In 1977 we showed that cells of a human lymphocytic leukaemia-derived T line (Molt-4) have receptors for Epstein-Barr virus (EBV). More recently, EBV-positive human T cell lymphomas have been ...recognized and human T cell lines containing the EBV genome have been established in vitro. To understand better the interaction of EBV with T cells, we decided to determine first whether human peripheral blood T lymphocytes express receptors for EBV. Using flow cytometry we examined the binding of both lymphocyte-transforming (B95-8) and non-transforming (P3HR-1) strains of EBV to T lymphocyte subpopulations, using a double labelling technique with T cell-specific phycoerythrinated monoclonal antibodies (Leu 2a) and fluoresceinated viral preparation. Our results suggest that, in general, about 50% of the CD8+ (or suppressor/cytotoxic) T cell subpopulation from both EBV-seropositive and -seronegative individuals can bind EBV. EBV receptor expression on these T cells was about 10 and 51 times less than that on Molt-4 and Raji (an EBV receptor-positive B cell line) cells, respectively. The specificity of this binding was demonstrated by the inhibition of attachment of viral preparations preincubated with a monoclonal antibody directed against the viral ligand (gp240/350), and by preincubating these target T cells with unlabelled virus. We were unable to detect EBV-induced antigens in infected T cells, suggesting that, as in Molt-4 cells, virus internalization may not occur in fresh T cells and/or that the virus receptor may not be completely functional. We were also unable to detect C3d (or CR2) receptors on these T cells, or to inhibit virus attachment by treating the targets with an anti-CR2 monoclonal antibody (OKB7), suggesting that the EBV receptor on CD8+ peripheral blood lymphocytes is different from that on B cells.
The Hox genes, first identified in Drosophila, are now recognized as key
determinants of mammalian development. Recent evidence of Hox gene expression in leukemic cell lines have suggested that these ...transcription factors also play important roles in regulation of hematopoiesis. Using an improved RT/PCR technique which enables the generation of extended-length and
representative cDNA from fewer than 1,000 cells, the expression pattern of Hox
genes in highly purified primitive hematopoietic subpopulations was examined.
Two different approaches were used. The first exploited the presence of a highly conserved region in the DNA-binding domain of Hox genes that could be targeted with degenerate primers for amplification from cDNA obtained from each purified population and the amplified produced were subsequently
subcloned and sequenced. Over 150 Hox sequence containing clones were
characterized. HOXA9, A6, A5, A4, A2, B3, B7 B9 and C9 were detected in a subpopulation which was highly enriched for very primitive cells detected as long-term culture-initiating cells (LTC-IC) and depleted for clonogenic progenitors. HOXA1O, A9, A7 A5, A4 and B7 were detected in a subpopulation highly enriched for clonogenic myeloid progenitors, and HOXA1O, A9, A7, A6,
A5, B7and C8 were found in an erythroid progenitor enriched subpopulation. These data suggested that HOX A cluster genes are widely expressed amongst early hematopoietic subpopulations; in contrast, some genes of the B cluster appear to be restricted to the more primitive LTC-lC containing subpopulation. To better assess the potential differential expression of Hox genes, the initial amplified cDNA from five purified CD34 subpopulations of bone marrow cells
was analyzed by Southern blot using probes for specific Hox genes. With this
approach, it was possible to show that expression of HOXB3 and B4 was markedly higher (up to 40 fold) in the most primitive subpopulation than in the more mature subpopulations whereas that of other Hox genes such as HOXA1O, A9 and B9 was constant in all populations. Taken together, these data suggest that many Hox genes are active in early hematopoiesis and that some
of them (i.e. HOXB3 and B4) are possible candidates for regulating stem cell
function. To gain further insight into the role Hox genes may play in early hematopoiesis, the effect of overexpression of HOXB4 was studied in a murine
model using a myeloproliferative sarcoma-based retroviral vector carrying the
human HOXB4 cDNA under the control of the 5’ viral long terminal repeat. Overexpression of HOXB4 had proliferative effects on clonogenic progenitors, day 12 CFU-S and cells with marrow repopulating ability (MRA) as assessed by colony replating or recovery from seven day liquid cultures (up to 200 fold over
neo-transduced control). To study the possible effects of HOXB4 overexpression on hematopoietic cells maintained for prolonged periods in vivo, HOXB4 or nec-transduced marrow cells were transplanted into lethally
irradiated syngeneic recipients and reconstitution of various hematopoietic
populations analyzed. At 20 weeks post-transplantation, recipients of HOXB4-
transduced marrow had -5 fold more clonogenic progenitors per femur than neo controls. To determine if HOXB4 overexpression affected the expansion of the earliest hematopoietic stem cells (HSC), their numbers were determined by using the competitive repopulation unit (CRU) assay. CRU numbers in
recipients of HOXB4-transduced bone marrow cells had recovered to 140% of
normal levels found in untransplanted mice or some 50 fold higher than in recipients of nec-transduced marrow; all recipients of HOXB4-transduced marrow however had normal peripheral blood counts. Southern blot analysis of unique proviral integration patterns in DNA isolated from bone marrow and
thymus of secondary recipients confirmed that HQXB4-transduced hematopoietic repopulating cells were totipotent and that they extensively self-renewed. This dramatic effect of HOXB4 on HSC self-renewal was maintained in serial transplantation experiments. Together, these results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell
proliferation. The ability of HOXB4 to reverse the severe decline in HSC
numbers suggest an exciting new approach to the controlled amplification of
genetically modified hematopoietic stem cell populations.
A study documents expression of multiple Hox genes in primitive normal hematopoietic cells and identifies striking differences in the patterns of expression of certain Hox genes within functionally ...distinct subpopulations of these cells.
This study was aimed at quantitating, by means of fluorescence-activated cell sorter (FACS), EBV binding to different types of target cells, and at learning about a possible relation between EBV ...receptor density and the fate of cell-surface bound virus. We used fluoresceinated virus preparations of two strains of EBV (B95-8: lymphocyte transforming strain; P3HR-1: non-transforming strain) to analyze quantitatively the expression and density of EBV receptors on different human lymphoid cell lines and on B lymphocytes from both EBV-seropositive and -seronegative donors. FACS analysis was also used as a tool to approximate the cell surface area of the different lymphoid cells examined. Our results indicate that: (a) after accounting for the difference in cell surface dimensios, the fluorescence intensity of EBV-bound Raji (a B line) cells was three to four times higher per unit area than that of EBV-bound fresh B lymphocytes from an EBV-seropositive donor; (b) Molt-4 (a T line) cells bound about 21-fold less P3HR-1 EBV and 6-fold less B95-8 EBV than Raji cells per unit area; (c) B lymphocytes from EBV-seronegative adult donors bound only about one third as much virus as B cells from seropositive individuals; (d) two B lymphocyte sub-populations can be identified in the peripheral blood in regard to their ability to bind EBV, regardless of the EBV antibody status of the donor; (e) the EBV receptor on Molt-4 cells appears structurally different from the one found on Raji cells since EBV binding to Molt-4 cells was not blocked by a monoclonal antibody (OKB7) specific to the complement receptor (CR2). Further, in contrast to Raji cells, Molt-4 expressed a differential binding activity for each of the two EBV strains used. Taken together, the important differences observed in regard to EBV attachment to various targets also appear to relate to the fate of cell-surface bound virus: i.e., virus penetration might be determined, at least in part, by the density of EBV receptors on the target cell surface; thus the receptor density may play a major role in viral infection.
Two homosexual HIV1-positive male patients with thrombotic thrombopenic purpura (TTP) were found to have past history of immune thrombocytopenia (ITP). The first patient had ITP 10 months prior to ...TTP and was successfully treated by splenectomy. The second patient had ITP 32 months before TTP. No specific treatment was given for his asymptomatic ITP. Association of HIV infection to TTP seems to be frequent. These two types of purpura have different pathogenic mechanisms but share a common altered immune response to antigenic challenge. The occurrence of ITP and TTP in HIV-positive patients may lead to errors in diagnosis and therapy.
Viral envelope glycoproteins are important in antiviral immunity; however, their precise role in both generating virus-neutralizing antibodies as well as in viral binding to target cell receptors ...remains largely unexplored. We studied nine monoclonal antibodies (MoAbs) to Epstein-Barr virus (EBV) envelope glycoproteins in order to define the role(s) of their respective epitopes in both EBV neutralization and binding to cellular receptors. Only four MoAbs neutralized EBV infectivity, one requiring complement, and only 1 of these 4 did inhibit EBV binding to target cell receptors. Our results suggest that (1) the epitopes recognized by a majority of these MoAbs play no role in viral neutralization and (2) that the majority of epitopes recognized by the neutralizing antibodies play no role in EBV binding to target cell receptors. This and previous studies would also suggest that only one epitope is involved in EBV binding to its receptor or that, until now, we were able to identify only one EBV-neutralizing MoAb (i.e., 72A1) which is specific for the receptor-binding viral epitope. It is probable that the epitope recognized by 72A1 MoAb is the only glycoprotein domain with a dual role, i.e., in EBV neutralization and binding to target cell receptors.
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