Sarit Samira

HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34 + stem cell recruitment to the liver

Hematopoietic stem cells rarely contribute to hepatic regeneration, however, the mechanisms governing their homing to the liver, which is a crucial first step, are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1), which attracts human and murine progenitors, is expressed by liver bile duct epithelium. Neutralization of the SDF-1 receptor CXCR4 abolished homing and engraftment of the murine liver by human CD34+ hematopoietic progenitors, while local injection of human SDF-1 increased their homing. Engrafted human cells were localized in clusters surrounding the bile ducts, in close proximity to SDF-1-expressing epithelial cells, and differentiated into albumin-producing cells. Irradiation or inflammation increased SDF-1 levels and hepatic injury induced MMP-9 activity, leading to both increased CXCR4 expression and SDF-1-mediated recruitment of hematopoietic progenitors to the liver. Unexpectedly, HGF, which is increased following liver injury, promoted protrusion formation, CXCR4 upregulation, and SDF-1-mediated directional migration by human CD34+ progenitors, and synergized with stem cell factor. Thus, stress-induced signals, such as increased expression of SDF-1, MMP-9, and HGF, recruit human CD34+ progenitors with hematopoietic and/or hepatic-like potential to the liver of NOD/SCID mice. Our results suggest the potential of hematopoietic CD34+/CXCR4+cells to respond to stress signals from nonhematopoietic injured organs as an important mechanism for tissue targeting and repair.

Delivery of cytokines by liposomes: hematopoietic and immunomodulatory activity of interleukin-2 encapsulated in conventional liposomes and in long-circulating liposomes.

Although liposomal delivery of interleukin-2 (IL-2) and other cytokines improves their pharmacokinetics and biologic activity in vivo, there are no comparative functional studies of various liposomal formulations as cytokine carriers. In the present investigation, recombinant human IL-2 was encapsulated in two formulations of large (mean diameter 0.75-1.5 microns) multilamellar vesicles (MLV, referred to as conventional liposomes) or in small (mean diameter, 60 nm), unilamellar, long-circulating liposomes (referred to as sterically stabilized liposomes, SSL). The biologic activity of the liposomal formulations and of free IL-2 was tested in parallel in vitro and in mice. The main observations were as follows: (a) All the liposomal IL-2 (Lip-IL-2) formulations were more efficient than soluble IL-2 in stimulating spleen cell proliferation and lymphokine-activated killer (LAK) cell activation in vitro, particularly at low cytokine doses (1-100 CU/mL). (b) After i.v. injection, the circulation time of MLV-IL-2 and SSL-IL-2 was 7 and 17 times greater, respectively, than that of soluble IL-2. (c) In comparison with IL-2, all Lip-IL-2 formulations caused a marked increase in the leukocyte levels in blood, spleen, and peritoneal exudate, especially in those of myeloid origin (neutrophils, eosinophils, immature granulocytes, and macrophages). (d) Although SSL-IL-2 exhibited the longest circulation time, MLV-IL-2 was more potent in elevating leukocyte levels and in triggering LAK cell activity in vivo. (e) The route of Lip-IL-2 administration greatly affected the immunomodulatory activity in the various compartments. (f) MLV-IL-2 proved to be a much more efficient immunoadjuvant than free IL-2 for influenza subunit vaccines as well as for tumor cell vaccines. These findings lend support to our previous studies in which we demonstrated the superior immunomodulatory activity of liposomal IL-2, and suggest that cytokine pharmacokinetics, biodistribution, and pharmacodynamics are markedly influence both by liposomal formulation and route of administration.

Tumor Necrosis Factor Promotes Human T‐Cell Development in Nonobese Diabetic/Severe Combined Immunodeficient Mice

A major problem after clinical hematopoietic stem cell transplantations is poor T‐cell reconstitution. Studying the mechanisms underlying this concern is hampered, because experimental transplantation of human stem and progenitor cells into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice usually results in low T–lymphocyte reconstitution. Because tumor necrosis factor α (TNFα) has been proposed to play a role in T‐lineage commitment and differentiation in vitro, we investigated its potential to augment human T‐cell development in vivo. Administration of TNF to irradiated NOD/SCID mice before transplantation of human mononuclear cells from either cord blood or adult G‐CSF–mobilized peripheral blood (MPBL) led 2–3 weeks after transplantation to the emergence of human immature CD4+CD8+ double‐positive T‐cells in the bone marrow (BM), spleen, and thymus, and in this organ, the human cells also express CD1a marker. One to 2 weeks later, single‐positive CD4+ and CD8+ cells expressing heterogenous T‐cell receptor αβ were detected in all three organs. These cells were also capable of migrating through the blood circulation. Interestingly, human T‐cell development in these mice was associated with a significant reduction in immature lymphoid human CD19+ B cells and natural killer progenitors in the murine BM. The human T cells were mostly derived from the transplanted immature CD34+ cells. This study demonstrates the potential of TNF to rapidly augment human T lymphopoiesis in vivo and also provides clinically relevant evidence for this process with adult MPBL progenitors.

Adjuvanted influenza vaccines

Influenza is one of the most common causes of human morbidity and mortality that is preventable by vaccination. Immunization with available vaccines provides incomplete protection against illness caused by influenza virus, especially in high-risk groups such as the elderly and young children. Thus, more efficacious vaccines are needed for the entire population, and all the more so for high-risk groups. One way to improve immune responses and protection is to formulate the vaccine with antigen carriers and/or adjuvants, which can play an important role in improving immune responses and delivery to antigen-presenting cells, especially for a vaccine like influenza that is based on protein antigens usually administered without a carrier or adjuvant. In this review, the authors present an overview of available vaccines, focusing on research and development of new adjuvants used in influenza vaccines, as well as adjuvanted influenza vaccines aimed to improve immune responses, protection and breadth of coverage for influenza.

Immunogenicity, protective efficacy and mechanism of novel CCS adjuvanted influenza vaccine.

We optimized the immunogenicity of adjuvanted seasonal influenza vaccine based on commercial split influenza virus as an antigen (hemagglutinin = HA) and on a novel polycationic liposome as a potent adjuvant and efficient antigen carrier (CCS/C-HA vaccine). The vaccine was characterized physicochemically, and the mechanism of action of CCS/C as antigen carrier and adjuvant was studied. The optimized CCS/C-HA split virus vaccine, when administered intramuscularly (i.m.), is significantly more immunogenic in mice, rats and ferrets than split virus HA vaccine alone, and it provides for protective immunity in ferrets and mice against live virus challenge that exceeds the degree of efficacy of the split virus vaccine. Similar adjuvant effects of optimized CCS/C are also observed in mice for H1N1 swine influenza antigen. The CCS/C-HA vaccine enhances immune responses via the Th1 and Th2 pathways, and it increases both the humoral responses and the production of IL-2 and IFN-γ but not of the pro-inflammatory factor TNFα. In mice, levels of CD4(+) and CD8(+) T-cells and of MHC II and CD40 co-stimulatory molecules are also elevated. Structure-function relationship studies of the CCS molecule as an adjuvant/carrier show that replacing the saturated palmitoyl acyl chain with the mono-unsaturated oleoyl (C18:1) chain affects neither size distribution and zeta potential nor immune responses in mice. However, replacing the polyalkylamine head group spermine (having two secondary amines) with spermidine (having only one secondary amine) reduces the enhancement of the immune response by ∼ 50%, while polyalkylamines by themselves are ineffective in improving the immunogenicity over the commercial HA vaccine. This highlights the importance of the particulate nature of the carrier and the polyalkylamine secondary amines in the enhancement of the immune responses against seasonal influenza. Altogether, our results suggest that the CCS/C polycationic liposomes combine the activities of a potent adjuvant and efficient carrier of seasonal and swine flu vaccines and support further development of the CCS/C-HA vaccine.

Cycling G1 CD34+/CD38+ Cells Potentiate the Motility and Engraftment of Quiescent G0 CD34+/CD38−/low Severe Combined Immunodeficiency Repopulating Cells

The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2‐ to 3‐day in vitro cytokine stimulation of human cord blood CD34+‐enriched cells induces the production of short‐term repopulating, cycling G1 CD34+/CD38+ cells with increased matrix metalloproteinase (MMP)‐9 secretion as well as increased migration capacity to the chemokine stromal cell–derived factor‐1 (SDF‐1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice. These cycling G1 cells enhance SDF‐1–mediated in vitro migration and in vivo homing of quiescent G0 CD34+ cells, which is partially abrogated after inhibition of MMP‐2/‐9 activity. Moreover, the engraftment potential of quiescent G0 SCID repopulating cells (SRCs) is also increased by the cycling G1 CD34+/CD38+ cells. This effect is significantly abrogated after incubation of cycling G1 cells with a neutralizing anti‐CXCR4 antibody. Our data suggest synergistic interactions between accessory cycling G1 CD34+/CD38+ committed progenitor cells and quiescent, primitive G0 CD34+/CD38−/low SRC/stem cells, the former increasing the motility and engraftment potential of the latter, partly via secretion of MMP‐9.