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J W Larrick

Publications and source records attributed to J W Larrick.

At least 55 records · Page 3Linked to original sources
PubMed1994

Rabbit CAP18 derived peptides inhibit gram negative and gram positive bacteria.

CAP18 (cationic antimicrobial protein of 18 kDa) was originally isolated from rabbit granulocytes using as an assay the agglutination of Re-lipopolysaccharide coated erythrocytes. The C-terminal 37 amino acids of CAP18 comprise the LPS-binding domain called RNIP, reactive nitrogen inhibitory peptide. Synthetic RNIP has broad antimicrobial activity versus both gram positive [IC50 = 130-200 nM] and gram negative bacteria [IC50 = 20-100 nM). Susceptible strains include Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes. Antimicrobial activity is highly dependent upon peptide structure. Although a 32 amino peptide resulting from truncation of five amino acids from the C terminus of RNIP is highly active, other fragments of RNIP including truncation of its N-terminus do not exhibit antimicrobial activity. Unlike previously characterized antimicrobial peptides derived from granulocyte proteins RNIP is active in serum. RNIP or a derivative peptide may have therapeutic potential for bacterial sepsis.

Amino Acid Sequence↗
PubMed1994

Endotoxin-binding synthetic peptides with endotoxin-neutralizing, antibacterial and anticoagulant activities.

Endotoxin(lipopolysaccharide = LPS), cell wall component of gram-negative bacteria, activates monocytes and macrophages to release cytokines, reactive nitrogen intermediates (RNI), and to generate tissue factor(TF) which initiate coagulation. We have purified 7kDa and 18kDa cationic antibacterial proteins (CAP-7 and CAP-18) with LPS-binding and LPS-neutralizing activities from rabbit granulocytes using as an assay the agglutination of erythrocytes coated with Re-LPS. From protein sequencing, CAP-7 was identified as the C-terminal 37 amino acid fragment of CAP-18. Synthetic peptide #197 (identical sequence to CAP-7, Gly1-Try37) and #36-1 (a truncation of CAP consisting of 32 amino acid residues, Gly1-Ala32) showed LPS-binding activity. Each peptide inhibited LPS-induced tissue factor(TF) generation by murine peritoneal macrophages, even added 1-3 hours after stimulation of cells with LPS. C57BL/6 mice treated with #197 were significantly protected from lethal LPS challenge. Peptide #36 also blocked the LPS-induced lethality. These peptides had antibacterial activity to gram-negative bacteria, such as E.coli, S.typhimurium, K.pneumonia, Ps.aeruginosa and also to gram-positive S.aureus (Methicillin sensitive and resistant strains). Both peptides inhibited TF- and Xa-induced plasma clotting. Using synthetic chromotogenic substrates, both CAP7 peptides blocked the coagulation cascade at two sites, activation of factor X to Xa and conversion of Factor II (prothrombin) to factor IIa (thrombin). In vivo treatment of peptide #197 prevented acute lethality in mice injected with tissue factor (rabbit brain thromboplastin). Two other peptides, #32(Gly1-Phe9) and #50(Ile13-Typ37) failed to demonstrate LPS-binding, LPS-neutralizing, antibacterial and anticoagulant activities. The active peptides but not the inactive peptide maintain a putative heparin binding domain at their N-termini. This heparin binding domain is participate in the LPS-binding, LPS neutralizing, antibacterial and anticoagulant activities of CAP7. These active peptides may have a therapeutic potential for treatment for DIC due to sepsis and endotoxin shock.

Amino Acid Sequence↗
PubMed1993

Antibody engineering by parsimonious mutagenesis.

The human monoclonal antibody (humAb) problem has largely been solved with the aid of the polymerase chain reaction (PCR) [Larrick et al., Bio/Technology 7 (1989a) 934-938; Larrick et al., Biochem. Biophys. Res. Commun. 160 (1989b) 1250-1256; Chiang et al., BioTechniques 7 (1989) 360-366]. Phage display has now made it possible to recover humAb with primary response level affinities (approx. 10(6) M-1) for virtually any antigen (including self antigens) from comprehensive libraries of B-cell repertoires from non-immunized humans [Marks et al., J. Mol. Biol. 222 (1991) 581-597; Marks et al., Bio/Technology 10 (1992) 779-783; Griffiths et al., EMBO J. 12 (1993) 725-734]. This means that the goal of therapeutic humAb without immunization is within reach. However, in order to achieve the affinities generally required for therapeutic use (> or = 10(9) M-1), reliable methods will be needed to complete the affinity maturation process in vitro. Available X-ray crystallographic data and energy calculations indicate that only a fraction of the substantial contact surface between the Ab and protein antigens contribute significantly to affinity. Thus, the remaining contact surface presents multiple opportunities to develop additional high-affinity contacts, needing only a means to identify them. To this end, we have developed a computer-assisted method for oligodeoxyribonucleotide-directed scanning mutagenesis, called parsimonious mutagenesis (PM), whereby all three complementarity-determining regions (CDR) of a variable region (V-region) gene can be simultaneously and thoroughly searched for improved variants in libraries of manageable size. These libraries are made with low-redundancy 'doping' codons and biased nucleotide (nt) mixtures designed to maximize the abundance of combining sites with predetermined proportions of preselected sets of alternative amino acids (aa). This allows the library to 'probe' the surface of the antigen one or a few aa residues at a time with a wide selection of aa side chains to search out and identify new high-affinity contacts. In addition to affinity maturation in vitro, PM can also be used to remove unwanted cross-reactivities and to 'reshape' rodent mAb for human therapeutic use.

Amino Acid Sequence↗
PubMed1993

Allotypic dependency of the specificity and avidity of human monoclonal IgM rheumatoid factors derived from rheumatoid synovial cells.

OBJECTIVE: To better understand the genetic derivation and pathogenicity of rheumatoid factor (RF) molecules in rheumatoid arthritis (RA), we have focused our studies on rheumatoid synovial cells (RSC). METHODS: Five monoclonal human IgM rheumatoid factor (mRF)-secreting hybridomas were produced from the RSC of an RA patient. Fine subclass specificities and avidities of these RSC mRFs were compared with several paraprotein monoclonal IgM RFs using direct binding (reactivity) and competitive inhibition (specificity and avidity) enzyme-linked immunosorbent assays. RESULTS: The following observations were made: 1) RSC mRF had greater avidity for IgG than did paraprotein mRF; 2) 4 of the 5 RSC RF were highly avid for IgG3; and, 3) the avidity of RSC RF binding for IgG3 was highest for IgG molecules expressing the G3m(5) allotype. CONCLUSION: We conclude that RSC RF have different specificities and avidities than do paraprotein RF. This may suggest an antigen-driven process in RA synovium, with the production of higher-avidity IgG3m(5)-specific RSC RF, which could have special pathogenetic importance.

Adult↗
PubMed1993

Role of protein phosphorylation in TNF-induced apoptosis: phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF-resistant U937 variants.

This study examined the role of protein phosphorylation in TNF induction of apoptosis in several tumor cell lines by testing the effects of agents that either stimulate or inhibit protein phosphorylation. The serine-threonine phosphatase inhibitors, okadaic acid (OKA) and calyculin A (CLA), synergistically augmented TNF-induced apoptosis in several TNF-sensitive tumor cell lines including the U937 histiocytic lymphoma, the BT-20 mammary carcinoma, and the LNCap prostatic tumor cell line. Furthermore, the phosphatase inhibitors completely reversed the TNF resistance of a variant (U9-TR) derived from U937. CLA also inhibited phosphatase activity in cell-free extracts from both U937 and U9-TR at the same concentrations (0.4-2.0 nM) that it synergized with TNF. In contrast, TNF treatment of U937 cells did not result in inhibition of phosphatase activity mediated by protein phosphatase 1 (PP1) and PP2A in cell extracts. Since the phosphatase inhibitors are known to increase the overall levels of protein phosphorylation in cells, this suggested that TNF may act by stimulating protein kinase (PK) activity. This hypothesis was supported by the results of testing a panel of relatively specific protein kinase inhibitors. TNF activation of DNA fragmentation was blocked by a potent inhibitor of myosin light chain kinase (MLCK) but was unaffected by inhibitors of cAMP or cGMP-dependent PKs. We postulate that a defect in the activation of MLCK or possibly some other as yet unknown PK may be responsible for the TNF resistance of U9-TR. Furthermore, this resistance may be circumvented by promoting protein phosphorylation with the serine-threonine-dependent phosphatase inhibitors.

Alkaloids↗
PubMed1993

Regulation of neutrophil interleukin 8 gene expression and protein secretion by LPS, TNF-alpha, and IL-1 beta.

Neutrophils are possibly involved in the pathogenesis of various lung diseases through the release of numerous mediators. In the present study, we studied the regulation of IL-8 gene induction and protein secretion in human blood neutrophils. Northern blot analysis revealed that LPS increased IL-8 mRNA levels in neutrophils, with a maximal fivefold increase by 2 h. IL-8 mRNa levels returned to baseline values within 12 h. In contrast, LPS-stimulated monocytes demonstrated a sustained increase of IL-8 mRNA levels for more than 24 h. TNF-alpha, IL-1 beta, and phorbol myristate acetate also increased IL-8 mRNA levels in neutrophils. Immunohistochemical analysis confirmed that IL-8 was localized within stimulated neutrophils. IL-8 secretion by neutrophils and monocytes was quantified using a specific ELISA for IL-8. Resting neutrophils secreted minimal IL-8 activity. However when cells were stimulated with LPS, TNF-alpha, or IL-1B, neutrophils secreted IL-8. IL-8 secretion was most marked during the first 2 h after stimulation and decreased thereafter. In contrast, monocytes maintained a high rate of IL-8 secretion over 12 h. Although a single monocyte secreted 70-fold more IL-8 than did a single neutrophil after 4 h of incubation, the high abundance of neutrophils in peripheral blood made the neutrophil-secreted IL-8 more significant. During the first 2 h, neutrophils secreted approximately 40% of the IL-8 released by monocytes in the same volume of blood. This ratio decreased to 9% after 12 h. Neutrophil-secreted IL-8 may play an autocrine or paracrine role during the initial stage of inflammation.

Cells, Cultured↗
PubMed1993

Antimicrobial activity of rabbit CAP18-derived peptides.

A cationic antimicrobial protein of 18 kDa (CAP18) was originally isolated from rabbit granulocytes by using as an assay the agglutination of Re-lipopolysaccharide-coated erythrocytes. The C-terminal 37 amino acids of CAP18 (CAP18(106-142)) make up the lipopolysaccharide-binding domain. Synthetic CAP18(106-142) has broad antimicrobial activity against both gram-positive (50% inhibitory concentration, 130 to 200 nM) and gram-negative (50% inhibitory concentration, 20 to 100 nM) bacteria. Susceptible strains include Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium. Antimicrobial activity is highly dependent on peptide structure. Although a 32-amino-acid peptide resulting from the truncation of 5 amino acids from the C terminus of CAP18(106-142) is highly active, other fragments of CAP18(106-142), including CAP18(106-142) with a truncated N terminus, do not exhibit antimicrobial activity. Unlike previously characterized antimicrobial peptides derived from granulocyte proteins, CAP18(106-142) is active in serum. CAP18(106-142) or a derivative peptide may have therapeutic potential for bacterial sepsis.

Agglutination Tests↗
PubMed1993

PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR.

A rapid and reliable method is described for preparing competitive DNA fragments for quantitative PCR. Synthetic DNAs complementary to previously established PCR primers are ligated together with the primers to both ends of a generic DNA fragment whose length differs from the natural target gene PCR product. After a short ligation step, the properly constructed ligation products (i.e., those that have the correct primer templates on opposite sides of the generic DNA fragment) are preferentially amplified by PCR. The generation of competitive PCR fragments, MIMICS, can be completed in a single day. To perform quantitative PCR, known quantities of PCR MIMICS are spiked into PCR amplification reactions containing the experimental cDNA samples. A visual or radioactive comparison of the PCR products can then be used to determine the initial quantity of target gene. We show that competitive PCR MIMICS can be used to accurately measure small changes in mRNA levels.

Base Sequence↗
PubMed1993

Nicotine inhibition of apoptosis suggests a role in tumor promotion.

Recent evidence indicates that cell death through apoptosis may be an important mechanism to prevent tumor development. Therefore, agents that inhibit apoptosis may function as tumor promoters. The purpose of this study was to determine the effects of nicotine on the process of apoptosis. The results demonstrate that nicotine inhibits apoptosis induced by diverse stimuli including tumor necrosis factor (TNF), UV light, chemotherapeutic drugs, and calcium ionophore. This phenomenon was observed in normal and transformed cells derived from a variety of species and tissues, including tumor cell types related to tobacco use. The major nicotine metabolite, cotinine, also inhibited apoptosis, whereas N-nitrosodiethylamine, a carcinogen found in tobacco, was without effect. Therefore, nicotine-mediated inhibition of apoptosis may contribute to the pathogenesis of tobacco-related cancer as well as decrease the efficacy of cancer therapies.

Animals↗
PubMed1992

Competitive PCR.

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Amino Acid Oxidoreductases↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗