Philip M. Wallace

Ctla4ig Treatment Ameliorates The Lethality Of Murine Graft-versus-host Disease Across Major Histocompatibility Complex Barriers

Graft-versus-host disease (GVHD), a pathological condition associated with BMT, results from activation of donor T lymphocytes by host tissues. CD28 and CTLA-4 are structurally related T cell receptors for members of the B7 (CD80) gene family, which transmit important costimulatory signals for T cell activation in vitro and in vivo. Here we have investigated the effects of CTLA4Ig, a soluble form of CTLA-4, on lethal GVHD in a murine model. Lethal GVHD was induced by transfer of parent C57BL/6 bone marrow and spleen cells into lethally irradiated (C57BL/6 x DBA/2)F1 recipients. Short courses of treatment with CTLA4Ig did not block engraftment, but prolonged survival of BMT recipients even when administration was delayed for 6 days after transplantation. CTLA4Ig-treated survivors of GVHD maintained body weight and did not exhibit visible signs of GVHD. However, treatment regimens that maximally prolonged survival did not detectably prevent T cell-mediated hematological abnormalities associated with GVHD, including pancytopenia and abnormal cellular composition of the spleen. Our data thus show that the lethality of acute GVHD in this model system is more dependent upon CD28/CTLA-4 costimulation than are other GVHD-associated abnormalities, and can be blocked for an extended period by brief treatment with CTLA4Ig.

CD34+ selected bone marrow grafts are radioprotective and establish mixed chimerism in dogs given high dose total body irradiation

BACKGROUND Canine stem cell transplantation models have provided important preclinical information for human clinical studies. The recent cloning of cDNA for canine CD34 and the production of monoclonal antibodies that recognize canine CD34 have been the basis for the development of techniques for the large-scale enrichment of canine hematopoietic progenitor cells. In this study, we evaluated the in vivo functional properties of canine bone marrow CD34+ cells after a myeloablative conditioning regimen. METHODS After 920 cGy total body irradiation, three dogs received infusion of autologous CD34+ selected cells from the marrow, three dogs CD34+ depleted autologous marrow cells, and two dogs received CD34+ autologous marrow cells that were immunomagnetically selected and then further purified by cell sorting. In addition, four dogs received allogeneic marrow enriched for CD34+ cells from dog leukocyte antigen-identical littermates to investigate long-term repopulating function of CD34+ cells. Chimerism studies were performed using polymerase chain reaction to detect highly polymorphic microsatellite markers. RESULTS In three recipients of autologous marrow enriched for CD34+ cells to between 29% and 70% (1.6 x 10(6) to 3.4x10(6) CD34+ cells/kg), prompt and full hematopoietic recovery occurred, whereas in three dogs that received marrow depleted of CD34+ cells (1 x 10(7) cells/kg), no hematopoietic recovery was achieved. In two dogs that received highly purified CD34+ cells (purity: 98% and 96%, 0.79x10(6) to 0.547x 10(6) CD34+ cells/kg), delayed but full hematopoietic recovery was seen. Three of four allograft recipients of 1.75x10(6) to 6.8x10(6) CD34+ cells/kg engrafted and showed full hematopoietic recovery, whereas one dog rejected the graft. The three long-term survivors showed stable mixed hematopoietic chimerism with predominantly donor hematopoiesis. CONCLUSION Transplantation of canine CD34+ cells after lethal total body irradiation provides radioprotection and gives rise to long-term hematopoietic reconstitution. Stable donor/host mixed chimerism was observed in allograft recipients most likely as a result of T-cell depletion of the grafts. Our findings suggest a future role for canine preclinical transplant studies involving in vitro manipulation of hematopoietic pro.

Activation of prodrugs by antibody-enzyme conjugates

Monoclonal antibodies (MAbs) against human tumor antigens have been the subject of extensive investigation as carriers of cytotoxic agents to tumor cells. Promising results, both in vitro and in vivo, have been obtained in studies involving immunoconjugates such as MAb-toxins (Vitetta et al., 1987; Blakey et al., 1988), MAb-drugs (Pietersz, 1990; Koppel, 1990), and radiolabeled MAbs (Goldenberg, 1990). Based on these findings, many such conjugates are currently being evaluated in the clinic.

Liver LDL Receptor mRNA Expression Is Decreased in Human ApoB/CETP Double Transgenic Mice and Is Regulated by Diet as Well as the Cytokine Oncostatin M

We have investigated liver LDL receptor mRNA expression in nontransgenic, human cholesteryl ester transfer protein (CETP) transgenic, and human apolipoprotein (Apo) B/CETP double transgenic mice fed a normal chow diet and a high fat, high cholesterol diet (HFHC). Three weeks of HFHC feeding increased total serum cholesterol 1.5-fold in the nontransgenic, 3.1-fold in the CETP transgenic, and 3.4-fold in the ApoB/CETP double transgenic mice. To examine the liver LDL receptor mRNA expression among the different groups of mice fed the normal diet or fed the HFHC diet, we developed a quantitative reverse-transcribed polymerase chain reaction assay in which the LDL receptor mRNA level was normalized with the beta-actin mRNA. The results show that on the normal chow diet, the LDL receptor mRNA expression levels were lower in the ApoB/CETP mice than in the nontransgenic mice and the human CETP transgenic mice. Liver LDL receptor gene expression was lower in all groups of mice fed the HFHC diet, with the lowest level of expression in the ApoB/CETP mice. Similar results were obtained by Northern blot analysis. In addition, we have previously shown that the cytokine oncostatin M (OM) increases LDL receptor gene expression in HepG2 cells. In this study, we used the ApoB/CETP mice as the model system to examine the in vivo activity of OM on liver LDL receptor gene expression. Our data show that OM increased the level of liver LDL receptor mRNA up to 80% to 90% when the animals were fed the HFHC diet. The results from these studies demonstrate that the expression of the liver LDL receptor in the ApoB/CETP mice is suppressed compared with nontransgenic mice and that the expression of the hepatic LDL receptor gene in these mice is subjected to the normal cholesterol feedback regulation. In addition, LDL receptor gene expression in these mice is also inducible by a positive regulator.

Sulfated etoposide and nitrogen mustard prodrugs and their activation by streptomyces arylsulfatase

AbstractEtoposide sulfate (ES) and p-di-2-chloroethylaminophenyl sulfate (CAPS) were designed as nontoxic anticancer prodrugs of etoposide and p-di-2-chloroethylaminophenol (CAP) that could be activated on tumor cell surfaces by monoclonal antibody (mAb)-arylsulfatase conjugates. In vitro assays indicated that CAPS and ES were nontoxic to the H2981 human lung adenocarcinoma cell line, while etoposide and CAP had IC50 values of 1–2 μM. Several commercially available arylsulfatases, as well as the arylsulfatases from human liver and urine, were either unable or poorly able to effect the hydrolysis of ES and CAPS. In contrast, arylsulfatase from Streptomyces (SAS) hydrolyzed ES and CAPS with specific activities of 3.0 and 4.8 μmol/min/ mg, respectively. SAS was conjugated to the L6 mono-clonal antibody and was able to activate ES in an immunologically specific manner on H2981 cells (L6 antigen positive). ES and CAPS were stable in mouse serum and were at least 9–13 times less toxic to mice on a molar basis t...

The Potential of Membrane-Acting Toxins for Targeted Cancer Therapy

Cell membrane-disrupting toxins (cytolysins) have great appeal as candidates for targeting to tumor cells by means of monoclonal antibodies, yet their use has been explored in only a few studies to date. Here, we appraise the potential of antibody-cytolysin conjugates, or ‘immunolysins’ for cancer therapy.