Suppression of B-cell proliferation to lipopolysaccharide is mediated through induction of the nitric oxide pathway by tumor necrosis factor- alpha in mice with acute graft-versus-host disease

Graft-versus-host disease (GVHD) is associated with impaired B-cell responses. We investigated the mechanism of impaired proliferation of B cells in response to the mitogen lipopolysaccharide (LPS) by analyzing the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which have independently been described as important effector mechanisms in the pathogenesis of acute GVHD. A threefold decrease of mature surface Ig-positive (slg+) B cells was observed in GVHD spleens isolated 2 weeks after transplant. However, proliferation of these cells in response to LPS was suppressed by more than 35-fold. Activated GVHD splenocytes secreted large amounts of TNF- alpha and NO in culture. Neutralization of TNF-alpha with anti-TNF- alpha antibody (Ab) both abrogated NO production and restored LPS- induced proliferation of B cells to levels found in non-GVHD control mice. The specific inhibition of NO synthesis with LG-monomethyl- arginine (NMMA) restored splenocyte responses but did not significantly reduce TNF-alpha levels, showing that TNF-alpha per se did not cause immunosuppression. These data show that, during GVHD, induction of the NO pathway is an important mechanism that mediates B-cell hyporesponsiveness to LPS and that this pathway is induced by TNF-alpha.     

Isolation of the full-length murine erythropoietin receptor using a baculovirus expression system

The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.     

Platelet crossmatching with lymphocytotoxicity test: an effective method in alloimmunized Chinese patients

Fifty-three patients receiving long-term platelet transfusions were regularly screened for platelet-associated antibodies by a platelet suspension immunofluorescence test (PSIFT) and a lymphocytotoxicity test (LCT). Subsequently, 24 patients became alloimmunized; all of their antibodies were of HLA specificity. Eighty-two single-donor platelet transfusions were given, and the clinical responses were considered satisfactory if the 18-hour corrected count increment was 7.5 x 10(3) per microL or higher. In the meantime, 82 pairs of patient sera and donor lymphocytes were crossmatched. Among 63 crossmatched transfusions, 53 (84%) resulted in a satisfactory increment, with a mean (+/- SEM) of 17.71 +/- 1.96 (x 10(3)/microL), and 10 did not result in a satisfactory increment. The increments after 19 unmatched transfusions and 25 random-donor (uncrossmatched) transfusions were 0.7 +/- 0.3 and 2.39 +/- 0.66, respectively. The difference was not significant (p greater than 0.05). The agreement between the LCT results and clinical response was 88 percent. Retrospectively, the corrected count increments showed no significant differences (p greater than 0.05) among three groups of HLA grading: the increments for A/BU/BX, C/D, and random HLA matches were 22.97 +/- 4.07, 15.1 +/- 1.97, and 14.85 +/- 2.04, respectively. These results suggest that platelet crossmatching by LCT is an effective method for use in alloimmunized patients, especially Chinese patients.     

Acute lymphoblastic leukemias with deletion of 11q23 or a novel inversion (11)(p13q23) lack MLL gene rearrangements and have favorable clinical features

Balanced translocations affecting the 11q23 region are among the most frequent chromosomal abnormalities in childhood acute lymphoblastic leukemia (ALL), comprising 5% to 6%. These cases consistently have a rearranged MLL gene and are associated with high-risk presenting features, hyperleukocytosis and younger age, and a poor treatment outcome. To assess the clinical and biologic significance of 11q23- associated structural chromosomal abnormalities other than translocations, we studied 17 cases of childhood ALL [14 with del(11)(q23) and 3 with inv(11)(p12q23)] that were identified among 785 cases with successful chromosome analysis. In contrast to reported cases with 11q23 and MLL gene rearrangement, our series was characterized by relatively low leukocyte counts (median, 15.1 x 10(9)/L), expression of CD10 antigen but not myeloid-associated CD15 and CDw65 antigens, a relatively high frequency of T-cell immunophenotypes, and a generally favorable prognosis. All 13 cases with interpretable molecular analysis lacked MLL gene rearrangements. We suggest that most cases with deletions or inversions affecting the 11q23 region represent clinically and biologically different entities as compared with those defined by 11q23 translocation.     

Radionuclide bone imaging in spondylolysis of the lumbar spine in children

Bone scintigraphy and radiography were performed in seven children with back pain. Six of the children with radiographic evidence of a pars interarticularis defect also had abnormal scintigrams. Increased uptake of the bone imaging agent occurred at six of the ten sites of radiographic pars interarticularis defects, implying increased bone metabolic activity. However, the location of scintigraphic abnormalities did not correspond to the location of radiographic abnormalities in several cases. Possible explanations for the discordant findings are: (a) normal bone metabolism at the site of an old spondylolysis and (b) radiographically inapparent stress fractures. Measurements of absorbed radiation dose indicate that plain radiography, including oblique views where appropriate, has a lower absorbed radiation dose than scintigraphy or tomography and should be performed prior to these studies.