Fabian D. Arditti

Human and porcine early kidney precursors as a new source for transplantation

Kidney transplantation has been one of the major medical advances of the past 30 years. However, tissue availability remains a major obstacle. This can potentially be overcome by the use of undifferentiated or partially developed kidney precursor cells derived from early embryos and fetal tissue. Here, transplantation in mice reveals the earliest gestational time point at which kidney precursor cells, of both human and pig origin, differentiate into functional nephrons and not into other, non-renal professional cell types. Moreover, successful organogenesis is achieved when using the early kidney precursors, but not later-gestation kidneys. The formed, miniature kidneys are functional as evidenced by the dilute urine they produce. In addition, decreased immunogenicity of the transplants of early human and pig kidney precursors compared with adult kidney transplants is demonstrated in vivo. Our data pinpoint a window of human and pig kidney organogenesis that may be optimal for transplantation in humans.

Induction of strong hepatitis B virus (HBV) specific T helper cell and cytotoxic T lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic HBV infection

Humanized BALB / c mice (termed trimera mice) conditioned by lethal total body irradiation and bone marrow transplantation from SCID mice have been described to support rapid engraftment of human peripheral blood mononuclear cells (PBMC) and the induction of strong B and T cell responses after immunization in vivo. Moreover, these mice can be infected with the hepatitis B and C viruses (HBV, HCV). The current study employed this model to study therapeutic vaccination approaches against the HBV. Thus, strong primary Th cell responses against the HBV core (HBc) and the Borrelia burgdorferi control antigen were induced by transfer of antigen‐loaded dendritic cells together with autologous PBMC from HBV‐naive donors as well as by vaccination with high doses of antigen or a DNA plasmid encoding for HBcAg. Moreover, primary peptide‐specific CTL responses against the immunodominant epitope HBc18 – 27 were induced by HBc particle or DNA vaccination of chimera engrafted with HBV‐naive PBMC. Finally, strong HBc‐specific Th cell and antibody responses were induced by HBc or DNA vaccination of mice reconstituted with PBMC from a chronic HBV patient. Thus, since HBc represents the immunodominant antigen in self‐limited HBV infection, HBc particles or DNA vectors are good candidates for therapeutic vaccination, that will be further studied in ourmodel and clinical studies.

Novel derivatives of 6-mercaptopurine: Synthesis, characterization and antiproliferative activities of S-allylthio-mercaptopurines

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

A novel small animal model for HIV-1 infection

Lethally irradiated normal BALB/c mice, reconstituted with murine SCID bone marrow and engrafted with human PBMC (Trimera mice), were used to establish a novel murine model for HIV‐1 infection. The Trimera mice were successfully infected with different clades and primary isolates of T‐ and M‐tropic HIV‐1, with the infection persisting in the animals for 4–6 wk. Rapid loss of the human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation accompanied the viral infection. All HIV‐1 infected animals were able to generate both primary and secondary immune responses, including HIV specific human humoral and cellular responses. In addition to testing the efficacy of new antiviral compounds, this new murine HIV‐1 model may be used for studying host‐virus interactions and, most importantly, for screening and developing potential HIV‐1 protective vaccines and adjuvants (Ayash‐Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04‐3185fje; doi:10.1096/fj.04‐3185fje.).

Human Colon Adenocarcinoma in the SCID/CB6 Radiation Chimera Is Susceptible to Adoptive Transfer of Allogeneic Human Peripheral Blood Mononuclear Cells

The aim of this study was to develop a murine model of human colon carcinoma (hCC) and to ascertain the potential of cellular immunotherapy in this model. Fragments of hCC obtained at surgery from 6 patients were transplanted under the kidney capsule of lethally irradiated CB6 mice radioprotected with severe combined immunodeficient (SCID) mice bone marrow. Tumor xenografts conserved their malignant behavior in the new environment, invading the mouse kidney parenchyma and expanding into the peritoneal cavity and adjacent tissues. Their growth was typically exponential, and they expanded to dimensions that allowed their subsequent fragmentation and passage to further preconditioned mice. Human carcinoembryonic antigen (hCEA) was detected on the implanted tumor and at occasionally spontaneous lung metastases. Most significantly, high levels of this tumor marker were detected in the sera of tumor-bearing mice, providing a useful tool, which allowed long-term experiments, monitoring of tumor progression, and its response to some treatment modalities. For instance, complete resection of the transplanted tumors, by means of nephrectomy, resulted in the disappearance of hCEA from mice sera within 2 weeks. Similarly, adoptive transfer of allogeneic human peripheral blood mononuclear cells (PBMC) into the peritoneum of tumor-bearing mice, resulted in their rapid engraftment, infiltration of tumor mass, and a significant drop of hCEA levels in mice serum, accounting for inhibition of tumor growth. We suggest that this novel model of human colon carcinoma affords the opportunity for in vivo evaluation of different preclinical treatment modalities, particularly, those involving manipulation with immune effector cells.

T cells from newborn humans are fully capable of developing into cytotoxic T lymphocyte effector cells in adoptive hosts.

BACKGROUND The finding of reduced incidence of graft-versus-host disease (GVHD) associated with cord blood transplantation, compared to unrelated allogeneic bone marrow, could be related to the lower number of T cells infused in the cord blood (CB) inoculum or it might represent an intrinsic property of CB T cells. We investigated the in vivo function of human cord blood mononuclear cells (CBMC) after their adoptive transfer into lethally irradiated BALB/c radioprotected with severe combined immunodeficiency (SCID) mouse bone marrow. METHODS The ability of human CBMC to engraft and produce antigen-specific alloreactive cytotoxic T lymphocytes (CTLs) and antibodies was determined by FACS, 51Chromium-release assay, and ELISA. RESULTS Recipients of human CBMC showed engraftment of high levels of both CD14+ and CD3+ cells. Human cells recovered from the peritoneal cavity of chimeric mice, 1 week after immunization with irradiated allogeneic cells, showed adult-level human CTL response against the immunizing cells, without further stimulation in vitro. In contrast, immunization with the tetanus (TT) antigen did not lead to the generation of anti-TT immunoglobulins (Ig) in the cord blood chimera. Furthermore, whereas the addition of purified adult T cells to the cord blood inoculum resulted in the enhancement of human Ig production of both IgG and IgM subclasses, it could not induce antigen-specific antibodies after immunization. CONCLUSION This report demonstrates in mice for the first time the generation of classical human alloreactive CTLs, derived from cord blood cells. The alloreactivity exhibited by the cord blood mononuclear cells is not different from that displayed by cells originating from adult blood. Any reduction in the observed GVHD associated with cord blood transplants might therefore represent a quantitative difference in the total number of T cells infused.

S-allyl derivatives of 6-mercaptopurine are highly potent drugs against human B-CLL through synergism between 6-mercaptopurine and allicin

S-allylthio-6-mercaptopurine and its ribose derivative were tested for anti-leukemic activity, using a human- mouse B-CLL model. The novel prodrugs contain two components, a purine analog, which interferes with DNA synthesis, and an S-allylthio, readily engaging in thiol-disulfide exchange reactions. The latter component targets the redox homeostasis which is more sensitive in leukemic cells, than in normal B-cells. Upon administration, the prodrug permeates cells, instantly reacts with free thiol, forming S-allyl mixed disulfides and releasing purine. Several cycles of thiol-disulfide exchange reactions occur, thus extending the duration of the prodrug effects. The concerted action of 2 components, as compared with purine alone, boosted in vitro apoptotis in B-CLL cells from 10% to 38%, and decreased in vivo engraftment of B-CLL from 30% to 0.7%.