Prospective study of one- vs two-unit umbilical cord blood transplantation following reduced intensity conditioning in adults with hematological malignancies.

As the threshold nucleated cell dose for one-unit umbilical cord blood (UCB) in adults has not to date been firmly established, we prospectively compared one- vs two-unit UCB transplantation after reduced intensity conditioning (RIC) in adult patients with hematological malignancies. Study design specified one-UCB unit if the cryopreserved total nucleated cell (TNC) dose was 2.5 × 10(7)/kg recipient weight, otherwise two units matched at minima of 4/6 HLA loci to the patient and 3/6 to each other were infused. A total of 27 patients received one unit; 23 patients received two units. Median time to ANC >500/µL was 24 days (95% confidence interval 22-28 days), 25 days for one unit and 23 days for two units (P=0.99). At day 100, ANC >500/µL was 88.4 and 91.3% in the one- and two-unit groups (P=0.99), respectively. Three-year EFS was 28.6% and 39.1% in the one- and two-unit groups (P=0.71), respectively. Infusion of two units was associated with a significantly lower relapse risk, 30.4% vs 59.3% (P=0.045). Infused cell doses (TNC, CD3(+), CD34(+) and CD56(+)CD3(neg)) did not impact on engraftment, OS or EFS. Taken together, one-unit UCB transplantation with a threshold cell dose 2.5 × 10(7)/kg recipient weight after RIC is a viable option for adults, although infusion of two units confers a lower relapse incidence.

Flow cytometry for the diagnosis of mycosis fungoides.

The diagnosis of mycosis fungoides (MF) remains difficult even with current immunohistochemical and molecular techniques. Flow cytometry is a technology widely used for the diagnosis of hematologic malignancies in all areas of hematopathology but has not been readily applied for the diagnosis of MF. In this article the Authors review two issues that affect the ability to diagnosis of MF by flow cytometry, lymphocyte cell recovery from skin biopsies and identifying T cell neoplasia through alteration of the normal T cell phenotype. They present data from their recently published study and others that demonstrate that cell recovery is, in fact, adequate for diagnosing MF in the vast majority of cases. Moreover, they illustrate techniques used to identify neoplastic T cells that help in the analysis of skin biopsy specimens suspected of harboring a T cell malignancy. Finally, they discuss the power of flow cytometry for the diagnosis of cutaneous T cell lymphomas (CTCL). They argue that the capabilities of current flow cytometric techniques for the diagnosis of MF should allow for enhanced diagnostic accuracy and classification of CTCLs.

Diminished CD3 expression is useful for detecting and enumerating Sézary cells.

We evaluated 44 peripheral blood, bone marrow, and lymph node specimens from 23 patients with the clinical diagnosis or suspicion of cutaneous T-cell lymphoma by 4-color flow cytometry for the presence of altered CD3 expression and compared the results with light microscopic evidence of involvement by mycosis fungoides (MF) or Sézary syndrome (SS). In 19 specimens (15 peripheral blood, 3 lymph node, 1 bone marrow) from 6 patients, we noted reduced CD3 expression compared with normal T lymphocytes. Diminished CD3 expression correlated with morphologic evidence of MF/SS cells in all cases, although the enumeration of MF/SS cells differed. Comparison of the enumeration of MF/SS cells in the peripheral blood by CD3+CD7- phenotyping with CD3dim phenotyping suggested that CD3dim phenotyping was the most accurate method for counting MF/SS cells. Therefore, diminished CD3 expression as shown by flow cytometry may be particularly useful for detecting and enumerating MF/SS cells in hematopoietic tissue of patients with cutaneous T-cell lymphoma.

Lymphocyte proliferation assays may underestimate antigen responsiveness in human immunodeficiency virus infection.

The objective of this study was to examine the relationships among lymphocyte proliferation, interferon-gamma (IFN-gamma) production, and apoptosis in peripheral blood mononuclear cells (PBMC) of HIV-1-infected patients and controls. PBMC were prepared from 19 HIV-1-infected patients and 16 healthy controls. Using tetanus toxoid (TT) as a recall antigen, we assessed lymphocyte proliferation using [(3)H]thymidine incorporation after 2, 4, 6, and 7 days' culture and IFN-gamma production in 48-h culture supernatants by ELISA. Apoptosis was measured using TdT-mediated dUTP nick-end labeling. Median stimulation indices (SIs) in HIV-1-infected patients were 2.8 and 3.7 as opposed to 24.9 and 25.1 in healthy controls after 6 and 7 days' culture, respectively (P < 0. 001). Among the controls, peak proliferation was seen after 7 days in culture whereas in patients, SIs peaked at 4 days and fell progressively by days 6 and 7. At 2 and 4 days of stimulation with tetanus, patients' T cells showed increased apoptosis (19 and 25%) vs 12 and 15% apoptosis seen in controls' cells, P < 0.05. Interferon-gamma in 48-h supernatants of TT-stimulated PBMC was comparable among patients and controls. Whereas in our system, 6 and 7 day assays of lymphocyte proliferation provide increasing responses to TT among healthy controls, these durations of culture may underestimate antigen responsiveness in HIV-1 infection. Cell death due to apoptosis may account for this phenomenon. Whether shorter term or longer term assays of lymphocyte responsiveness more accurately reflect in vivo immune competence is unknown. Nonetheless, shorter duration assays may provide a more realistic estimate of the frequency of antigen-reactive cells in persons with HIV-1 infection.

Non-glycosylated human B7-1(CD80) retains the capacity to bind its counter-receptors.

Though the cell surface-associated costimulator B7-1(CD80) is known to be highly N-glycosylated, the functional significance of this N-glycosylation has not been evaluated. Two experimental approaches were taken to assess the influence of N-glycosylation on human B7-1 function. First, stable K562 transfectants expressing human B7-1 were treated with the N-glycosylation inhibitor tunicamycin. This treatment reduced the levels of B7-1 at the cell surface as judged by both indirect immunofluorescence/flow cytometry and immunoprecipitation analyses. Significantly, the non-glycosylated cell surface-associated B7-1 on tunicamycin-treated cells retained the capacity to bind CTLA-4 x Ig, a soluble derivative of the CTLA-4(CD152) counter-receptor. Second, experiments were performed with bacterially-produced non-glycosylated derivatives of human B7-1, comprising either the complete B7-1 extracellular domain (hB7-1 x ed) or the membrane-proximal IgC-homologue domain of B7-1 in isolation (hB7-1 x IgC). While the hB7-1 x IgC derivative failed to bind to CTLA-4, the larger hB7-1 x ed derivative associated with CTLA-4 x Ig in cell-free binding assays. Futhermore, recombinant hB7-1 x ed effectively blocked B7-1-mediated costimulation in an in vitro T cell proliferation assay, suggesting that this soluble non-glycosylated B7-1 derivative is capable of engaging CD28, the B7 counter-receptor implicated in T cell activation. Taken together, these data indicate that the N-glycosylation of B7-1 is not required for its association with counter-receptors. Moreover, the findings pave the way for the therapeutic use of recombinant bacterial B7-1 derivatives as competitive inhibitors of B7-mediated signals.

Transient increase in blasts mimicking acute leukemia and progressing myelodysplasia in patients receiving growth factor.

Previous studies of the hematologic effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have emphasized the morphologic changes induced by these growth factors, but few have reported increases in blasts. Here, we report six cases in which growth factor treatment resulted in a marked but temporary increase in peripheral and bone marrow blasts that led to diagnostic confusion with acute leukemia and high-grade myelodysplastic syndromes. Five of the six patients were receiving treatment for hematologic malignant neoplasms, and one patient had an optic nerve germinoma. Growth factor treatment included single agent therapy with G-CSF (three patients), GM-CSF (one patient), or simultaneous therapy with G-CSF and GM-CSF (two patients). In two patients, there was a dramatic increase in blasts in the peripheral blood (39% and 20%), whereas four had substantial increases in blasts on the aspirate smear (8%-41%). One patient had a medium-sized blast cluster shown on the core biopsy specimen. The blasts decreased after removal of growth factor in all patients. The findings indicate that growth factor therapy can cause a substantial transient increase in blasts in the bone marrow and peripheral blood that may be confused with relapse of acute leukemia or progression of a myelodysplastic syndrome.

Functional dissociation of CD8 alpha's Ig homologue and connecting peptide domains.

The contribution of the CD8 alpha.IgV homologue domain to class I MHC binding was evaluated using a series of chimeric human CD8 alpha:Fc polypeptides incorporating alternative CD8 alpha extracellular domain components. Using a nonisotopic cellfree physical binding assay, those Fc chimeras encompassing the CD8 alpha.IgV homologue domain only (dissociated from the 48-amino acid CD8 alpha connecting peptide) were shown to retain the capacity of the complete CD8 alpha extracellular domain to bind to a recombinant soluble class I MHC alpha 3 domain unit or to intact class I MHC. The specificity of the CD8 alpha:class I MHC alpha 3 domain interaction was verified by mAb and soluble polypeptide blocking experiments. Furthermore, co-precipitation of an Fc chimera incorporating only the CD8 alpha.IgV homologue domain and a recombinant soluble class I MHC alpha 3 domain unit was accomplished. In addition, a glycosylphosphatidylinositol (GPI)-modified variant of the CD8 alpha.IgV homologue domain was generated via chimerization with the GPI signal sequence from decay-accelerating factor. GPI anchorage for this truncated CD8 alpha polypeptide was verified, and its capacity to promote intercellular adhesion through class I MHC binding was shown in a cell:cell binding assay. The findings indicate that the CD8 alpha.IgV homologue domain acts as an independent structural unit when dissociated from the CD8 alpha connecting peptide, and in so doing retains class I MHC binding capacity. This further establishes the principle that Ig superfamily domains from receptor:counter-receptor pairs can interact with each other as isolated units, providing an experimental path for tailoring therapeutically useful IgSF protein derivatives.