Novel tetrameric tip motifs (APGK and VPGK) in the V3 loop of HIV type 1 envelope sequences in blood and brain from two injection drug users in Miami, Florida.
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Amino Acid Sequence↗
Biomedical subjects
D M Segal
Publications and source records attributed to D M Segal.
Immunocompromised tumor-bearing mice show a selective loss of STAT5a/b expression in T and B lymphocytes.
Impaired immune responses are frequently observed in tumor-bearing hosts during progression of tumor growth, but the molecular basis of these functional defects remains unclear. To investigate tumor-induced immunosuppression, we first established that lymphocytes from mice bearing s.c. mammary adenocarcinoma (TS/A) tumors were severely impaired in their ability to generate cellular and humoral Ag-specific responses. Lymphocytes from these mice were then screened for abnormalities in the expression of signal transducing proteins known to be involved in the regulation of cellular immunity. Interestingly, purified T and B cells isolated from immunocompromised tumor-bearing mice displayed a marked decrease in the transcription activators STAT5a and -b at the protein level and to a lesser extent at the mRNA level. By contrast, no change in the expression of STAT1, -3, and -6 or of the TCR itself were detected. The correlation in the loss of T and B cell function with the selective decrease in STAT5a/b expression suggests that regulation of the STAT5 signaling pathway may provide a molecular mechanism for modulating the immune system.
Animals↗
Multiple dimeric forms of human CD69 result from differential addition of N-glycans to typical (Asn-X-Ser/Thr) and atypical (Asn-X-cys) glycosylation motifs.
CD69 is expressed on the surface of all hematopoietically derived leukocytes and is suggested to function as a multipurpose cell-surface trigger molecule important in the development and activation of many different cell types. Human CD69 contains only a single consensus sequence for N-linked oligosaccharide addition within its extracellular domain (Asn-Val-Thr), yet exists as two distinct glycoforms that are assembled together into disulfide-linked homodimers and heterodimers. The molecular basis for human CD69 heterogeneity has remained elusive. In the current report we show that human CD69 glycoforms are generated before the egress of CD69 proteins from the endoplasmic reticulum to the Golgi and are synthesized under conditions where Golgi processing is inhibited, effectively ruling out the possibility that CD69 heterogeneity results from the differential processing of a single glycosylation site in the Golgi complex. Importantly, these data demonstrate that contrary to current belief, not one but two sites for N-glycan addition exist within the human CD69 extracellular domain and identify the second, "cryptic" CD69 N-glycan attachment site as the atypical Cys-containing glycosylation motif, Asn-Ala-Cys. The results in this study provide a molecular basis for human CD69 heterogeneity and show that multiple dimeric forms of human CD69 result from the variable addition of N-glycans to atypical and typical glycosylation motifs within the CD69 extracellular domain.
Antigens, CD↗
Signaling pathways regulating CD44-dependent cytolysis in natural killer cells.
CD44 is a cytotoxic triggering molecule on activated, but not fresh natural killer (NK) cells. In the current study, metabolic pathways used in CD44-directed lysis (CD44DL) were examined using activated human NK cells as effectors. We found that CD44 expressed by activated NK cells was indistinguishable in isoform and molecular weight from CD44 on unactivated cells. However, de novo protein expression was required for the induction of CD44DL, suggesting that activated NK cells contain proteins not present in fresh NK cells that couple CD44 to the lytic machinery. Concanimycin A, a selective inhibitor of perforin-based cytolysis, totally blocked CD44DL, natural cytotoxicity, and antibody-dependent cell-mediated cytolysis (ADCC). Moreover, studies in which kinase inhibitors were added during the effector phase of lysis indicated that protein-tyrosine and ser/thr kinases were required for all three cytolytic activities and that protein kinase C played a nonessential role in lysis. By contrast, wortmannin totally inhibited CD44DL, but failed to block natural cytotoxicity and only partially blocked ADCC, suggesting that phosphatidylinositol 3-kinase (PI 3-kinase) is required at an early, receptor-specific stage of CD44DL. Finally, cytochalasin B enhanced CD44DL, but not ADCC, indicating that CD44DL is modulated by actin polymerization. Taken together, our data suggest that CD44 in NK cells interacts with proteins induced during interleukin-2 activation in a triggering pathway that induces perforin release, requires PI 3-kinase, and is modulated by the cytoskeleton.
Androstadienes↗
Up-regulation of D3 dopamine receptor mRNA in the nucleus accumbens of human cocaine fatalities.
The effects of chronic cocaine use on D3 receptor mRNA expression in the human nucleus accumbens was assessed by reverse transcription-polymerase chain reaction. D3 receptor/cyclophilin mRNA ratios in the nucleus accumbens were increased 6-fold in cocaine overdose victims as compared to age-matched and drug-free control subjects. This finding demonstrates that chronic cocaine exposure leads to adaptive increases in the expression of D3 receptor mRNA in a critical reward center in brain.
Autopsy↗
Targeted adenovirus-mediated gene delivery to T cells via CD3.
T cells are primary targets in numerous gene therapy protocols. However, the use of subgroup C adenovirus serotype 2 or 5 (Ad2 or Ad5) as a vector to transduce T cells is limited by its poor transduction efficiency for these cells. In this report we show that poor T-cell transduction results from these cells lacking both the primary Ad2-Ad5 receptor, used in attachment, and the secondary Ad receptor, which mediates entry of most adenovirus serotypes. These deficiencies were overcome by using a bispecific antibody (bsAb) with specificities for human CD3 and for a FLAG epitope genetically introduced into Ad5 (Ad.FLAG) to redirect the virus to human T cells. The anti-FLAG x anti-CD3 bsAb increased Ad.FLAG binding 30-fold, induced the efficient uptake of Ad.FLAG into the cells, and led to a 100- to 500-fold increase in the transduction of resting T cells. Moreover, fluorescence-activated cell sorter analysis showed that 25 to 90% of the T cells were transduced by the bsAb-complexed Ad.FLAG at multiplicities of infection between 20 and 100 active particles per cell. These results demonstrate that bsAbs can target Ad to non-Ad receptors on cells that are normally resistant to Ad, resulting in their efficient and specific transduction.
Adenoviruses, Human↗
Recombinant mouse Bcl-2(1-203). Two domains connected by a long protease-sensitive linker.
Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity but whose mechanism of action is poorly understood. The purpose of this paper was to obtain large amounts of soluble Bcl-2 protein for structural and functional studies. Mouse Bcl-2(1-203) (missing the COOH-terminal hydrophobic tail) was produced in bacterial inclusion bodies, solubilized in guanidine, and refolded by dialysis. The resulting protein was monomeric in nondenaturing solution and was active in protecting mouse T hybridoma cells from glucocorticoid-induced apoptosis. Refolded Bcl-2(1-203) showed no tendency to homodimerize by gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2(1-203) which was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1beta-converting enzyme family) protease activity. The protease-sensitive sites were located within a 50-residue stretch that contained most of the nonconserved and proline residues of Bcl-2(1-203). Trypsin-cleaved Bcl-2(1-203) eluted in the same position as the undigested protein on gel filtration in nondenaturing solution, indicating that the two portions of the molecule connected by the protease-sensitive region associate stably and noncovalently. The solution properties of Bcl-2(1-203) suggest that it consists of two noncovalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-xL, which has been determined by x-ray and NMR analysis.
Amino Acid Sequence↗
CD44 is a cytotoxic triggering molecule on human polymorphonuclear cells.
In this study, we present evidence that CD44 is a cytotoxic triggering molecule on freshly isolated polymorphonuclear cells (PMN). PMN constitutively express high levels of CD44 as determined by FACS analysis, and immunoprecipitation studies using PMN lysates and an anti-CD44 mAb show a band of 80 to 90 kDa that migrates slightly faster than CD44 from PBL. A bispecific Ab consisting of anti-CD44 Fab cross-linked to anti-DNP Fab (anti-CD44(Fab) x anti-DNP(Fab)) induces PMN to lyse DNP-coated tumor cells in an 18-h assay, and this lysis is specifically inhibited by a polyclonal anti-CD44 F(ab')2. A second bispecific Ab, anti-CD16(Fab) x anti-DNP(Fab), that binds to Fc(gamma)RIIIb on PMN does not induce lysis, indicating that the bridging of target cells to PMN per se is not sufficient for killing. Moreover, CD44-directed killing by PMN results in the lysis of bystander cells, suggesting that the mechanisms of tumor cytolysis by CD44-targeted PMN does not require cell-cell contact. Lastly, PMN lyse target cells coated with hyaluronic acid (HA), the principal ligand for CD44, and this cytolytic activity is specifically blocked by the polyclonal anti-CD44 F(ab')2 and by an anti-CD44 mAb. We suggest that the interaction of HA with CD44 on neutrophils might initiate cytotoxic or inflammatory responses in vivo when neutrophils encounter high amounts of HA, for example on tumor cells, or in the extracellular matrix.
Animals↗
A single-chain bispecific Fv2 molecule produced in mammalian cells redirects lysis by activated CTL.
Single-chain Fv (sFv) molecules consist of the two variable domains of an antibody (Ab) connected by a polypeptide spacer and contain the binding activities of their parental antibodies (Abs). In this paper we have attached the C-terminus of 2C11-sFv (anti-mouse CD3 epsilon-chain) to the N-terminus of OKT9-sFv (anti-human transferrin receptor [TfR]) through a 23 amino acid inter-sFv linker consisting primarily of CH1 region residues from 2C11, to form a single-chain bispecific Fv2 [bs(sFv)2] molecule. The bs(sFv)2 was expressed in COS-7 cells, and was secreted at the same rate as the two parental sFvs. The secreted protein had both anti-CD3 and anti-TfR binding activities. Essentially all of the secreted bs(sFv)2 molecules bound TfR and the binding affinity of the bs(sFv)2 was comparable to that of OKT9 sFv and Fab. Thus, the attachment of the inter-sFv linker to the N-terminus of OKT9-sFv did not impair its binding function. The bs(sFv)2 retained both binding specificities after long-term storage at 4 degrees C or overnight incubation at 37 degrees C. It redirected activated mouse CTL to specifically lyse human TfR+ target cells at low (ng/ml) concentrations and was much more active than a chemically cross-linked heteroconjugate prepared from the same parental mAbs. Because bs(sFv)2 molecules secreted by mammalian cells are homogeneous proteins containing two binding sites in a single polypeptide chain, they hold great promise as an easily obtainable, economic source of a bispecific molecule suitable for in vivo use.
Amino Acid Sequence↗
Linguistic complexity of protein sequences as compared to texts of human languages.
A notion and a measure of linguistic complexity introduced earlier (Trifonov, 1990) were originally used for analysis of nucleotide sequences. This measure was shown to reflect multiplicity of codes (messages) of different natures superimposed in the sequences. Unlike human language texts, genetic texts are 'read' by cellular mechanisms in several different ways, each time using a different selection of the characters of the same text while skipping others (Trifonov, 1989). Human texts are read in one way only, sequentially and involving all characters (one code). The conceptual significance and essence of the idea on the multiplicity of overlapping codes in genetic sequences, as opposed to human languages, is discussed. The linguistic complexity technique allows a calculation to be made of the structural complexity of any linear sequence of characters irrespective of whether the text is cognized or presently undeciphered. The texts (sequences) are compared exclusively from the point of view of their structural complexity with no reference to the meaning of the texts which is beyond the scope of this article. Results of such a comparison of protein sequences with various texts, written in English, Italian and Welsh are presented. The human texts are found to be structurally simpler than genetic (protein) texts, reflecting, apparently, a difference in the reading modes: single code versus many codes.
Amino Acid Sequence↗
Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.
A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer.
Adenoviridae↗
Retargeting of CTL by an efficiently refolded bispecific single-chain Fv dimer produced in bacteria.
A single-chain bispecific Fv dimer (bs(sFv)2) having specificity for mouse CD3 epsilon chain and human transferrin receptor was produced in bacterial inclusion bodies. To overcome difficulties associated with in vitro protein folding, we used a novel renaturation approach to obtain active bs(sFv)2. The protein was dissolved in the weak ionic detergent sodium lauroylsarcosine, and disulfides were formed by oxidation in air. After oxidation, the bs(sFv)2 exhibited very little covalent aggregation and migrated as a single species in nonreducing SDS-PAGE, suggesting that disulfides were correctly paired. The detergent was removed using an ion exchange resin and the protein fractionated by size exclusion chromatography. The recovered 65-kDa protein was monomeric in non-denaturing solvent, homogeneous by SDS-PAGE, and comprised 15 to 20% of material applied to the gel filtration column. This protein bound specifically to both mouse CD3 epsilon chain and human transferrin receptor with affinities indistinguishable from those of the parental Fabs or single-chain Fvs. The bs(sFv)2 specifically redirected mouse cytotoxic T cells to lyse target cells expressing human transferrin receptor at picomolar concentrations. Bacterially produced and detergent oxidized bs(sFv)2 molecules may therefore provide the abundant amounts of homogeneous active material required to redirect cytotoxic cells against tumors and other unwanted cells in animal models and in patients.
Amino Acid Sequence↗
Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo.
Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally synergeic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 epsilon-chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV+ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargeting T cells with bispecific antibodies against syngeneic breast cancer.
Adenocarcinoma↗
Redirection of cellular cytotoxicity. A two-step approach using recombinant single-chain Fv molecules.
In this article the authors discuss an indirect system for redirecting cellular cytotoxicity, which utilizes a "universal" bispecific antibody to redirect T-cells to kill cells targeted with single-chain Fv (sFv) fusion proteins that carry a peptide tag recognized by the bispecific antibody. This approach has a number of theoretical advantages in the immunotherapy of cancer.
Antibodies, Bispecific↗
Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies.
In vitro folding of denatured proteins has remained an inefficient and empirical process that has limited the use of bacterially expressed recombinant proteins. In this paper we show that in vitro folding of recombinant single-chain Fv (sFv) proteins is markedly facilitated when disulfide bonds are formed in detergent solution. sFv proteins from three different antibodies were expressed as bacterial cytoplasmic inclusion bodies and solubilized in the weak ionic detergent, sodium lauroylsarcosine (SLS). Upon oxidation in air in the presence of metal ion catalysts, all three sFvs quantitatively formed intrachain disulfide bonds which ran as a single band in SDS-polyacrylamide gel electrophoresis under non-reducing conditions. By contrast, oxidation from 6 M urea gave large amounts of disulfide linked aggregates, and three closely spaced bands of monomeric protein. Detergent was removed from the oxidized sFvs by addition of 6 M urea and absorption with an ion exchange resin. After dialysis and gel filtration in non-denaturing solution, moderate to high yields of monomeric sFv were obtained, depending upon the sFv. All three sFvs gave single bands on isoelectric focussing and SDS-PAGE gels and had similar or identical binding specificities and affinities as the parental Fabs, implying that the final products contained correctly paired disulfide bonds. The correct disulfide pairing suggests that the disulfide loops within the detergent-solubilized sFvs adopt a native-like structure.
Animals↗
Construction of a functional disulfide-stabilized TCR Fv indicates that antibody and TCR Fv frameworks are very similar in structure.
Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by an interchain disulfide bond engineered between structurally conserved framework positions. We now design and produce a disulfide-stabilized Fv of a T cell receptor. It is composed of V alpha and V beta variable domains of the 2B4 TCR stabilized by a disulfide bond between framework residues of the TCR Fv at a site corresponding to that used for disulfide stabilization of antibody Fvs. For ease of production and detection, the TCRdsFv was fused to a truncated form of Pseudomonas exotoxin (PE38). The TCR(dsFv) retains its native conformation and is much more stable than a TCR scFv. Moreover, it is functional in biological assays. Because successful disulfide stabilization of the TCR Fv by the positions used for antibody Fv stabilization would not occur unless the mutated residues in TCR Fv are at positions closely similar to those in antibody Fvs, most likely within less than 1.5 A, these results provide very strong experimental evidence for the structural similarity between immunoglobulin and TCR antigen-binding variable domains.
Amino Acid Sequence↗
Alternative triggering molecules and single chain bispecific antibodies.
Although T cell receptors and Fc receptors are the best known cytotoxic triggering molecules, a number of adhesion molecules recently have been identified as alternative triggering molecules. We discuss how CD44, an adhesion molecule found on all leukocytes and many other cell types, becomes a triggering molecule on NK cells following stimulation with IL-2. We also describe a genetically engineered single chain bispecific antibody, produced in mammalian cells and in bacteria, that is capable of redirecting lysis in the nanogram per milliliter range.
Animals↗
A bispecific antibody prolongs survival in mice bearing lung metastases of syngeneic mammary adenocarcinoma.
In the present study we tested whether T cells retargeted with a bispecific antibody (bsAb) could block the growth of lung metastases of syngeneic mammary adenocarcinoma in immunocompetent mice. BALB/c mice were injected i.v. with tumor and i.p. with a genetically engineered bispecific F(ab')2 [bs(Fab')2] having specificity for murine CD3 epsilon chain and for the gp52 mouse mammary tumor viral glycoprotein, which is expressed on the tumor cells. The bs(Fab')2 was physically stable in blood and serum, was removed from the body with a half-time of 12-15 h, and accumulated in lymphoid tissue where it bound to T cells. We show that treatment of tumor bearing mice with the bs(Fab')2 significantly prolonged their survival relative to untreated controls. Two other genetically engineered bs(Fab')2s having specificity for murine CD3 epsilon chain and irrelevant antigens did not inhibit tumor growth. In addition, survival was not affected by bsAb therapy using a variant tumor cell line that expressed low levels of the gp52 target antigen. Inhibition of tumor growth was even more evident by histologic analysis. Treatment with the relevant bs(Fab')2 resulted in a marked reduction of tumor burden in lung sections taken on days 7, 9 and 11. This is the first report demonstrating that a bsAb can inhibit the growth of syngeneic solid tumor metastases in mice without addition of T cell activators.
Adenocarcinoma↗