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To determine whether cellular components of tissue-engineered cardiovascular structures are derived from cells harvested and seeded onto an acellular scaffold, or from cells originating from surrounding tissue (e.g., proximal and distal anastomosis), cellular retroviral transfection with green fluorescent protein (GFP) was used. Ovine endothelial cells (ECs) were transfected with a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector expressing GFP. Transfection was evaluated by fluorescence microscopy and fluorescence-activated cell sorting. The rate of transfection of the primary cells was 33.4% for ECs, 48 hours after transfection. Stable transfection could be observed for at least 25 subsequent passages. Retroviral transfection with GFP enables stable and reliable long-term labeling of ovine ECs. This approach might offer an attractive pathway to study tissue development, with emphasis on distinguishing between cellular components initially seeded onto a construct and those occurring as a result of cell ingrowth from surrounding tissue.

The human CD7 antigen (gp40) is a cell surface glycoprotein found on thymocytes and mature T‐cells. It is one of the earliest antigens to appear on cells of the T‐lymphocyte lineage, and the most reliable clinical marker of T‐cell acute lymphocytic leukemia. This report describes the isolation and nucleotide sequence of a full length CD7 cDNA, and of a cDNA for an unusual intron‐bearing precursor. The DNA sequence of the clone predicts a highly glycosylated membrane protein with homology to members of the immunoglobulin superfamily, and no relationship to known oncogenes. Over‐expression of CD7 RNA was observed in only one T‐cell tumor line, and genomic DNA rearrangement was not observed in any lines. Prompted by a recent suggestion that CD7 plays a role in IgM binding, COS cells expressing CD7 were tested and found not to bind IgM or IgM immune complexes.

CD44 is a polymorphic integral membrane protein which recognizes hyaluronate and whose proposed roles encompass lymphocyte activation, matrix adhesion and the attachment of lymphocytes to lymph node high endothelial venules (HEVs). Immunochemical and RNA blot data have supported the existence of two forms of CD44: a hematopoietic form expressed by cells of mesodermal origin (and by some carcinoma cell lines) and an epithelial form weakly expressed by normal epithelium but highly expressed by carcinomas. This report describes the isolation of a cDNA encoding a distinct CD44 polypeptide expressed by epithelial cells. Re‐expression of each form of CD44 in a B cell line allowed cells transfected with the hematopoietic but not the epithelial form to bind to viable rat lymph node HEV cells in primary culture.

The CD95 (Fas/APO‐1) and tumor necrosis factor (TNF) receptor pathways share many similarities, including a common reliance on proteins containing ‘death domains’ for elements of the membrane‐proximal signal relay. We have created mutant cell lines that are unable to activate NF‐kappaB in response to TNF. One of the mutant lines lacks RIP, a 74 kDa Ser/Thr kinase originally identified by its ability to associate with Fas/APO‐1 and induce cell death. Reconstitution of the line with RIP restores responsiveness to TNF. The RIP‐deficient cell line is susceptible to apoptosis initiated by anti‐CD95 antibodies. An analysis of cells reconstituted with mutant forms of RIP reveals similarities between the action of RIP and FADD/MORT‐1, a Fas‐associated death domain protein.

B cells and primary carcinomas express a surface molecule, Bp50 (CDw40), absent from other hematopoietic cells and from normal epithelium, and thought to play a regulatory role in B‐cell maturation and epithelial neoplasia. In this work the sequence of a cDNA clone encoding Bp50 was analyzed by a newly derived transition matrix method. Among several interesting relationships with known receptors was found an extensive homology with the nerve growth factor receptor. The mRNA is induced by gamma‐interferon in both B cells and epithelial neoplasms, suggesting a role for the molecule in the development of carcinomas at sites of chronic inflammation.

An emerging theme in immunology finds receptors which initiate cellular effector programs forming multichain complexes in which the ligand recognition elements associate with one or more ‘trigger molecules’ whose aggregation initiates a signal transduction cascade. The sequence motifs constituting the active sites of these trigger molecules are found in the T cell and B cell antigen receptors, and some Fc receptors, and appear to be central to effector function activation. For example, of the many molecules that mimic or potentiate the action of the T cell antigen receptor (TCR), none have yet been found to initiate effector programs autonomously in cells lacking TCR. We have devised two strategies to study activation mediated by myeloid Fc receptors, which appear not to associate with trigger molecules: the use of primary human cytolytic T cells as surrogate effector cells for genetically delivered receptors, and the use of vaccinia virus vectors to introduce genetically modified receptors into primary human monocytes. Using these approaches, we have found that the cytoplasmic domains of two Fc receptors show comparable function to equivalent domains of the trigger molecule family, but are not homologous to members of that family.

Two cDNA clones encoding different but related receptors for immunoglobulin G constant domains were isolated from cDNA expression libraries by a ligand‐mediated selection procedure (‘affinity cloning’). Because both of the receptors encoded by the cDNAs react with CDw32 monoclonal antibodies, and both show the appropriate IgG binding affinity, both appear to be forms of the receptor formerly thought to be a single species called FcRII. The extracellular domains encoded by the isolated clones are closely related to the murine IgG2b/1 beta receptor extracellular domains, but the intracellular domains are unrelated. The receptors expressed in COS cells show a preference for IgG1 among IgG subtypes and no affinity for IgM, IgA or IgE. Abundant expression of the RNAs was detected in myeloid cell lines and placenta.

A cDNA clone encoding the human monocyte antigen CD14 was isolated by transient expression in COS cells of a cDNA library prepared from phorbol diester-treated HL60 cells. RNA blot analysis showed abundant expression of a single mRNA species in mature monocytes and an increased expression of the mRNA following induction of differentiation in leukemic cell lines. The DNA blot hybridization pattern was consistent with a single-copy gene. The predicted amino acid sequence lacks the characteristic transmembrane domain and stop transfer motif of conventionally anchored membrane proteins. COS cells transfected with the CD14 cDNA released virtually all CD14 protein in soluble form following treatment with glycosyl phosphatidylinositol-specific phospholipase C, and CD14 immunoreactivity was absent from the affected monocytes of a patient with paroxysmal nocturnal hemoglobinuria (PNH). The data show that, to the limit of experimental sensitivity, all monocyte CD14 is joined to the plasma membrane by a phosphatidylinositol phospholipid.

Phagocytosis is a phylogenetically primitive mechanism adapted by specialized cells of the immune system to ingest particulate pathogens. Recent evidence suggests that the program of specific cytoskeletal rearrangements that underlies phagocytosis may share elements with the antigen receptor signaling pathway in lymphocytes. Tyrosine phosphorylation, necessary for both lymphocyte effector function and phagocytosis, is thought to allow cytoskeletal elements to couple to the intracellular domains of antigen and Fc receptor subunits. We show here that the intracellular domains of the receptors are not inherently required for cytoskeletal coupling. Chimeric transmembrane proteins bearing syk but not src family tyrosine kinase domains are capable of autonomously triggering phagocytosis and redistribution of filamentous actin in COS cells. These responses cannot be initiated by a receptor chimera bearing a point mutation in the syk catalytic domain, and the kinase domain alone is sufficient for initiating cytoskeletal coupling.

The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.

Three major difficulties have hindered the general application of in vivo recombination techniques to library screening: (i) the original selection could not be applied to libraries prepared in phage vectors lacking amber mutations, (ii) nonirradiated packaging extracts gave high backgrounds even when amber mutated vectors were used, and (iii) most red- vectors lacked rap, a recently discovered phage gene promoting phage-plasmid recombination. Here we describe a selection scheme for phage bearing suppressor tRNA plasmids, which relies upon an Escherichia coli host bearing an amber mutation in the dnaB gene. The selection is tight enough to allow library screening by recombination, is applicable to almost every phage vector in common use, and overcomes the background associated with nonirradiated packaging extracts. We also describe an ancillary plasmid that supplies the rap gene function in trans, permitting the recombination level to be raised fruitfully in phage libraries lacking endogenous rap. Finally, we demonstrate simple procedures for preparing and detecting phages that have lost integrated suppressor tRNA plasmids by homologous recombination.

We describe the synthesis of a family of arylamine-substituted papers which can be diazotized and coupled with nucleic acids. The synthesis is simple and uses readily obtainable starting materials. A partial characterization of the nucleic acid binding activity is reported, as well as a demonstration of the utility of the activated paper for the detection of electrophoretically separated RNA by blot transfer and hybridization.

To define the role of the CD2-lymphocyte function-associated antigen 3 (LFA-3) interaction in T-cell activation, we have expressed a cDNA encoding the human CD2 molecule in a murine antigen-specific T-cell hybridoma. Expression of the CD2 molecule greatly enhances T-cell responsiveness to antigen; this enhancement is inhibited by anti-CD2 and anti-LFA-3 monoclonal antibodies (mAbs). CD2+ hybridomas produce interleukin 2 in response to combinations of anti-CD2 mAbs 9.6 and 9-1 and, in the presence of mAb 9-1, to sheep erythrocytes or to the LFA-3 antigen. Furthermore, hybridomas expressing a mutant CD2 molecule that has lost mAb 9.6 binding do not exhibit the enhanced response to antigen or the ability to respond to LFA-3 plus mAb 9-1, but these hybridomas retain the ability to respond to combinations of anti-CD2 mAbs. The role of the CD2-LFA-3 interaction in T-cell activation and the potential for other physiologic ligands for CD2 are discussed.

The Syrinx 2A phage and pi AN13 plasmid were designed for screening of DNA libraries by homologous recombination in vivo. Syrinx 2A carries multiple cloning sites and a recently identified lambda gene, rap (recombination adept with plasmid), required for efficient phage-plasmid recombination. We describe a rapid, reliable, and technically easy method to screen Syrinx 2A libraries, expand the resulting phage-plasmid cointegrates, and subclone plasmid in as little as 2 days. Recombination screening allows one specific member of a closely related multigene family to be isolated selectively.

CD28 (Tp44) is a human T-cell-specific homodimer surface protein that may participate in T-cell activation. We have isolated a cDNA clone encoding CD28 by a simple and highly efficient cloning strategy based on transient expression in COS cells. Central to this strategy is the use of an efficient method to prepare large plasmid cDNA libraries. The libraries are introduced into COS cells, where transient expression of surface antigen allows the isolation of cDNAs by way of monoclonal antibody binding. The CD28 cDNA encodes a highly glycosylated membrane protein with homology to the immunoglobulin superfamily and directs the production of a homodimer in transfected COS cells.

The isolation and expression of a full-length cDNA clone encoding the B cell-specific glycoprotein CD19 is reported. The sequence of the cDNA predicts a glycosylated integral membrane protein with a precursor molecular weight of 51.8 x 10(3) and an extracellular domain organized into three contiguous Ig-like sub-domains. The cytoplasmic domain bears significant relatedness to two proteins encoded by the Epstein-Barr virus and the int-1 oncogene. CD19 transcripts are restricted to members of the B cell lineage, being most abundant in pre-B cell lines and least abundant in plasmacytomas.

Comparison of NH2-terminal protein sequence from the rat OX-44 antigen with the sequence of the human CD37 antigen deduced from a cDNA clone shows that these antigens are species homologues. The CD37 sequence is 244 amino acids in length and lacks a conventional leader sequence. The molecule is likely to have an NH2-terminal cytoplasmic domain followed by three transmembrane sequences that lie within the first 110 amino acids. The rest of the molecule is hydrophillic and contains three sites for N-linked glycosylation.

The isolation, characterization, and expression of a full-length cDNA encoding the human T cell glycoprotein CD6 is described. COS cells transfected with the CD6 clone express a 90-kD protein that reacts with all available anti-CD6 monoclonal antibodies. RNA blot hybridization analysis indicates that CD6 transcripts are predominantly restricted to cells in the T lineage. The predicted CD6 sequence is 468 amino acids long, with the typical features of a type I integral membrane protein. The cytoplasmic domain of CD6 contains two serine residues, one or both of which are substrates for phosphorylation during T cell activation. The extracellular domain of CD6 is significantly related to the extracellular domain of the human and mouse T cell antigen CD5, the cysteine-rich domain of the bovine and mouse type I macrophage scavenger receptor, the extracellular domain of the sea urchin spermatozoa protein that crosslinks the egg peptide speract, the mammalian complement factor 1, and the human lung tumor antigen L3. These molecules, therefore, constitute a new gene superfamily that is well conserved across species boundaries.

We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

Two cDNA clones encoding the pan-B cell CD20 antigen were isolated from a COS cell expression library. The two clones bear identical coding sequences and differ only in the length of the 3' untranslated region. The predicted CD20 sequence is 297 residues long and contains three hydrophobic domains, one of which is long enough to span the membrane twice. COS cells transfected with either CD20 clone express an immunoreactive protein of 33 kD.

A cDNA encoding the CD2 antigen has been isolated by a highly efficient technique based on transient expression in COS cells and adherence of cells expressing surface antigen to antibody-coated dishes. COS cells expressing a CD2 cDNA isolated by this method readily formed rosettes with sheep as well as human and other mammalian erythrocytes. Pretreatment of transfected COS cells with anti-CD2 antibody, or pretreatment of human erythrocytes with anti-LFA-3 antibody, abolished rosette formation.