Platelets from WAS patients show an increased susceptibility to ex vivo phagocytosis.

The thrombocytopenia of Wiskott-Aldrich syndrome (WAS) is thought to be due to both reduced platelet production and accelerated platelet consumption. We have previously demonstrated that platelets from WASP-deficient mice are consumed more rapidly in vivo than are WT platelets, and that opsonization accelerates their uptake by bone marrow- derived macrophages more than it does that of WT platelets. Here we asked whether platelets from WAS patients show similar features. We show that ex vivo phagocytosis by activated THP-1 cells of DIO-labeled platelets from a series of WAS or XLT patients is increased in comparison to that of normal control platelets. Using a numerical analysis method, we distinguish this effect from a concurrent effect on the amount of detectable fluorescent signal transferred to the macrophage per phagocytosed platelet. We show that the latter quantity is reduced by platelet WASP deficiency, as might be expected if the fluorescence transferred from these smaller platelets is more rapidly quenched. We are unable to detect a differential effect of opsonization with anti-CD61 antibody on the uptake of WASP(-) vs. WT platelets. However, the high probability of phagocytosis per adsorbed WASP(-) platelet could limit the sensitivity of the assay in this case. We also see no effect of sera from WAS patients on the uptake of normal control platelets, suggesting that in vivo opsonization is not the cause of increased uptake of WASP(-) platelets. Finally, we show little, if any, increase in the reticulated platelet fraction in WAS patients, suggesting that impaired production of reticulated platelets contributes to the thrombocytopenia. Our findings suggest that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. They also demonstrate the feasibility of routinely performing functional assays of phagocytosis of small numbers of platelets obtained at remote locations, a method which should be applicable to the study of other types of thrombocytopenia such as ITP.

Nonmyeloablative hematopoietic stem cell transplantation corrects the disease phenotype in the canine model of leukocyte adhesion deficiency.

OBJECTIVE: The aim of this study was to test a nonmyeloablative hematopoietic stem cell transplant regimen applicable to children with leukocyte adhesion deficiency (LAD) who have a histocompatible sibling donor by using the canine model of LAD, namely canine leukocyte adhesion deficiency or CLAD. METHODS: Thirteen CLAD pups received a hematopoietic stem cell transplant from a dog leukocyte antigen (DLA)-matched littermate donor after pretransplant nonmyeloablative conditioning with 200 cGy total-body irradiation and posttransplant immunosuppression with cyclosporine and mycophenolate mofetil. Donor chimerism following transplant was assessed by flow cytometry for the presence of donor CD18 peripheral blood leukocytes and leukocyte subsets. RESULTS: Eleven of the 13 transplanted animals achieved stable mixed donor chimerism and reversal of the severe CLAD phenotype without graft-vs-host disease. The level of donor chimerism ranged from 3.9 to 95.5% at 1 year following transplant. There was one early death 3 weeks after transplant from thrombocytopenia and hemorrhage, and one dog with donor microchimerism (0.5% CD18+ donor leukocytes) who had attenuation of the CLAD phenotype. CONCLUSION: These results demonstrate that a nonmyeloablative transplant regimen from a DLA-matched littermate donor leads to mixed chimerism and reversal of the severe disease phenotype in dogs with CLAD, and provides support for the use of this approach in children with LAD who possess a histocompatible sibling donor.

Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy.

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent, life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs, therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment, and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific, large animal model using 2 clinically applicable conditioning regimens, and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD.

Nonmyeloablative conditioning with busulfan before matched littermate bone marrow transplantation results in reversal of the disease phenotype in canine leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD)-1, a primary immunodeficiency disease caused by molecular defects in the leukocyte integrin CD18 molecule, is characterized by recurrent, life-threatening bacterial infections. Myeloablative hematopoietic stem cell transplantation is the only curative treatment for LAD-1. Recently, canine LAD (CLAD) has been shown to be a valuable animal model for the preclinical testing of nonmyeloablative transplantation regimens for the treatment of children with LAD-1. To develop new allogeneic transplantation approaches for LAD-1, we assessed a nonmyeloablative conditioning regimen consisting of busulfan as a single agent before matched littermate allogeneic bone marrow transplantation in CLAD. Three CLAD dogs received busulfan 10 mg/kg intravenously before infusion of matched littermate bone marrow, and all dogs received posttransplantation immunosuppression with cyclosporin A and mycophenolate mofetil. Initially, all 3 dogs became mixed chimeras, and levels of donor chimerism sufficient to reverse the CLAD phenotype persisted in 2 animals. The third dog maintained donor microchimerism with an attenuated CLAD phenotype. These 3 dogs have all been followed up for at least 1 year after transplantation. These results indicate that a nonmyeloablative conditioning regimen with chemotherapy alone is capable of generating stable mixed chimerism and reversal of the disease phenotype in CLAD.

Myeloid ELF1-like factor is a potent activator of interleukin-8 expression in hematopoietic cells.

Myeloid ELF1-like factor (MEF), also known as ELF4, is a member of the ETS family of transcription factors which is expressed in hematopoietic cells. MEF-deficient mice have defects in natural killer cell and natural killer T cell development, suggesting a role for MEF in regulating innate immunity. MEF also functions in myeloid cells, where it can transactivate target genes. To identify MEF target genes in a "myeloid" environment, we created an inducible expression system and used oligonucleotide microarrays to examine the transcript profile of HEL cells after induction of MEF expression. Sixteen genes were reproducibly turned on or off more than 2-fold, 8 h after induction of MEF expression, and we examined one of the genes, interleukin-8 (IL-8), in greater detail. IL-8 is a CXC chemokine involved in neutrophil chemoattraction, angiogenesis, and stem cell mobilization. It is expressed by several tumor types, and its expression is regulated primarily transcriptionally. The IL-8 promoter contains three ETS binding sites, and we identified the specific site that binds MEF and is required for MEF responsiveness. MEF, but not the closely related ETS factors PEA3, ETS1, ETS2, ELF1, or PU.1, strongly activates the IL-8 promoter. MEF overexpression is sufficient to induce IL-8 protein expression, and reduction in MEF expression (using RNA interference) results in decreased IL-8 levels. These data demonstrates that MEF is an important regulator of IL-8 expression.

Leukocyte adhesion deficiency in children and Irish setter dogs.

Children with the genetic immunodeficiency disease leukocyte adhesion deficiency, or LAD, develop life-threatening bacterial infections as a result of the inability of their leukocytes to adhere to the vessel wall and migrate to the sites of infection. Recently, the canine counterpart to LAD, known as canine leukocyte adhesion deficiency, or CLAD, has been described in Irish setter dogs. This review describes how the clinical phenotype of dogs with CLAD closely parallels that of children with the severe deficiency phenotype of LAD, thus enabling the CLAD dog to provide a disease-specific, large-animal model for testing novel hematopoietic stem cell and gene therapy strategies before their translation to children with LAD.