[ESTABLISHING A REGISTRY FOR INFLAMMATORY BOWEL DISEASE PATIENTS IN MACCABI HEALTHCARE SERVICES - JOINT PROJECT BETWEEN HOSPITALS, EPI-IIRN GROUP AND COMMUNITY MEDICINE].

INTRODUCTION: Inflammatory bowel diseases (IBD) are becoming a significant cause for chronic long term complex morbidity, particularly among adolescents and young adults. IBD patients require multidisciplinary management and considerable health resources. Recent advances and developments in the diagnostics and therapeutic options require identification and tight monitoring of these patients at both hospital and community level for better management and care. AIMS: To establish at Maccabi Healthcare Services (MHS) a dedicated registry for inflammatory bowel disease patients for long term monitoring in order to optimize care, better use of health resources and to promote high quality research. METHODS: A national project, initiated and headed by a team from Shaare Zedek Medical Center aimed to resolve the complexity in identifying IBD patients at the community setting. The project included data from all Israeli HMOs and major hospitals, that was incorporated into various algorithms to determine prevalence and incidence and to distinguish between Crohn's disease and ulcerative colitis diagnoses. Eventually, an algorithm that includes the number of diagnoses, number of purchases and duration of IBD-related medications showed the best results for separating those that suffer from IBD and those that do not. This algorithm was further validated by chart review. RESULTS: According to the established registry criteria there were 14488 IBD patients in MHS, 13000 active. Additionally we have established an ongoing platform for ongoing monitoring of clinical, therapeutic, laboratory and imaging information. DISCUSSION: Establishing an IBD registry in MHS was enabled by a national project that combined deep professional knowledge of the disease by leading academic centers together with advanced informatics and community large data. We now move on to operate the registry in real life, together with live monitoring of various parameters in order to promote excellent care, communication with patients, management and control and to enable prospective high quality research.

Hyaluronan grafted lipid-based nanoparticles as RNAi carriers for cancer cells.

RNA interference (RNAi), a natural cellular mechanism for RNA-guided regulation of gene expression could in fact become new therapeutic modality if an appropriate efficient delivery strategy that is also reproducible and safe will be developed. Numerous efforts have been made for the past eight years to address this challenge with only mild success. The majority of these strategies are based on cationic formulations that condense the RNAi payload and deliver it into the cell cytoplasm. However, most of these formulations also evoke adverse effects such as mitochondrial damage, interfering with blood coagulation cascade, induce interferon response, promote cytokine induction and activate the complement. Herein, we present a strategy that is devised from neutral phospholipids and cholesterol that self-assembled into lipid-based nanoparticles (LNPs). These LNPs were then coated with the glycosaminoglycan, hyaluronan (HA). HA-LNPs bound and internalized specifically into cancer cells compared with control, non-coated particles. Next, loaded with siRNAs against the multidrug resistance extrusion pump, p-glycoprotein (P-gp), HA-LNPs efficiently and specifically reduced mRNA and P-gp protein levels compared with control particles and with HA-LNPs loaded with control, non-targeted siRNAs. In addition, no cellular toxicity or cytokine induction was observed when these particles were cultured with human Peripheral Blood Mononuclear Cells (PBMCs). The HA-LNPs may offer an alternative approach to cationic lipid-based formulations for RNAi delivery into cancer cells in an efficient and safe manner.

Pulmonary thrombosis in the mouse following intravenous administration of quantum dot-labeled mesenchymal cells.

Quantum dots (QDs) are emerging as novel diagnostic agents. Yet, only a few studies have examined the possible deleterious effects of QD-labeled stem cells. We assessed the potential toxic effects of QD-labeled human embryonic palatal mesenchymal (QD-HEPM) cells in male NOD/SCID mice for six months, following the administration of a single intravenous injection. Control animals were administered with non-labeled HEPM cells. No treatment-related clinical signs, hematological, or biochemical parameters were found in the QD-HEPM animals in comparison to control animals. Histologically, multifocal organizing thrombi were noted in the pulmonary arteries of all QD-HEPM animals from the one-week study group and in one animal from the one-month group. Additionally, increased severity of perivascular inflammation was noted at the injection sites of QD-HEPM animals from the one-week group. This is the first study reporting histopathological evidence for pro-thrombotic adverse effects mediated by QD labeling.

The myelopoietic supportive capacity of mesenchymal stromal cells is uncoupled from multipotency and is influenced by lineage determination and interference with glycosylation.

Cultured bone marrow stromal cells create an in vitro milieu supportive of long-term hemopoiesis and serve as a source for multipotent mesenchymal progenitor cells defined by their ability to differentiate into a variety of mesodermal derivatives. This study aims to examine whether the capacity to support myelopoiesis is coupled with the multipotency. Our results show that the myelopoietic supportive ability of stromal cells, whether from the bone marrow or from embryo origin, is not linked with multipotency; cell populations that possess multipotent capacity may or may not support myelopoiesis, whereas others, lacking multipotency, may possess full myelopoietic supportive ability. However, upon differentiation, the ability of multipotent mesenchymal progenitors to support myelopoiesis is varied. Osteogenic differentiation did not affect myelopoietic supportive capacity, whereas adipogenesis resulted in reduced ability to support the maintenance of myeloid progenitor cells. These differences were accompanied by a divergence in glycosylation patterns, as measured by binding to lectin microarrays; osteogenic differentiation was associated with an increased level of antennarity of N-linked glycans, whereas adipogenic differentiation caused a decrease in antennarity. Inhibition of glycosylation prior to seeding the stroma with bone marrow cells resulted in reduced capacity of the stromal cells to support the formation of cobblestone areas. Our data show that myelopoietic support is unrelated to the multipotent phenotype of cultured mesenchymal progenitors but is dependent on the choice of differentiation pathway and upon correct glycosylation of the stromal cells.

Targeting the bone marrow with activin A-overexpressing embryonic multipotent stromal cells specifically modifies B lymphopoiesis.

In vitro and in vivo studies implicate a series of cytokines in regulation of lymphohematopoiesis. However, direct indications for a local role of most of these cytokines within the bone marrow is lacking. In the present study, we aimed to test the contribution of a specific cytokine, activin A, a member of the transforming growth factor-beta (TGF-beta) family, to lymphohematopoiesis in mouse bone marrow. We show that mouse embryonic fibroblasts (MEFs) are indistinguishable from multipotent stromal cells (MSCs). Such MEFs overexpressing activin A, supported in vitro myelopoiesis in long-term bone marrow cultures as effectively as control MEFs. In contrast, activin A-overexpressing MEFs interfered with the in vitro generation of B lineage cells in such cultures. Thus, excessive expression in vitro of activin A, by supportive stromal cells, causes preferential maturation of myeloid rather than lymphoid cells. Moreover, the activin A-overexpressing MEFs caused an increased incidence in vivo of relatively immature B lineage cells; upon transplantation through the spleen route, MEFs engrafted the bone marrow specifically. Activin A-overexpressing MEFs accumulated in the bone marrow compartment and slowed down the progression of B cell precursors along the differentiation pathway, while sparing the myeloid population. The assay system described in this paper provides a means to assess the contribution of a wide range of molecules to hematopoiesis without perturbing the constitution of other organs.

Toll-like receptors and their ligands control mesenchymal stem cell functions.

Mesenchymal stem cells (MSCs) are widespread in adult organisms and may be involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. Thus, it is important to discover the factors controlling MSC renewal and differentiation. Here we report that adult MSCs express functional Toll-like receptors (TLRs), confirmed by the responses of MSCs to TLR ligands. Pam3Cys, a prototypic TLR-2 ligand, augmented interleukin-6 secretion by MSC, induced nuclear factor kappa B (NF-kappaB) translocation, reduced MSC basal motility, and increased MSC proliferation. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We show herein that Pam3Cys inhibited MSC differentiation into osteogenic, adipogenic, and chondrogenic cells while sparing their immunosuppressive effect. Our study therefore shows that a TLR ligand can antagonize MSC differentiation triggered by exogenous mediators and consequently maintains the cells in an undifferentiated and proliferating state in vitro. Moreover, MSCs derived from myeloid factor 88 (MyD88)-deficient mice lacked the capacity to differentiate effectively into osteogenic and chondrogenic cells. It appears that TLRs and their ligands can serve as regulators of MSC proliferation and differentiation and might affect the maintenance of MSC multipotency.

Reorganization of specific chromosomal domains and activation of silent genes in plant cells acquiring pluripotentiality.

The transition from leaf cells to protoplasts (plant cells devoid of cell walls) confers pluripotentiality coupled with chromatin reorganization. Here, we sought to identify remodeled chromosomal domains in Arabidopsis protoplasts by tracking DNA sequences undergoing changes in DNA methylation and by identifying up-regulated genes. We observed a reduction in DNA methylation at a pericentromeric region of chromosome 1, and up-regulation of several members of the NAC (NAM/ATAF1/CUC2) domain family, two of which are located near the telomeric region of chromosome 1. Fluorescence in situ hybridization (FISH) analysis demonstrated that both pericentromeric and telomeric subdomains underwent chromatin decondensation. This decondensation is subdomain-specific inasmuch as centromeric repeats remained largely unchanged, whereas the 18S rDNA underwent condensation. Within the pericentromeric subdomain, VIP1, a gene encoding a b-Zip nuclear protein required for Agrobacterium infectivity, was transcriptionally activated. Overexpression of this gene in tobacco resulted in growth retardation and inhibition of differentiation and shoot formation. Altogether, our data indicate that acquisition of pluripotentiality involves changes in DNA methylation pattern and reorganization of specific chromosomal subdomains. This change leads to activation of silent genes whose products are involved in acquisition or maintenance of pluripotentiality and/or the ensuing fate of the cell.