Immune status of human immunodeficiency virus seropositive and seronegative hemophiliacs infused for 3.5 years with recombinant factor VIII. The Kogenate Study Group

Recent studies suggest that treatment of hemophiliacs with highly purified factor VIII concentrates may preserve immune function. To test this hypothesis, we prospectively studied 51 hemophilic patients (21 human immunodeficiency virus [HIV] seropositive and 30 seronegative) who were on home therapy exclusively with recombinant factor VIII (Kogenate, Miles Laboratory, Berkeley, CA) for 3.5 years. Patients, all of whom had been previously treated with plasma-derived factor VIII concentrates, were monitored every 6 months with T-lymphocyte subsets and beta 2-microglobulin levels. Mean rate of change in absolute CD4 cell counts, calculated from regression slopes for individual patients, showed a small but statistically significant decrease over the 3.5-year study period for HIV seropositive hemophiliacs. No decrease in CD4 cell counts was seen in HIV seronegative hemophiliacs when the data for children under age 6 years were excluded from the analysis. beta 2- microglobulin levels and CD8 cell counts remained unchanged. These data show stability of immunologic parameters in HIV seronegative hemophiliacs, and a small decrease in CD4 cell counts in HIV seropositive hemophiliacs treated with recombinant factor VIII.     

Low salt intake down-regulates the guanylin signaling pathway in rat distal colon

BACKGROUND & AIMS: Guanylin, an endogenous gastrointestinal peptide, causes the translocation of NaCl from interstitial fluid to the intestinal lumen. The aim of this study was to examine whether changes in dietary salt intake lead to compensatory changes in expression of the guanylin signaling pathway. METHODS: Rats received low-, normal-, or high-sodium diets for 1 week. Colonic guanylin expression was evaluated by Western and Northern blotting, rates of guanylin secretion by measuring biologically active guanylin released into the medium from colon explants, and expression of the guanylin receptor (C-type guanylate cyclase) by Northern blotting and bioassay. RESULTS: By every criterion, the low-salt diet reduced expression of guanylin to 30%-40% of the level found in control animals. Guanylin receptor expression was also decreased, although less dramatically and with a lower statistical significance. For both guanylin and guanylin receptor, the high-salt diet had no significant effect on expression. CONCLUSIONS: The data support the hypothesis that the guanylin pathway is down-regulated as an adaptive response to salt restriction. (Gastroenterology 1996 Dec;111(6):1714-21)     

A mechanism for the hypoprothrombinemia of the acquired hypoprothrombinemia-lupus anticoagulant syndrome

Antibodies that bind prothrombin without neutralizing its coagulant activity were demonstrated in the plasma of two patients with the acquired hypoprothrombinemia-lupus anticoagulant syndrome. The first patient's plasma contained less than 1% prothrombin activity and no detectable prothrombin antigen. The second patient's plasma contained about 6% of both prothrombin activity and antigen. Neither patient's plasma neutralized the prothrombin coagulant activity of normal plasma or of purified prothrombin added in vitro. Nevertheless, double immunodiffusion studies and binding experiments utilizing 125I- prothrombin demonstrated the presence of prothrombin antibodies in each patient's plasma. A Scatchard analysis of the binding data obtained with different concentrations of 125I-prothrombin and the first patient's plasma indicated the presence of a high affinity antibody, apparent Kd approximately 10(-10)M, and a lower affinity antibody, apparent Kd approximately 10(-9)M. Studies utilizing purified cleavage products of prothrombin suggested that the antibodies were directed against an epitope or epitopes located on the carboxyl-terminal, latent thrombin segment of the prothrombin molecule. We postulate that the acquired hypoprothrombinemia in these two patients and in other reported patients with the acquired hypoprothrombinemia-lupus anticoagulant syndrome stems from rapid clearance from the circulation of prothrombin antigen-antibody complexes.     

Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: analysis of gene transduction of long-lived progenitors

We have developed a novel cotransplantation system in which gene- transduced human CD34+ progenitor cells are transplanted into immunodeficient (bnx) mice together with primary human bone marrow (BM) stromal cells engineered to produce human interleukin-3 (IL-3). The IL- 3-secreting stroma produced sustained circulating levels of human IL-3 for at least 4 months in the mice. The IL-3-secreting stroma, but not control stroma, supported human hematopoiesis from the cotransplanted human BM CD34+ progenitors for up to 9 months, such that an average of 6% of the hematopoietic cells removed from the mice were of human origin (human CD45+). Human multilineage progenitors were readily detected as colony-forming units from the mouse marrow over this time period. Retroviral-mediated transfer of the neomycin phosphotransferase gene or a human glucocerebrosidase cDNA into the human CD34+ progenitor cells was performed in vitro before cotransplantation. Human multilineage progenitors were recovered from the marrow of the mice 4 to 9 months later and were shown to contain the transduced genes. Mature human blood cells marked by vector DNA circulated in the murine peripheral blood throughout this time period. This xenograft system will be useful in the study of gene transduction of human hematopoietic stem cells, by tracing the development of individually marked BM stem cells into mature blood cells of different lineages.     

Transduction of human melanoma cell lines with the human interleukin-7 gene using retroviral-mediated gene transfer: comparison of immunologic properties with interleukin-2

Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2- transduced melanoma cell lines grew in athymic mice, whereas one IL-7- transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.     

Macrophages can recognize and kill tumor cells bearing the membrane isoform of macrophage colony-stimulating factor

NBXFO hybridoma cells produced both the membrane and secreted isoforms of macrophage colony-stimulating factor (M-CSF). Murine bone marrow cells stimulated by the secreted form of M-CSF (sM-CSF) became Mac1+, Mac2+, Mac3+, and F4/80+ macrophages that inhibited the growth of NBXFO cells, but not L1210 or P815 tumor cells. In cytotoxicity studies, M- CSF activated macrophages and freshly isolated macrophages killed NBXFO cells in the presence of polymyxin B, eliminating the possibility that contaminating lipopolysaccharide (LPS) was responsible for the delivery of the cytotoxic signal. Retroviral-mediated transfection of T9 glioma cells with the gene for the membrane isoform of M-CSF (mM-CSF), but not for the secreted isoform of M-CSF, transferred the ability of macrophages to kill these transfected T9 cells in a mM-CSF dose- dependent manner. Macrophage-mediated killing of the mM-CSF transfected clone was blocked by using a 100-fold excess of recombinant M-CSF. Catalase, superoxide dismutase, and the nitric oxide inhibitor, N-omega- nitro-arginine methyl ester (NAME), did not effect macrophage cytotoxicity against the mM-CSF transfectant T9 clones. T9 parental cells when cultured in the presence of an equal number of the mM-CSF transfectant cells were not killed, indicating specific target cell cytotoxicity by the macrophages. Electron microscopy showed that macrophages were capable of phagocytosizing mM-CSF bearing T9 tumor cells and NBXFO hybridoma cells; this suggested a possible mechanism of this cytotoxicity. This study indicates that mM-CSF provides the necessary binding and triggering molecules through which macrophages can initiate direct tumor cell cytotoxicity.     

Retroviral-mediated gene expression in human myelomonocytic cells: a comparison of hematopoietic cell promoters to viral promoters

Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.     

Specific toxicity of 2-chlorodeoxyadenosine toward resting and proliferating human lymphocytes

2-Chlorodeoxyadenosine (CdA), an adenosine-deaminase-resistant purine deoxynucleoside, is markedly toxic toward human T-lymphoblastoid cell lines in vitro and is an effective agent against L1210 leukemia in vivo. The present studies have examined the toxicity, and in some cases, metabolism, of CdA in (1) multiple established human cell lines of varying phenotype, (2) leukemia and lymphoma cells taken directly from patients, (3) normal bone marrow cells, and (4) normal peripheral blood lymphocytes. Nanomolar concentrations of CdA blocked the proliferation of lymphoblastoid cell lines with a high ratio of deoxycytidine kinase to deoxynucleotidase. The drug had virtually no effect on the growth of cell lines derived from solid tissues. The CdA inhibited the spontaneous uptake of tritiated thymidine by many T and non-T, non-B acute lymphoblastic leukemia cell specimens at concentrations less than or equal to 5 nM. The same concentrations did not impair either thymidine uptake or granulocyte-monocyte colony formation by normal bone marrow cells. In common with deoxyadenosine, but unlike several other agents affecting purine and purine metabolism, CdA was lethal to resting normal T lymphocytes and to slowly dividing malignant T cells. In both resting and proliferating lymphocytes, the CdA was phosphorylated by deoxycytidine kinase and entered a rapidly turning over nucleotide pool. Dividing lymphocytes also incorporated abundant CdA into DNA. The selective toxicity of CdA toward both dividing and resting lymphocytes may render the drug useful as an immunosuppressive or antileukemic agent.     

Carrier detection in the Wiskott Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by immunodeficiency and severe thrombocytopenia in affected males, but no demonstrable clinical abnormalities in carrier females. Through analysis of the methylation patterns of X-linked genes that display restriction fragment length polymorphisms (RFLPs), we studied the pattern of X-chromosome inactivation in various cell populations from female relatives of patients with WAS. The peripheral blood T cells, granulocytes, and B cells of eight obligate WAS carriers were found to display specific patterns of X-chromosome inactivation clearly different from these of normal controls. Thus, carriers of WAS could be accurately identified using this analysis.