Bone marrow CD34(+) cells and megakaryoblasts secrete beta-chemokines that block infection of hematopoietic cells by M-tropic R5 HIV.

CD34(+) cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34(+) cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34(+) cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34(+) cells and CD34(+)KIT(+) cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1alpha, and MIP-1beta and that IFN-gamma stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34(+)KIT(+) cells, even by RT-PCR. Conditioned media from CD34(+) cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34(+) cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34(+) cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.

Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines.

The aim of this study was to learn more about the role of the HIV-related chemokine-chemokine receptor axes in human hematopoiesis. To address this issue we phenotyped 35 selected hematopoietic cell lines for the expression of CD4, CXCR4 and CCR5. We next evaluated the functionality of these chemokine receptors by calcium flux and chemotaxis assays, and by the ability of SDF-1, MIP-1alpha, MIP-1beta and RANTES to influence the growth of the cells expressing CXCR4 and/or CCR5. Lastly, we examined whether human hematopoietic cell lines may secrete some HIV-related chemokines, and whether endogenously secreted chemokines might interfere with the infectability. of hematopoietic cells by X4 and R5 HIV strains. These results demonstrate that: (1) HIV-related receptors are widely expressed on human hematopoietic cell lines; (2) stimulation of CXCR4 by SDF-1 induces calcium flux and chemotaxis in several hematopoietic cell lines more efficiently than stimulation of CCR5 by receptor-specific beta-chemokines; (3) chemokines do not regulate proliferation of the hematopoietic cells; and finally (4) infectability of the hematopoietic cells by HIV-1 may be auto-modulated by endogenously secreted chemokines. These data shed more light on the role of HIV-related chemokine-chemokine receptors axes in human hematopoiesis and interaction of hematopoietic cells with HIV.

The limited infectability by R5 HIV of CD34(+) cells from thymus, cord, and peripheral blood and bone marrow is explained by their ability to produce beta-chemokines.

The resistance of human bone marrow (BM) CD34(+) cells to human immunodeficiency virus (HIV) infection is at this point not fully understood. Recently we reported that the chemokines MIP-1alpha, MIP-1beta, and RANTES secreted by BM-derived CD34(+) cells may compete with the macrophagotropic HIV (R5 HIV) strain for the CCR5 coreceptor.In this study we extended our previous observations and examined various lympho-hematopoietic CD34(+) cells isolated from thymus (Th), cord blood (CB), mobilized peripheral blood (mPB), and BM for the expression of beta-chemokines binding to CCR5, i.e., MIP-1alpha, MIP-1beta, RANTES, MCP-2, MCP-3, and MCP-4, and the alpha chemokine SDF-1 (binding to CXCR4) as these chemokines may compete with the R5 and X4 HIV strains, respectively, for entry into cells. We found that Th-, CB-, mPB-, and BM-derived CD34(+) cells express mRNA transcripts for all the beta-chemokines tested but not for SDF-1. Using sensitive ELISA assays we found that although MIP-1alpha and MIP-1beta proteins were secreted by all the lympho-hematopoietic CD34(+) cells tested, RANTES was detectable only in media conditioned by BM- and CB-derived CD34(+) cells and not Th-derived cells. However, media conditioned by BM-, mPB- and Th-derived CD34(+) cells protected the T lymphocytic cell line (PB-1) from infection by the R5 but not the X4 HIV strain. Hence this study demonstrates that beta-chemokines are secreted by lympho-hematopoietic CD34(+) cells originating from various sources and that these endogenously secreted chemokines may limit entry of the R5 HIV strain into the cells by competing for the CCR5 coreceptor.

Posttranslational modifications of the amyloid precursor protein : glycosylation.

Many studies have demonstrated the importance of amyloid precurser protein (APP) in the pathogenesis of Alzheimer's disease. Nonetheless, the exact mechanism by which APP contributes to the pathogenesis of Alzheimer's disease is still not clear. Because APP is a glycoprotein, and because glycosylation can be important in the cell biology of individual glycoproteins (for review, see refs. 1 and 2), it is possible that changes in APP glycosylation during development and aging are important in APP biosynthesis, proteolysis, and degradation. However, few studies have addressed this issue (3 -8). This chapter provides methods for analyzing the glycosylation of APP that is actively synthesized by living cells in tissue culture. These methods can be applied to primary cultures, continuous cell lines, and transfected cell lines expressing recombinant APP.

Influence of immunosuppressive pretreatment on the survival of mice challenged with Ehrlich ascites carcinoma (EAC).

Mice were pretreated with immunosuppressive doses of cyclophosphamide (CY), cortisone acetate (CA) or X-irradiation. 24 h later all mice were given 1 x 10(5) EAC cells intraperitoneally. In another experiment mice were splenectomized 1 h before injection of EAC cells. Neither CY nor CA pretreatment had significant influence on the survival of tumor-inoculated mice. On other hand, X-irradiation or splenectomy strengthened tumor resistance.

In vivo activity of spleen cells from untreated syngeneic mice against Ehrlich ascites carcinoma.

Inbred CFW/L1 or BALB/c male mice were injected intraperitoneally with 1 X 10(5) EAC cells. They died in 98.6 or 100%, respectively, within 100 days of observation. Intraperitoneal injection of 6 X 10(7) syngeneic spleen cells from 6--9-week-old CFW/L1 or BALB/c donors 2.5--4 h after tumour inoculation significantly prolonged MST and 16.7 or 23.5% of mice, respectively, survived for 100 days. Smaller amounts of spleen cells, thymus cells, mixed spleen and thymus cells, or spleen cells from older donors did not show this effect. Homogenized spleen cells were also inactive. Antitumour activity of spleen cells was still evident when they were isolated from irradiated (2 000R) donors or were given 24 h after tumour inoculation. Spleen cells used for in vivo studies showed spontaneous cytotoxic activity in vitro against EAC cells in the 51Cr-release cell-mediated cytotoxicity test. The possible participation of NK cells in the observed in vivo antitumour activity of normal spleen cells is discussed.

Recombinant blood group antigens are useful for studying the fine specificity of antibodies against glycophorin A encoded antigens.

Recombinant wild-type and variant forms of glycophorin A (GPA) were expressed in wild-type and glycosylation-defective Chinese hamster ovary cells. The binding to recombinant GPA of antibodies submitted to the Third International Workshop was assessed by immunoprecipitation and indirect immunofluorescence. This is a powerful approach for determining the fine specificity of antibodies to blood group antigens. The advantages and limitations of this approach are discussed.

Comparison of the different strategies for cryopreserving and storage of the bone marrow CD34+ cells. Possibility of unprogrammed rate freezing and storage at -80 degrees C mechanical freezer.

We have compared the efficiency of the different strategies allowing for a long term storage of a human CD34+ bone marrow cells. Accordingly, the aliquots of CD34+ cells isolated from bone marrow were frozen using: controlled rate freezing equipment, or freezer unprogrammed in a -80 degrees C mechanical freezer. After freezing, CD34+ cells were subsequently stored for one month in a liquid nitrogen tank at -196 degrees C or in mechanical freezer at -80 degrees C. We have found that both the viability and the recovery of clonogeneic progenitors of CD34+ cells samples stored at different temperature were similar. Therefore, regarding the costs and simplicity, we recommend the unprogrammed freezing and storage of human CD34+ cells at -80 degrees C in a mechanical freezer as a convenient, inexpensive, and reliable method for storing marrow for transplantation. This data also indirectly indicate that the aliquots of the CD34+ cells can be shipped frozen on dry ice (-80 degrees C), and that these cells will maintain viability under this conditions. Furthermore, in this study we have confirmed validity of our earlier observation that human CFU-Meg progenitors are more sensitive to cryopreservation.

Evidence that human haematopoietic stem cells (HSC) do not reside within the CD34+KIT- cell population.

Contradictory reports are published concerning the c-kit receptor (KIT) expression on human haematopoietic stem cells (HHSC). Therefore, the aim of this study was to reevaluate the expression of KIT on human early haematopoietic cells, and to study the distribution of HHSC among bone marrow mononuclear KIT+ and KIT- cells. First, we found that the detection sensitivity of the KIT expression on human HEL cells as well as CD34+ depends on the type of fluorochrome employed for the immunostaining (Cy5 > PE > FITC). Based on this observation, in our strategy for isolating human HHSC we employed a Cy-5 conjugated alpha-KIT MoAbs, which stained CD34+ cells in our preliminary studies the brightest. Accordingly, we labeled human BMMNC with PE-alpha-CD34 and Cy5-alpha-KIT MoAbs and subsequently sorted various subsets of labeled cells (CD34+KIT+, CD34+KIT- and CD34-KIT-). Cells sorted by FACS were then evaluated for their ability to engraft in the immunodeficient SCID mice model. We report here that only CD34+KIT+ cells, but not CD34+KIT- or CD34-KIT- were able to establish a human-murine haematopoietic chimerism in these animals. We found that SCID mice transplanted with CD34+ KIT+ cells, possessed approximately 5-11% of mononuclear cells, which expressed human CD45 antigen 4-5 weeks after transplantation in their bone marrow and, more importantly, early human haematopoietic progenitors from the myeloid and B-lymphoid lineages. Based on these results we conclude that KIT (CD117) is a very useful marker for identifying HHSC, and that HHSC, at least in our hands, are found in the KIT+ population of CD34+ cells.

[Thrombocytopenia in a patient with hemophilia and HIV infection treated with intravenous immunoglobulin and splenectomy]. / Maloplytkowosc u chorego na hemofilie z zakazeniem HIV leczona dozylnie immunoglobulina i splenektomia.

In a patient with haemophilia B who had developed immune thrombocytopenia following HIV infection, high doses of intravenous immunoglobulin were administered. The therapy brought about only a temporary rise in platelet counts. No beneficial effects were also obtained during a 3-month treatment with corticosteroids. Since there was no response to the conservative therapy, the patient was splenectomized under the cover of factor IX concentrate and the surgical procedure resulted in a complete remission of the thrombocytopenia.

[Effect of thioproline in vitro on selected functions of peripheral blood lymphocytes from healthy persons and patients with lympho- proliferative syndromes]. / Wplyw tioproliny in vitro na wybrane funkcje limfocytów krwi obwodowej osób zdrowych i chorych z zespolami limfoproliferacyjnymi.

Thioproline (thiazolidino-4-carboxylic acid) is one of promising antineoplastic preparations arousing interest since several years. In view of controversies with respect to the mechanism of thioproline action it was tried to study the effects of the preparation on certain functions of lymphocytes. The experimental model included cultures of T and B lymphocytes obtained from 20 healthy blood donors. The cultures were exposed to various concentrations of thioproline for determining the incorporation of 3H-thymidine into the DNA of these cells, their viability and intracellular concentrations of cAMP and cGMP. The lymphocytes from these healthy donors served also as a control for B-cells isolated from 24 patients with lymphoproliferative syndromes. It was found that at low concentrations thioproline added to the culture of T-cells from healthy donors caused a slight increase of 3H-thymidine incorporation, but at high concentrations a strong cytotoxic effect on T-cells was noted. B-cells isolated from the peripheral blood of healthy subjects and from the patients were highly sensitive to thioproline. These observations demonstrated a cytotoxic effect of thioproline on T and B lymphocytes.

[Preliminary results of the treatment of acute leukemia with mitoxantrone]. / Wstepne wyniki leczenia ostrych bialaczek mitoksantronem.

Mitoxantrone is a new anthracenodione derivative with a high antineoplastic activity in proliferative diseases of the haemopoietic system. In the Institute of Haematology in Warsaw and in the Department of Haematology, Silesian Medical Academy in Katowice this agent was used in combination with cytarabine in 49 cases of acute leukaemia (35 with acute myeloid leukaemia and 14 with acute lymphoblastic leukaemia). The preparations used were Mitoxantrone (POLFA Works in Jelenia Góra) and Novantrone (Lederle). These agents were given intravenously in doses of 10-20 mg/m2 for 3 days in combination with cytarabine in three doses: 100 mg/m2 on days 1 through 7, and 1 g/m2 or 3 g/m2 every 12 hours on days 1 through 4 of the treatment. Complete remission was obtained in 17 cases (35%), including 13 with acute myeloid leukaemia (37%) and 4 with acute lymphoblastic leukaemia (29). The most frequent side effects were: long-lasting pancytopenia (in 100% of cases), hair loss (39%) and gastrointestinal toxicity (33%). No significant differences were noted in the effectiveness and toxicity between these two preparations. In the light of the presented results it may be accepted that the combination of mitoxantrone with cytarabine is an important advance in the treatment of acute leukaemias.

New T-lymphocytic cell lines for studying cell infectability by human immunodeficiency virus.

We identified five human T-lymphoid cell lines (PB-1, Sez-4, C19PL, HUT 102B and ATL-2) which highly express CD4 in addition to CXCR4 and CCR5. In order to evaluate if these cells are infectabile by human immunodeficiency virus (HIV) and could be employed as a model in HIV research we exposed these cell lines to X4 (T-cell tropic) and R5 (macrophage tropic) and subsequently tried to correlate their infectability with (i) level of chemokine coreceptor (CXCR4 and CCR5) expression, (ii) coreceptor functionality (calcium flux, chemotaxis and phosphorylation of MAPK p42/44 and AKT) and (iii) endogenous expression and secretion of HIV-related chemokines which compete with the virus for binding to CXCR4 (SDF-1/CXCL12) or CCR5 (MIP-1beta/CCL4, MIP-1alpha/CCL3, RANTES/CCL5, MCP-2/CCL8, MCP-3/CCL7 and MCP-4/CCL13). We demonstrated that while PB-1 cells are infectable by both X4 and R5 HIV, Sez-4, C91PL, HUT 102B and ATL-2 cells were infected by X4 HIV only. Moreover, we noticed that the susceptibility of these cells to HIV did not correspond either with the level of surface expression or with the functionality of CXCR4 or CCR5; however, it was modulated to some degree by the endogenously secreted HIV-related chemokines. Thus all five mature T-cell lines described here may provide useful new models for studying various aspects of HIV infection. In addition we demonstrate that the infectability of cells by HIV is modulated by so far unidentified intrinsic factors as well as some already known endogenously secreted chemokines. The identification of these factors may be important for developing new strategies to protect cells from HIV infection.