Competitive PCR.
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Amino Acid Oxidoreductases↗
Biomedical subjects
J W Larrick
Publications and source records attributed to J W Larrick.
Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells.
The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.
Benzamides↗
Message amplification phenotyping (MAPPing)--principles, practice and potential.
Message amplification phenotyping (MAPPing) is a sensitive polymerase chain reaction (PCR)-based technique for the rapid determination of the mRNA phenotype of small numbers of cells. Isolated mRNA is reverse-transcribed (RT) into DNA, and then the DNA fragments corresponding to proteins or genes of interest are specifically amplified by PCR. The technique, also called RT-PCR, has wide application in biology and medicine, and comparison with bioassays demonstrates that MAPPing saves significant time and material. The technique should be applicable to analyse mRNAs of virtually any tissue or cell type.
Animals↗
Native cytokine antagonists.
Cytokines orchestrate the complex homeostasis of cells and tissues by acting in both an autocrine and paracrine fashion. The processes responsible for regulation of cytokines is not well understood. This chapter has summarized what is known about antagonism and inhibition of the action of cytokines. Several concepts have emerged from work in this area. At least two cytokines (IL-1 alpha and IL-1 beta) have an endogenous receptor antagonist, the IL-1 receptor antagonist. This is the first example of one endogenous molecule directly blocking the binding of another molecule to its receptor: most forms of regulation occur through independent receptors. Several cytokines, including TNF, IFN-gamma, IL-2 and IL-4, are inhibited by soluble receptors. Several cytokines, including IL-10, TGF-beta and MDF, act to inhibit other cytokines. It is likely that these inhibitors will be found to have pleiotropic actions in vivo. Finally, we describe antibody inhibition of cytokines. Detailed studies will be required to understand the complex interplay of the aforementioned cytokine inhibitors and the processes they regulate.
Animals↗
Therapeutic human antibodies derived from PCR amplification of B-cell variable regions.
Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the development of immunodeficient mice (McCune et al. 1988) for the reconstitution of the human immune system ex vivo, immortalization of antigen-specific human B cells remains the limiting step in the generation of human monoclonal antibodies. Typically this is performed with the aid of Epstein-Barr virus transformation followed by subcloning, confirmation of antigen binding and hybridization of the B lymphoblasts to a suitable fusion partner such as GLI-H7. This general approach is effective and widely used; however, it is time-consuming with erratic results. These were the immediate reasons we and others devised methods to directly obtain the variable regions from small numbers of human B cells (Larrick et al. 1987). The success of the PCR-based approach is illustrated above. In the present studies we successfully captured and stably produced antibodies from the V regions of two potent human anti-tetanus antibodies secreted by heteromyelomas that were too unstable for scale-up production. Although further preclinical evaluation of these antibodies is in progress, results to date indicate that the recombinant antibodies produced in myeloma-based cell lines or CHO cells are equivalent in binding specificity and activity to the native heteromyeloma-derived antibodies. Recent studies from this laboratory indicate that effective anti-tetanus protection will require a cocktail of anti-tetanus antibodies. Details of this work will be the subject of a future communication (Lang et al., in preparation).
Amino Acid Sequence↗
Modulation of viral infections by cytokines.
The survival of a host challenged by viral infection depends on many factors. Some are specific, such as antiviral T-cell and B-cell responses. Others are nonspecific, such as intrinsic cellular resistance, macrophage activation, and activation of humoral protective mechanisms (e.g., complement, coagulation, etc.). Cytokines are important mediators and regulators of both types of host response. Furthermore, orchestration of specific humoral and cellular immune responses requires the participation of many cytokines. Details of the complex and overlapping roles that cytokines play in specific immune responses is beyond the scope of this review. Instead, this review will focus on the nonspecific antiviral effects of cytokines.
Animals↗
Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes.
Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.
Amino Acid Sequence↗
Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein.
CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).
Amino Acid Sequence↗
Human monoclonal antibodies specific for capsular polysaccharides of Klebsiella recognize clusters of multiple serotypes.
We report the generation and the characterization of a set of human monoclonal antibodies (HmAb) specific for Gram-negative bacteria of Klebsiella pneumoniae. The eight human hybridomas secrete either IgM kappa, IgA1 kappa, or IgA2 kappa antibodies. One HmAb binds bacteria of only one serotype. Five HmAb recognize non-overlapping clusters of 2, 3, or 10 different serotypes. The remaining two HmAb both recognize three serotypes. Two serotypes are recognized by both HmAb, and in addition both HmAb bind one more nonidentical serotype. These results suggest that in man, epitopes are immunodominant, different from serotype-specific determinants detected by conventional rabbit antisera. Screening of clinical isolates revealed that the HmAb recognize not only representative typing strains but also most isolates of the corresponding serotype. In addition, most of the isolates that were non-typable by polyclonal antisera were recognized by one of the HmAb. Fine specificity analyses revealed that all HmAb are highly specific for the isolated capsular polysaccharides (CPS) of bacteria within the corresponding cluster of serotypes. However, the avidity of a HmAb for the different CPS can differ significantly. Taken together, our results suggest that the unequivocal interactions between HmAb and CPS offer the basis for an alternative, better defined classification system, and that passive immunization with a limited number of HmAb may provide a feasible strategy for the protection against the majority of fatal, nosocomial infections with multidrug-resistant strains of K. pneumoniae.
Antibodies, Bacterial↗
Human neutrophil granule cationic protein CAP37 is a specific macrophage chemotaxin that shares homology with inflammatory proteinases.
Cationic antimicrobial protein CAP37 (Mr = 37 kD) is derived from the azurophilic granules of human PMN. In vitro and in vivo studies demonstrate that CAP37 is a novel monocyte-specific chemoattractant. The N-terminal amino acid sequence of CAP37 shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, substitutions in the catalytic triad (serine for a histidine at position 41 and glycine for a serine at position 175), may account for its lack of serine protease activity. A full length cDNA for CAP37 was identified in an HL60 cDNA library screened with oligonucleotide probes designed from the N-terminal amino acid sequence. Sequencing of the cDNA reveals a protein of 225 amino acids with significant nucleotide homology to cathepsin G and human neutrophil elastase.
Amino Acid Sequence↗
Tumor necrosis factor induces activation of mitochondrial succinate dehydrogenase.
We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed. In contrast, TNF does not activate SDH in TNF resistant variants derived from U937 and WEHI-164.
Animals↗
The methyl xanthine hypothesis: does tea consumption by Tibetan natives blunt the effects of high altitude?
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Altitude↗
Recombinant antibodies.
Many fundamental advances in our understanding of the structure and function of eukaryotic genes were derived from the study of antibody genes. Examples include mRNA splicing and rearrangement to generate antibody diversity. The capacity to immortalize an individual B cell using cell fusion permitted the generation of monoclonal antibodies. Monoclonal antibodies have had wide application in many fields of the life sciences and beyond. Recent advances permitting manipulation of antibody genes using recombinant DNA techniques offer many advantages over conventional somatic cell hybridization techniques. Rodent monoclonals can be "humanized" and antibody isotype readily changed. Grafting of the complementarity determining regions from rodent to human framework regions demonstrated the importance of these hypervariable portions of the immunoglobulin to the integrity of the antibody combining site. Recombinant monoclonal antibodies (rMAb) or fragments thereof have been successfully produced in both prokaryotic and eukaryotic hosts at levels equal to those produced by hybridomas. Successful efforts to express rMAbs in plants and other large capacity systems suggest that rMAbs can be produced inexpensively. Use of antibody catalysis and antibodies mimicking various receptors or ligands have numerous applications. Technology developed to immortalize the heavy and light chain repertoire permits the generation in vitro of recombinatorial libraries of antibodies. The capacity to artificially generate high-affinity antibodies in vitro using the methods of recombinant DNA technology has enormous pharmaceutical and industrial potential.
Animals↗
In vitro immunization of human B lymphocytes. MAPPing of lymphokine specific mRNA and the effect of recombinant factors.
The kinetics of lymphokine-specific DNA transcription during in vitro immunization of human peripheral blood lymphocytes and splenocytes were studied using the polymerase chain reaction. The levels of specific mRNA were shown to be down regulated by cytolytic L-leucyl-leucine methyl ester-sensitive lymphocytes. In in vitro immunizations using L-leucyl-leucine methyl ester-treated human PBL or splenocytes, the lymphokine mRNA expression pattern indicated an active gene transcription during the entire stimulation period, especially for the IL-2 and IL-5 genes. Transcription of IL-6 and TNF beta started on day 4, whereas IFN gamma mRNA reached its maximum level on day 4. In vitro immunizations of cells not treated with L-leucyl-leucine methyl ester revealed a transient transcription of lymphokine DNA that was declining already after day 2. Exogenously added recombinant IL-2, IL-4, and IL-6 all exhibited a positive immunoregulatory effect on Ig secretion, whereas IL-5 was not found to have any effect on immunoglobulin secretion during the in vitro culture. These results present the first information useful for designing in vitro immunization systems based on recombinant lymphokines and antisense DNA for gene regulation.
B-Lymphocytes↗
Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR.
To facilitate the rapid cloning and sequencing of rearranged murine heavy-chain variable regions, we have designed a set of universal primers using conserved sequences of leader (signal peptide), framework one and constant regions of the immunoglobulin heavy-chain genes. RNA was extracted from the mouse hybridoma cells secreting monoclonal antibodies: IOR-T3 (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), CB-Fib.1 (anti-human fibrin) and CB-Hep.2 (anti-hepatitis B surface antigen). First-strand cDNA was synthesized and amplified using PCR. The primers successfully amplified correct size fragments from cDNA prepared from all hybridomas. These methods will facilitate the cloning and sequencing of mouse immunoglobulin variable regions.
Animals↗
Natural killer lines and clones with apparent antigen specificity.
Fresh CD3-, CD16+ lymphocytes that adhered to selected allogeneic lymphoblastoid cell lines (LCL) were cultured with LCL in the presence of IL-2-containing medium. The resulting lines as well as clones derived from these lines expressed CD16 and/or CD56, but lacked detectable CD3 or TCR-alpha/beta or TCR-gamma/delta complexes on the cell surface. Northern blot analysis failed to detect CD3 epsilon or TCR-beta transcripts, but revealed the presence of a TCR-gamma chain transcript in one of these lines. In addition to displaying potent cytolytic activity against K562 erythroleukemia cells (a classical NK target), the vast majority of these lines and clones lysed their specific stimulator LCL to a significantly greater extent than irrelevant LCL. This selective killing was inhibited by the addition of cold stimulator LCL or K562 cells, or anti-LFA 1 mAbs, but not by irrelevant LCL or mAbs to CD3, class I or class II MHC antigens. These results indicate that some CD3- lymphocytes, phenotypically indistinguishable from NK cells, can recognize and lyse allogeneic targets in a specific manner.
Antibodies, Monoclonal↗
Serologic and molecular characterization of a human monoclonal rheumatoid factor derived from rheumatoid synovial cells.
Molecular characterization of rheumatoid factors (RF) in rheumatoid arthritis (RA) has been hampered because of their polyclonality. To overcome this problem, we generated monoclonal RF-secreting hybridomas from rheumatoid synovial cells. Among the RF-secreting hybridomas, HAF10 secreted an IgM-RF that was monospecific for human IgG. It bound well to IgG1 and IgG2, but not to IgG3 and IgG4. Sequence analysis of its heavy and light chains showed that it contained a VH1 heavy chain and a V lambda light chain that did not belong to any known lambda light chain subgroup, and therefore, probably represented a new lambda subgroup. These results indicated that both the heavy and light chains of a monoclonal IgM-RF from rheumatoid synovial cells were quite different from the reported variable region sequences of several monoclonal RF derived mainly from patients with mixed cryoglobulinemia. Further studies of additional monoclonal RF from RA patients are warranted to define precisely their genetic basis and to further our understanding of the immunopathology of RA.
Amino Acid Sequence↗
CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases.
We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity.
Amino Acid Sequence↗