A randomized controlled trial of pentoxifylline for the prevention of regimen-related toxicities in patients undergoing allogeneic marrow transplantation

This study evaluated the effect of pentoxifylline (PTX) on the incidence of regimen-related toxicity in patients receiving allogeneic marrow transplants from related donors. All patients received a regimen of methotrexate and cyclosporine as prophylaxis against acute graft- versus-host disease (GVHD). Patients were randomized to receive PTX or a placebo for 70 days and the outcome was examined in a blinded fashion. Forty-four patients were evaluate in each study arm. PTX had no significant effect on engraftment, the incidence of GVHD, venocclusive disease of the liver, infection, the need for oxygen, posttransplant survival, or the duration of hospitalization. Patients receiving PTX were significantly more likely to develop major elevations of serum creatinine levels. PTX was poorly tolerated and induced significantly more vomiting than the placebo. PTX as administered in this randomized study was associated with significant toxicity and offered no benefit in reducing transplant-related morbidity or mortality.     

Reconstitution of normal megakaryocytopoiesis and immunologic functions in Wiskott-Aldrich syndrome by marrow transplantation following myeloablation and immunosuppression with busulfan and cyclophosphamide

Three patients with Wiskott-Aldrich syndrome received transplants of marrow from their HLA-A, B, C, D identical siblings after myeloablation with busulfan, 2 mg/kg/day x 4 days, followed by immunosuppression with cyclophosphamide, 50 mg/kg/day x 4. Sustained engraftment of lymphoid and hematopoietic elements was documented in each case. Platelet counts in excess of 100,000/cu mm were restored 20--50 days posttransplant and remain in the normal range 6--12 mo later. Platelets exhibit normal size and in vitro aggregation. The patients produce isoagglutinins and antibodies to other polysaccharides. The use of busulfan in moderate dosages as a myeloablative agent, coupled with cyclophosphamide, may offer an improved alternative to the use of lethal total body irradiation as a preparative regimen for complete correction of Wiskott- Aldrich syndrome by marrow transplantation.     

Stromal cell progeny of murine bone marrow fibroblast colony-forming units are clonal endothelial-like cells that express collagen IV and laminin

Studies of human and murine bone marrow explants have demonstrated the existence of stromal cell precursors that give rise to colonies of adherent cells in short-term cultures. Because previous data suggested that these colonies were composed of fibroblasts, the precursor cells were termed fibroblast colony-forming units (CFU-F). However, we have recently shown that the stromal cells which support hematopoiesis in murine long-term bone marrow cultures (LTBC) express collagen IV and laminin, markers associated with an endothelial cell lineage, but are negative for collagen I and III, markers associated with a fibroblast cell lineage. Because these conflicting results suggest major functional differences between the stromal cells observed in long-term cultures and the short-term assay, we re-examined the lineage of CFU-F- derived stromal cells. Using two-color immunofluorescence, we characterized virtually all of the cells comprising individual "CFU-F" colonies derived from mouse radiation chimeras. Identification of donor (hematopoietic) or host (stromal) origin was based on surface staining for strain-specific H-2 surface antigens, and, for endothelial or fibroblast properties, on cytoplasmic staining for laminin and collagen IV, or collagens I and III, respectively. The results demonstrate that a large proportion of the cells in CFU-F colonies are donor-derived and fail to stain with any of the antisera specific for nonhematopoietic cells. In addition, these donor-derived cells exhibit marked phagocytic capacity and stain positively with monoclonal antibodies characteristic of the monocyte-macrophage hematopoietic cell lineage (anti-T200, anti- Mac-1, F4/80). However, the remainder of the cells are host-derived cells that stain positively with antisera to collagen IV and laminin. In contrast, stains for collagen types I and III were negative under conditions that allowed for strong staining of control skin fibroblasts. In separate studies, using mixtures of two genetically distinct bone marrows, the cells expressing collagen IV were further shown to be clonal in origin within individual colonies, directly demonstrating that the CFU-F assay provides a quantitative measure of the numbers of marrow stromal cell precursors. Thus, the current studies establish a remarkable similarity between the hematopoietic microenvironment in the short-term CFU-F assay and the long-term culture system: the majority of adherent cells are hematopoietic cells of the monocyte-macrophage lineage, while the remainder are stromal cells whose precise lineage remains uncertain, but whose pattern of collagen expression is more consistent with an endothelial rather than a fibroblast cell origin.     

The bcl-2 oncogene in Hodgkin's disease arising in the setting of follicular non-Hodgkin's lymphoma

Expression of the bcl-2 proto-oncogene on chromosome 18 is deregulated by the 14; 18 chromosomal translocation, an abnormality that is consistently associated with follicular non-Hodgkin's lymphomas (NHL). Because bcl-2 is believed to function by prolonging cell survival rather than by increasing proliferation, the presence of t(14; 18) in Hodgkin's disease (HD) would have profound implications for the pathogenesis of this neoplasm. We evaluated 32 cases of HD for t(14; 18) by polymerase chain reaction (PCR). These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with the presence of Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence of t(14; 18) in only 2 HD cases (6%), both of which were associated with a prior history of follicular lymphoma, and both of which were among the 7 cases (22%) with strong bcl-2 expression in Hodgkin cells. In at least 1 of the cases, the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed strong bcl-2 staining in Hodgkin cells. Although EBV was detected in 6 of 30 cases, it was not associated with t(14; 18) and usually not with strong bcl-2 expression. These results suggest that a small proportion of HD cases might evolve from follicular NHL, possibly through molecular events superimposed on the t(14; 18). High-level bcl-2 expression in Hodgkin cells is a potentially useful but not definitive marker for these cases.     

Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification

A considerable number of deletions of variable size and position that involve the beta-globin gene complex on chromosome 11 are associated with the clinical entities of hereditary persistence of fetal hemoglobin (HPFH) and delta beta thalassemia. Specific deletions appear to be associated with consistent phenotypes and some are known to be recurrent. To facilitate the molecular diagnosis of uncharacterized patients with HPFH and delta beta thalassemia, oligonucleotide primers have been designed to enzymatically amplify deletion-specific products for nine known deletions, which include those responsible for HPFH-1, HPFH-2, HPFH-3, Spanish (delta beta)zero thalassemia, hemoglobin (Hb) Lepore, Sicilian (delta beta)zero thalassemia, Chinese G gamma(A gamma delta beta)zero thalassemia, Asian-Indian inversion-deletion G gamma(A gamma delta beta)zero thalassemia, and Turkish inversion-deletion (delta beta)zero thalassemia. Using this approach, we have successfully characterized the molecular basis for delta beta thalassemia in 23 individuals from 16 families of diverse ethnic origins. Thirteen individuals from this group were shown to be heterozygous for the 13.4- kb Sicilian deletion, two were heterozygous for the 100-kb Chinese G gamma(A gamma delta beta)zero deletion, four were heterozygous for the Turkish form of inversion-deletion delta beta thalassemia, and three were heterozygous for the Asian-Indian form of inversion-deletion G gamma(A gamma delta beta)zero thalassemia. One Vietnamese subject was heterozygous for a 12.6-kb deletion, which we have fully characterized at the molecular level. Sequence analysis of the breakpoint regions of the Chinese deletion and the Turkish rearrangement indicates that, in each case, the mutation is likely to have arisen from a single origin. This hypothesis is supported by the evident geographical clustering of the various deletions described here.     

The hematopoietic stem cell antigen, CD34, is not expressed on the malignant cells in multiple myeloma

Autologous stem cell transplantation has become an important therapy in multiple myeloma (MM). To develop adequate autograft purging methods, it is necessary to determine whether antigens expressed on early hematopoietic progenitors exist on malignant cells. The Ig heavy chain produced by the MM cells shows evidence of prior somatic mutation without intraclonal diversity. As a result, this sequence can be used as a specific marker to detect all members of the malignant clone. The Ig heavy chain sequence expressed by the MM cells was obtained in five patients with advanced disease. Patient specific oligonucleotide primers were designed based on the complementarity determining regions (CDR) of each MM Ig sequence and used to amplify DNA by polymerase chain reaction for the detection of malignant cells. A highly purified collection of CD34+ cells was obtained after passage of the initial bone marrow cells through an immunoadsorption column and fluorescence- activated cell sorting. Despite an assay sensitivity of 1 tumor cell in 2,500 to 44,000 normal cells, none of the CD34+ samples showed product with the myeloma-specific CDR primers. Therefore, positive selection for cells bearing this antigen should yield a tumor-free autograft capable of providing hematopoietic recovery after myeloablative chemotherapy.     

In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of antithrombins produced by active site mutagenesis of human alpha 1-antitrypsin expressed in yeast

Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.     

Lack of evidence of prolonged human immunodeficiency virus infection before antibody seroconversion

Recently, considerable concern has been raised regarding the possibility that antibody-based screening tests for the human immunodeficiency virus (HIV) may fail to detect certain high-risk individuals for prolonged periods of time. It has been proposed that testing for HIV-related antigen may be a necessary procedure to detect such individuals. To address this issue, we longitudinally studied two groups of homosexual men: direct sexual partners of acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) patients and individuals who ultimately sero-converted. There was no evidence of prolonged infection with HIV in the absence of detectable antibody in these two groups. It appears at this time that, even among subjects at very high risk for HIV infection, currently available antibody-based assays are sufficient to identify infected individuals.