Induction of complete and molecular remissions in acute myeloid leukemia by Wilms' tumor 1 antigen-targeted dendritic cell vaccination.

Active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far, most dendritic cell trials have been performed in end-stage cancer patients with high tumor loads. Here, in a phase I/II trial, we investigated the effect of autologous dendritic cell vaccination in 10 patients with acute myeloid leukemia (AML). The Wilms' tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two patients in partial remission after chemotherapy were brought into complete remission after intradermal administration of full-length WT1 mRNA-electroporated dendritic cells. In these two patients and three other patients who were in complete remission, the AML-associated tumor marker returned to normal after dendritic cell vaccination, compatible with the induction of molecular remission. Clinical responses were correlated with vaccine-associated increases in WT1-specific CD8+ T cell frequencies, as detected by peptide/HLA-A*0201 tetramer staining, and elevated levels of activated natural killer cells postvaccination. Furthermore, vaccinated patients showed increased levels of WT1-specific IFN-gamma-producing CD8+ T cells and features of general immune activation. These data support the further development of vaccination with WT1 mRNA-loaded dendritic cells as a postremission treatment to prevent full relapse in AML patients.

A murine antibacterial ortholog to human bactericidal/permeability-increasing protein (BPI) is expressed in testis, epididymis, and bone marrow.

The bactericidal/permeability-increasing protein (BPI), stored in human neutrophil granulocytes, is cytotoxic against Gram-negative bacteria. Several genes related to BPI cluster on human chromosome 20 and on mouse chromosome 2, but expression and characterization of a BPI ortholog in the mouse have not been reported. We asked whether BPI is structurally and functionally conserved between humans and mice and whether murine BPI might be synthesized in neutrophils as well as in other tissues. We report the isolation of a murine full-length cDNA encoding a 54-kDa protein, showing 53% amino acid identity and 71% similarity, to human BPI. The murine BPI and human BPI genes show a similar exon-intron organization. Murine BPI mRNA was detected in testis, epididymis, and bone marrow, as well as in Sertoli and promyelocytic cell lines. Although levels of BPI mRNA in human and murine testis were comparable, expression in murine bone marrow cells was low as compared with that in human bone marrow. BPI protein showed a cytoplasmic, granular localization in mature neutrophils. BPI gene expression in Sertoli and promyelocytic cells was enhanced several-fold by all-trans retinoic acid. Overexpression of murine BPI in human embryonic kidney 293 cells resulted in antibacterial activity against Escherichia coli, comparable with that obtained with human BPI. In conclusion, it was demonstrated that mouse neutrophils store BPI with antibacterial activity and that murine BPI is also expressed in testis and epididymis.

AML-1, PU.1, and Sp3 regulate expression of human bactericidal/permeability-increasing protein.

Bactericidal/permeability-increasing protein (BPI) is an antimicrobial protein in neutrophils, stored in azurophil granules. Expression of BPI is absent in neutrophils of newborns and patients with secondary granule deficiency (SGD), possibly contributing to dysfunction of neutrophils. We report two alternative transcription start sites at 52 and 22bp upstream of the translation start. A proximal 222bp promoter conferring expression in myeloid cells was identified, and critical cis-acting sites for myeloid expression were contained within the 159bp upstream of translation start. Within this region, direct binding and transactivation by AML-1, PU.1, and Sp3 were demonstrated, as judged by electrophoretic mobility shift analysis. Moreover, transient transfections of C/EBPalpha or C/EBPepsilon to HeLa cells resulted in increased promoter activity, indicating a direct or indirect role for C/EBP. In conclusion, we provide evidence for AML-1, PU.1, and Sp3 cooperatively and directly mediating BPI-expression during myeloid differentiation.

Heat elution chromatography of immunoglobulins.

A tremendous increase has taken place over the last decades in the biochemical and clinical use of antibodies. Unfortunately, the constantly growing demand has not been matched by a corresponding easy access to pure immunoglobulin, as purification procedures tend to be either laborious, expensive, or inefficient. We present a new and simplified method to obtain pure antibody based on the special thermal properties of the streptococcal M proteins, a family of cell-surface exposed coiled-coil molecules which bind different sets of host plasma proteins. The coiled-coil structure is already destabilized at low temperatures and the M proteins unfold reversibly, usually below 40 degrees C. We demonstrate the use of this property to purify immunoglobulin G from rabbit serum with protein H from the AP1 strain of Streptococcus pyogenes. Recombinant protein H is linked to nickel-agarose via a C-terminal histidine tag. After mixing with rabbit serum and washing at room temperature, pure IgG can be eluted from the gel with a moderately heated buffer. In this case, protein H has been used to purify rabbit IgG, but the principle should be applicable to other M protein-ligand pairs.