Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.

Induction of "tissue" transglutaminase in HIV pathogenesis: evidence for high rate of apoptosis of CD4+ T lymphocytes and accessory cells in lymphoid tissues.

Peripheral blood mononuclear cells and lymphoid tissues from HIV-infected individuals display high levels of "tissue" transglutaminase (tTG) with respect to seronegative persons. In asymptomatic individuals, > 80% of the circulating CD4+ T cells synthesize tTG protein and the number of these cells matches the level of apoptosis detected in the peripheral blood mononuclear cells from the same patients. In HIV-infected lymph nodes tTG protein is localized in large number of cells (macrophages, follicular dendritic cells, and endothelial cells), showing distinctive morphological and biochemical features of apoptosis as well as in lymphocytes and syncytia. These findings demonstrate that during the course of HIV infection, high levels of apoptosis also occur in the accessory cells of lymphoid organs. The increased concentration of epsilon(gamma-glutamyl)lysine isodipeptide, the degradation product of tTG cross-linked proteins, observed in the blood of HIV-infected individuals demonstrates that the enzyme accumulated in the dying cells actively cross-links intracellular proteins. The enhanced levels of epsilon(gamma-glutamyl)lysine in the blood parallels the progression of HIV disease, suggesting that the isodipeptide determination might be a useful method to monitor the in vivo rate of apoptosis.

A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.

Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections. The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported. The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells. The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C. The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor. Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding. Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP. These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells. Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae. Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan.

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.

Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells.

Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene.

The possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenium metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)-containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminal-deduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val-Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved C-terminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system.

Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells.

Because of their known myelopoietic activities, both interleukin (IL)-3 and IL-1 are often used in combination with other cytokines for in vitro (ex vivo) expansion of stem cells. We have investigated the effects of IL-3 and IL-1 on in vitro expansion of murine hematopoietic stem cells with long-term engraftment capabilities, using a highly purified progenitor population. Lineage-negative, Ly-6A/E+, c-kit+ bone marrow cells from male mice were cultured in suspension in the presence of stem cell factor, IL-6, IL-11, and erythropoietin with or without IL-3 or IL-1. Kinetic studies revealed an exponential increase in total nucleated cells and about 10-fold enhancement of nucleated cells by IL-3 during the initial 10 days. Addition of IL-3 hastened the development but significantly suppressed the peak production of colony-forming cells. Addition of IL-1 also significantly suppressed the numbers of colony-forming cells. The reconstituting ability of the cultured cells was tested by transplanting the expanded male cells into lethally irradiated female mice. The cells expanded from enriched cells in the absence of IL-3 and IL-1 revealed engraftment at 2, 4, 5, and 6 months, whereas addition of IL-3 or IL-1 to the cultures significantly reduced the reconstituting ability. The results suggest that these cytokines may have a modulatory role on the self-renewal of stem cells and further indicate that the use of IL-3 and IL-1 for in vitro expansion of human stem cells needs to be cautiously evaluated.

Recombinant human uteroglobin suppresses cellular invasiveness via a novel class of high-affinity cell surface binding site.

The mechanism(s) that regulates invasion of trophoblasts through the uterine epithelium during embryo implantation and nidation in hemochorial placental mammals is poorly understood. While limited trophoblast invasion is essential for the establishment of normal pregnancy, dysregulation of this process may contribute to the pathogenesis of choriocarcinoma, a highly invasive and lethal form of cancer arising from the trophoblasts. We have previously demonstrated that rabbit uteroglobin (UG), a cytokine-like, antiinflammatory protein, produced by the endometrial epithelium during pregnancy, has a potent antichemotactic effect on neutrophils and monocytes in vitro. Here, we report that recombinant human UG (hUG) dramatically suppresses invasion of human trophoblasts and NIH 3T3 cells through an artificial basement membrane (Matrigel) in vitro but has no effect on that of human choriocarcinoma cells. We identified a previously unreported high-affinity, high molecular weight (approximately 190 kDa), nonglycosylated hUG-binding protein, readily detectable on human trophoblasts and NIH 3T3 cells but totally lacking on choriocarcinoma cells. Taken together, these results raise the possibility that (i) hUG plays a critical role in regulating cellular invasiveness, at least in part, via its previously unrecognized cell surface binding site, and (ii) some of the numerous biological activities of proteins of the UG family, reported so far, may be mediated via this binding site.

Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus.

The restriction of phosphatidylserine (PtdSer) to the inner surface of the plasma membrane bilayer is lost early during apoptosis. Since PtdSer is a potent surface procoagulant, and since there is an increased incidence of coagulation events in patients with systemic lupus erythematosus (SLE) who have anti-phospholipid antibodies, we addressed whether apoptotic cells are procoagulant and whether anti-phospholipid antibodies influence this. Apoptotic HeLa cells, human endothelial cells, and a murine pre-B-cell line were markedly procoagulant in a modified Russell viper venom assay. This procoagulant effect was entirely abolished by addition of the PtdSer-binding protein, annexin V, confirming that it was PtdSer-dependent. The procoagulant effect was also abolished by addition of IgG purified from the plasma of three patients with anti-phospholipid antibody syndrome, but not IgG from normal controls. Confocal microscopy of apoptotic cells stained with fluorescein-isothiocyanate-conjugated-annexin V demonstrated (Ca2+)-dependent binding to the surface of membrane blebs o apoptotic cells, but not to intracellular membranes. Recent data indicate that the surface blebs of apoptotic cells constitute an important immunogenic particle in SLE. We propose that the PtdSer exposed on the outside of these blebs can induce the production of anti-phospholipid antibodies, which might also enhance the immunogenicity of the bleb contents. When apoptosis occurs in a microenvironment in direct contact with circulating plasma, the unique procoagulant consequences of the apoptotic surface may additionally be expressed. This might explain the increased incidence of pathological intravascular coagulation events that occur in some lupus patients who have anti-phospholipid antibodies.

Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins.

The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.

Characterization of GMP-17, a granule membrane protein that moves to the plasma membrane of natural killer cells following target cell recognition.

Cytotoxic lymphocytes are characterized by their inclusion of cytoplasmic granules that fuse with the plasma membrane following target cell recognition. We previously identified a cytotoxic granule membrane protein designated p15-TIA-1 that is immunochemically related to an RNA-recognition motif (RRM)-type RNA-binding protein designated p40-TIA-1. Although it was suggested that p15-TIA-1 might be derived from p40-T1A-1 by proteolysis, N-terminal amino acid sequencing of p15-TIA-1 immunoaffinity purified from a natural killer (NK) cell line by using monoclonal antibody (mAb) 2G9 revealed that p15-T1A-1 is identical to the deduced amino acid sequence of NKG7 and GIG-1, cDNAs isolated from NK cells and granulocyte-colony-stimulating factor-treated mononuclear cells, respectively. Epitope mapping revealed that mAb 2G9 recognizes the C terminus of p15-T1A-1 and p40-T1A-1. The deduced amino acid sequence of p15-T1A-1/NKG7/GIG-1 predicts that the protein possesses four transmembrane domains, and immuno-electron microscopy localizes the endogenous protein to the membranes of cytotoxic granules in NK cells. Given its subcellular localization, we propose to rename-this protein GMP-17, for granule membrane protein of 17 kDa. Immunofluorescence microscopy of freshly isolated NK cells confirms this granular localization. Target cell-induced NK cell degranulation results in translocation of GMP-17 from granules to the plasma membrane, suggesting a possible role for GMP-17 in regulating the effector function of lymphocytes and neutrophils.

Osmotic regulation of cytokine synthesis in vitro.

These studies were undertaken to investigate the therapeutic mechanism of saturated solutions of KI, used to treat infectious and inflammatory diseases. The addition of 12-50 mM KI to cultured human peripheral blood mononuclear cells resulted in 319-395 mosM final solute concentration and induced interleukin (IL)-8 synthesis. Maximal IL-8 production was seen when 40 mM salt was added (375 mosM) and was equal to IL-8 induced by endotoxin or IL-1 alpha. However, there was no induction of IL-1 alpha, IL-1 beta, or tumor necrosis factor to account for the synthesis of IL-8; the effect of KI was not due to contaminating endotoxins. Hyperosmolar NaCl also induced IL-8 and increased steady-state levels of IL-8 mRNA similar to those induced by IL-1 alpha. IL-8 gene expression was elevated for 96 hr in peripheral blood mononuclear cells incubated with hyperosmolar NaCl. In human THP-1 macrophagic cells, osmotic stimulation with KI, NaI, or NaCl also induced IL-8 production. IL-1 signal transduction includes the phosphorylation of the p38 mitogen-activated protein kinase that is observed following osmotic stress. Using specific blockade of this kinase, a dose-response inhibition of hyperosmolar NaCl-induced IL-8 synthesis was observed, similar to that in cells stimulated with IL-1. Thus, these studies suggest that IL-1 and osmotic shock utilize the same mitogen-activated protein kinase for signal transduction and IL-8 synthesis.

Human naive and memory T lymphocytes differ in telomeric length and replicative potential.

The present study has assessed the replicative history and the residual replicative potential of human naive and memory T cells. Telomeres are unique terminal chromosomal structures whose length has been shown to decrease with cell division in vitro and with increased age in vivo for human somatic cells. We therefore assessed telomere length as a measure of the in vivo replicative history of naive and memory human T cells. Telomeric terminal restriction fragments were found to be 1.4 +/- 0.1 kb longer in CD4+ naive T cells than in memory cells from the same donors, a relationship that remained constant over a wide range of donor age. These findings suggest that the differentiation of memory cells from naive precursors occurs with substantial clonal expansion and that the magnitude of this expansion is, on average, similar over a wide range of age. In addition, when replicative potential was assessed in vitro, it was found that the capacity of naive cells for cell division was 128-fold greater as measured in mean population doublings than the capacity of memory cells from the same individuals. Human CD4+ naive and memory cells thus differ in in vivo replicative history, as reflected in telomeric length, and in their residual replicative capacity.

In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.