saline by 20 minutes of local dwelling. Fluorescence-labeled CMMCs were detected in all vessel layers 4 weeks after delivery. Colonies of cells that localized to the lumen and stained for endothelial markers were also identified. Local CMMC administration at the time of balloon injury accelerated reendothelialization at 4 weeks compared with saline (P <0.05). Moreover, CMMC delivery markedly improved endothelium-dependent vasoreactivity at 4 weeks compared with saline (P <0.005). Finally, CMMC treatment reduced neointimal formation by 55% at 4 weeks (P <0.05). Conclusions—These data demonstrate that delivery of CMMCs to balloon-injured arteries is associated with accelerated reendothelialization, enhanced endothelium-dependent vasoreactivity, and reduced neointimal formation. Thus, delivery of autologous CMMCs represents a novel vasculoprotective approach to attenuate the response to acute vascular injury.

Validation of cell-based fluorescence assays: Practice guidelines from the ICSH and ICCS – part V – assay performance criteria

Multi‐color flow cytometry is a unique technology, which enables the analysis of heterogeneous cellular systems and provides multiparametric information on a cell‐by‐cell basis. A variety of factors contribute to the complexity of validating cell‐based flow cytometric methods, including the lack of fully characterized cellular reference materials and the difficulty in obtaining, or creating, samples with varying levels of a given cell type or varying levels of expression of a given antigen. This document summarizes validation requirements and describes validation strategies for quasi‐quantitative and qualitative cell‐based flow cytometric assays.

Regulation of NK Cell-Mediated Cytotoxicity by the Adaptor Protein 3BP2

Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95vav and phospholipase C-γ isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.

Gene Therapy to Manipulate Effector T Cell Trafficking to Tumors for Immunotherapy

Strategies that generate tumor Ag-specific effector cells do not necessarily cure established tumors. We hypothesized that the relative efficiency with which tumor-specific effector cells reach the tumor is critical for therapy. We demonstrate in this study that activated T cells respond to the chemokine CCL3, both in vitro and in vivo, and we further demonstrate that expression of CCL3 within tumors increases the effector T cell infiltrate in those tumors. Importantly, we show that adenoviral gene transfer to cause expression of CCL3 within B16ova tumors in vivo increases the efficacy of adoptive transfer of tumor-specific effector OT1 T cells. We additionally demonstrate that such therapies result in endogenous immune responses to tumor Ags that are capable of protecting animals against subsequent tumor challenge. Strategies that modify the “visibility” of tumors have the potential to significantly enhance the efficacy of both vaccine and adoptive transfer therapies currently in development.

Interference of CD40L-Mediated Tumor Immunotherapy by Oncolytic Vesicular Stomatitis Virus

Oncolytic virotherapy can be achieved in two ways: (1) by exploiting an innate ability of certain viruses to selectively replicate in tumor tissues, and (2) by using viruses to deliver toxic or immunostimulatory genes to tumors. Vesicular stomatitis virus (VSV) selectively replicates in tumors lacking adequate type I interferon response. The efficacy of oncolytic virotherapy using VSV against B16 melanomas in C57BL/6 mice is dependent on CD8(+) T and natural killer cells. Because immunotherapies that prime specific CD8(+) T cells against melanocyte/melanoma antigens can generate significant therapeutic responses, we hypothesized that engineering VSV to express the potent T cell costimulatory molecule CD40 ligand (VSV-CD40L) would enhance virotherapy with concomitant priming of melanoma-specific T cells. However, we observed no difference in antitumor efficacy between the parental VSV-GFP and VSV-CD40L. In contrast, intratumoral injection of a replication-defective adenovirus expressing CD40L (Ad-CD40L) consistently produced significantly greater therapy than either replication-competent VSV-GFP or VSV-CD40L. The Ad-CD40L-mediated tumor regressions were associated with specific T cell responses against tumor-associated antigens (TAAs), which took several days to develop, whereas VSV-CD40L rapidly induced high levels of T cell activation without specificity for TAAs. These data suggest that the high levels of VSV-associated immunogenicity distracted immune responses away from priming of tumor-specific T cells, even in the presence of potent costimulatory signals. In contrast, a replication-defective Ad-CD40L allowed significant priming of T cells directed against TAAs. These observations suggest that an efficiently primed antitumor T cell response can produce similar, if not better, therapy against an established melanoma compared with intratumoral injection of a replication-competent oncolytic virus.

Intratumoral expression of a fusogenic membrane glycoprotein enhances the efficacy of replicating adenovirus therapy

We describe here a novel strategy to enhance the in vivo efficacy of replicating adenovirus therapy, using coinjection of plasmid DNA encoding a fusogenic viral glycoprotein. The combination of fusogenic membrane glycoprotein (FMG)-induced tumor cell fusion and infection with replicating adenovirus effectively treats even large established tumors at doses of plasmid DNA and virus that alone are ineffective. Adenoviral infection appears to increase the transduction of the tumor cells to a modest degree thereby boosting the FMG-mediated component of the therapy. Simultaneously, syncytial formation enhances the therapeutic effects of viral infection by increasing spread of adenoviral particles through the tumor cell population and by increasing titer of virus released from the tumor cells. This effect is due probably to release of intracellular viral particles upon tumor cell death and also to increased levels of E1A protein within syncytia, whose increased metabolic rate is associated with enhanced levels of protein expression. Cotransduction of tumor cells with replicating adenovirus and FMG-expressing vectors could either be combined within single replicating vectors or could be used in strategies using separate administration of two components, both at lower doses than required for either therapy alone.

Diagnosis and Management of Waldenström Macroglobulinemia: Mayo Stratification of Macroglobulinemia and Risk-Adapted Therapy (mSMART) Guidelines 2016.

Importance Waldenström macroglobulinemia (WM), an IgM-associated lymphoplasmacytic lymphoma, has witnessed several practice-altering advances in recent years. With availability of a wider array of therapies, the management strategies have become increasingly complex. Our multidisciplinary team appraised studies published or presented up to December 2015 to provide consensus recommendations for a risk-adapted approach to WM, using a grading system. Observations Waldenström macroglobulinemia remains a rare, incurable cancer, with a heterogeneous disease course. The major classes of effective agents in WM include monoclonal antibodies, alkylating agents, purine analogs, proteasome inhibitors, immunomodulatory drugs, and mammalian target of rapamycin inhibitors. However, the highest-quality evidence from rigorously conducted randomized clinical trials remains scant. Conclusions and Relevance Recognizing the paucity of data, we advocate participation in clinical trials, if available, at every stage of WM. Specific indications exist for initiation of therapy. Outside clinical trials, based on the synthesis of available evidence, we recommend bendamustine-rituximab as primary therapy for bulky disease, profound hematologic compromise, or constitutional symptoms attributable to WM. Dexamethasone-rituximab-cyclophosphamide is an alternative, particularly for nonbulky WM. Routine rituximab maintenance should be avoided. Plasma exchange should be promptly initiated before cytoreduction for hyperviscosity-related symptoms. Stem cell harvest for future use may be considered in first remission for patients 70 years or younger who are potential candidates for autologous stem cell transplantation. At relapse, retreatment with the original therapy is reasonable in patients with prior durable responses (time to next therapy ≥3 years) and good tolerability to previous regimen. Ibrutinib is efficacious in patients with relapsed or refractory disease harboring MYD88 L265P mutation. In the absence of neuropathy, a bortezomib-rituximab–based option is reasonable for relapsed or refractory disease. In select patients with chemosensitive disease, autologous stem cell transplantation should be considered at first or second relapse. Everolimus and purine analogs are suitable options for refractory or multiply relapsed WM. Our recommendations are periodically updated as new, clinically relevant information emerges.

Atrophic autoimmune pangastritis : A distinctive form of antral and fundic gastritis associated with systemic autoimmune disease

The 2 major recognized forms of atrophic gastritis are autoimmune and environmental atrophic gastritis. These differ in their topographical distribution in the stomach, histologic features, and etiology. Autoimmune atrophic gastritis results from immune-mediated destruction of specialized oxyntic glands, is restricted to the body and fundus, and shows characteristic neuroendocrine hyperplasia. Environmental atrophic gastritis is associated with long-standing Helicobacter pylori infection and preferentially involves antrum and transition zone mucosa. In this study, we describe a distinctive form of atrophic gastritis that differs markedly from both of these classic variants. This gastritis is characterized by: (1) intense mucosal inflammatory infiltrates, persisting even into the phase of severe glandular atrophy, (2) pangastric distribution with diffuse involvement of both body and antrum, (3) lack of association with H. pylori, and (4) lack of neuroendocrine hyperplasia. The 8 patients presented ranged from 1 to 75 years and showed a slight female predominance (5F:3M). All had systemic autoimmune and/or connective tissue diseases including autoimmune enterocolitis (4 cases), systemic lupus erythematosus, refractory sprue, autoimmune hemolytic anemia, and disabling fibromyalgia. Positive serum autoimmune markers were documented in 7 of 8 (87%) patients, but serologies for antiparietal cell and anti-intrinsic factor antibodies were undertaken in only 1 patient each and were negative. We propose that the distinctive histology of this form of atrophic pangastritis and its association with systemic autoimmune disease suggests an autoimmune process directed against multiple cell lineages in the stomach. The development of multifocal low-grade dysplasia in 1 patient, a 19-year-old woman, suggests that this condition might have neoplastic potential.

CD5+ B-cell lymphoproliferative disorders: Beyond chronic lymphocytic leukemia and mantle cell lymphoma

CD5 positivity in B-cell lymphoproliferative disorders (LPD) is usually considered characteristic of either chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). However, other neoplastic B-LPDs may express CD5, albeit infrequently. In this study we have reviewed the tissue pathology of CD5+ B-LPDs that do not fulfill diagnostic criteria for CLL or MCL on flow cytometric studies of peripheral blood or bone marrow. Our results indicate that although CD5 positivity is most commonly associated with CLL and MCL, a significant minority of cases do not fall into these two categories. Phenotypically unusual CLL, marginal zone lymphoma and lymphoplasmacytic lymphoma were the most common diagnoses in this group of patients. Applying strict flow cytometry criteria, using genetic studies, and deferring to a lymph node/tissue diagnosis in non-classical cases are critical for accurate diagnosis and classification of CD5+ B-cell LPD.