Construction of cDNA libraries from small numbers of cells using sequence independent primers.
Explore the source record for details and available documents.
Cell Line↗
Biomedical subjects
J W Larrick
Publications and source records attributed to J W Larrick.
Activated Langerhans cells release tumor necrosis factor.
Langerhans cells act as antigen-presenting cells in immune reactions in the skin. What other roles they may play in inflammation is less well defined. We have tested whether these cells can produce TNF-alpha, an important mediator of inflammation. Resting Langerhans cells produce less than 0.1 U TNF-alpha/ml. Langerhans cells stimulated with phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) release 4-5 U TNF-alpha/ml. Specificity of the released TNF-alpha in an L929 cytotoxicity assay was confirmed by using neutralizing anti-TNF-alpha monoclonal antibodies, and the identity of TNF-alpha was further confirmed by Northern blot hybridization with an TNF-alpha oligomer DNA probe. Activated Langerhans cells may contribute to inflammation in the skin by releasing TNF-alpha, which is known to effect fibroblast growth, endothelial cell activation, and lymphocyte function.
Humans↗
Therapeutic applications of oncogenes.
Explore the source record for details and available documents.
Genetic Markers↗
Molecular biology: concepts and techniques.
Explore the source record for details and available documents.
Animals↗
Native interleukin 1 inhibitors.
Much information has accumulated on the isolation and characterization of a heterogeneous group of molecules that inhibit one or more of the bioactivities of interleukin 1. In this review James Larrick discusses the biological and biochemical data available on several of these native interleukin 1 inhibitors, some of which have additional, unexpected activities.
Animals↗
Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta.
Cultured TA1 adipocytes treated with tumor necrosis factor alpha (TNF) lose intracytoplasmic lipid and, over a period of days, come to resemble their predifferentiated progenitors (preadipocytes). To examine the extent to which this phenotypic reversion represents a return to a less differentiated cell, we examined three major characteristics that distinguish preadipocytes from adipocytes: (a) pattern of gene expression; (b) hormonal requirement for accelerated adipogenesis; and (c) pattern of protein synthesis. We found that within hours of TNF addition to adipocytes, mRNAs for genes whose expression is augmented during adipogenesis decreased to predifferentiated levels; in addition, like preadipocytes, TNF-treated adipocytes required exposure to hormones to accelerate adipogenesis. Further, the pattern of protein synthesis seen on polyacrylamide gels reverted to that seen before differentiation. Transforming growth factor-beta (TGF-beta) also caused a rapid decrease in expression of adipose genes when added to fully differentiated cells, an effect that was achieved by treatment with either TGF-beta 1 or TGF-beta 2. These effects were seen in the absence of a demonstrable proliferative response to either TNF or TGF-beta. Thus characteristics that define the "terminally" differentiated state in adipocytes are subject to modulation by environmental influences.
Adipose Tissue↗
Isolation and characterization of a human monoclonal antibody that recognizes epitopes shared by Pseudomonas aeruginosa immunotype 1, 3, 4, and 6 lipopolysaccharides.
A hybridoma line secreting a human monoclonal antibody (HMAb) capable of recognizing Fisher immunotype (IT) 1, 3, 4, and 6 lipopolysaccharide (LPS) in vitro was isolated. Peripheral blood lymphocytes (PBL) were obtained from volunteers immunized with an experimental Pseudomonas aeruginosa O polysaccharide-toxin A vaccine. PBL-expressing surface antibodies able to bind to P. aeruginosa LPS were isolated by adsorption onto LPS-coated plastic wells. Such PBL were transformed with Epstein-Barr virus. Lymphoblastoid cell lines secreting anti-P. aeruginosa LPS antibodies were identified by an enzyme-linked immunosorbent assay and fused to the F3B6 heteromyeloma line. A hybridoma line producing a HMAb (2-8AH79) able to bind IT-1, IT-3, IT-4, and IT-6 LPS was identified by an enzyme-linked immunosorbent assay. This HMAb was found to bind to the O-polysaccharide regions of IT-1, IT-3, IT-4, and IT-6 LPS, as determined by immunoblotting. By using an immunofluorescence microscopy assay, the cell surfaces of IT-3 and IT-4 bacteria were strongly stained by HMAb 2-8AH79, whereas those of IT-1 and IT-6 bacteria were weakly stained. This HMAb was found to promote the uptake and killing of IT-3 and IT-4 bacteria, but not IT-1 or IT-6 organisms, by human polymorphonuclear leukocytes. Similarly, the passive transfer of HMAb 2-8AH79 to mice afforded significant protection only against a challenge with IT-3 and IT-4 bacteria.
Animals↗
Pentoxifylline prevents tumor necrosis factor-induced lung injury.
Human tumor necrosis factor-alpha (TNF) is a monokine produced by mononuclear cells after many stimuli, including bacterial endotoxin. Full exploration of its antineoplastic effects has been limited by side effects. We have previously shown that the administration of TNF to guinea pigs is associated with a syndrome similar to gram-negative septic shock, which includes capillary permeability lung injury. In this study, we measured the effects of pentoxifylline (PTX) on parameters of TNF-induced lung injury including: lung wet-to-dry weight ratio, the ratio of lung-to-plasma 125I-labeled albumin (albumin index), bronchoalveolar lavage (BAL) and peripheral leukocyte counts, and serial measurements of mean arterial pressure (MAP). Four groups of animals were studied: a TNF group received 3.75 x 10(6) U/kg TNF; a PTX group received a 20-mg/kg bolus of PTX followed by an infusion of 6 mg/kg/h; the PTX-TNF group received both; and the final group was a saline control. ANOVA analysis revealed significant elevations of lung wet-to-dry ratio only in the TNF group (5.9 [5.6 to 6.3], p less than 0.001), expressed as the mean followed by 95% confidence intervals). Lung albumin index was elevated only in the TNF group (0.24 [0.19 to 0.29], p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of anti-C5a antibodies on human polymorphonuclear leukocyte function: chemotaxis, chemiluminescence, and lysosomal enzyme release.
Previous investigation has demonstrated that in vivo complement activation can produce acute lung injury. Complement component C5a has been implicated as a key factor in this damage. In addition, C5a is thought to play a central role in mediating polymorphonuclear leukocyte (PMN) function. Studies suggest that administering antibodies to C5a might play a role in attenuating lung injury in animal models of sepsis. To evaluate further the effects of anti-C5a antibodies, we compared the effects of anti-human C5a des-Arg monoclonal (MAb) and polyclonal (PAb) antibodies on PMN functions including chemotaxis, chemiluminescence, and lysosomal release. PMN chemotaxis was assayed in Boyden chambers using 0.5% zymosan-activated serum (ZAS) as a source of C5a and 0.5% normal human serum (NHS) as a control. PMN chemiluminescence was measured by scintillation counting using ZAS as a stimulant and NHS as control. In addition, the lysosomal marker enzyme beta-D-glucuronidase was spectrophotometrically determined to assess lysosomal release. The PMN chemotactic response to ZAS was completely abolished with MAb and PAb anti-C5a antibodies (p less than 0.01). Control antibodies had no effect on ZAS-stimulated chemotaxis. The anti-C5a MAb markedly inhibited PMN chemotaxis at concentrations ranging from 20 to 0.2 microgram/ml, and was approximately 30 times more potent than the PAb. ZAS-stimulated PMN chemiluminescence was markedly decreased in response to monoclonal antibodies to C5a. In contrast, the control antibody did not inhibit ZAS-stimulated PMN chemiluminescence. Anti-C5a antibodies also significantly attenuated the release of the lysosomal enzyme beta-D-glucuronidase from ZAS-stimulated PMN. Anti-C5a antibody treatment did not cause a significant lytic effect when incubated with PMN, as demonstrated by the absence of the cytoplasmic marker lactate dehydrogenase in the supernatant. These studies suggest that in states of complement activation, MAbs and PAbs may decrease PMN functions including chemotaxis, chemiluminescence, and lysosomal enzyme release.
Antibodies↗
Direct cDNA cloning of the rearranged immunoglobulin variable region.
A major problem in the study of multigene families is the effort required to clone and sequence these genes. We describe a method to rapidly clone and sequence immunoglobulin variable region gene sequences without constructing cDNA libraries. Because immunoglobulin variable-region genes are flanked by conserved sequences, we have been able to apply the polymerase chain reaction (PCR) to clone and sequence both the light- and heavy-chain rearranged immunoglobulin genes from small numbers of hybridoma cells. This method will greatly facilitate the construction of chimeric mouse/human monoclonal antibodies for immunoglobulin structural studies as well as for therapeutic use.
Animals↗
Mechanisms that regulate the production and effects of tumor necrosis factor-alpha.
Macrophage-derived tumor necrosis factor (TNF) is increasingly being recognized as an important monokine possessing multifunctional activities. Current evidence has demonstrated that TNF can induce a number of pleomorphic effects in both physiological and immunological systems. Historically, the biological effects and nomenclature of TNF centered around the induction of hemorrhagic necrosis of specific solid murine tumors. This effected function has been greatly expanded upon, and TNF is now recognized as an important peptide mediator involved in various facets of cell activation. This is exemplified by the central role that TNF plays in endotoxemia, shock and multiple organ failure syndromes. Although TNF has been incriminated as the molecular signal mediating number of pathophysiological derangements, the regulatory mechanisms that control TNF expression at the cellular and molecular levels, as well as the modulation of the in vivo activity of preformed TNF, has not been full addressed. In this review, a detailed description of the mechanisms that regulate the production and effects of TNF is presented.
Animals↗
Potential of monoclonal antibodies as pharmacological agents.
Explore the source record for details and available documents.
Animals↗
Probing the normal and autoimmune B cell repertoire with Epstein-Barr virus. Frequency of B cells producing monoreactive high affinity autoantibodies in patients with Hashimoto's disease and systemic lupus erythematosus.
The frequency of cell precursors producing Ig of different classes and Ag-binding activities were determined, using EBV-infection and limiting dilution assays, in healthy subjects and patients with autoimmune disease. A large proportion of circulating B cells from healthy subjects were committed to the production of IgM antibodies that were polyreactive and bound a variety of self- and exogenous Ag, i.e., IgG Fc fragment, ssDNA, thyroglobulin, thyroid microsomal Ag, insulin, and tetanus toxoid. Similar frequencies of these polyreactive antibody-producing cells were found in patients with Hashimoto's disease and SLE. In contrast, significantly higher frequencies of cell precursors producing monoreactive IgG autoantibodies to thyroid Ag (thyroglobulin and thyroid microsomal Ag) and ssDNA were found in Hashimoto's disease and SLE patients, respectively. Calculation of the Kd revealed that monoclonal polyreactive antibodies were in general low affinity (Kd, 10(-3) to 10(-7) mol/liter), whereas monoclonal monoreactive autoantibodies were high affinity (Kd, 10(-9) to 10(-11) mol/liter). The detected frequency and high affinity of the monoreactive autoantibodies in Hashimoto's disease and SLE patients were comparable to those of anti-tetanus toxoid and anti-insulin IgG mAb produced by B cell clones from vaccinated healthy subjects and insulin-treated patients with insulin-dependent diabetes mellitus, respectively. These findings support the hypothesis that the autoimmune B cell repertoire in patients with organ-specific and systemic autoimmunity is shaped by Ag-driven responses rather than merely reflecting a polyclonal B cell activation.
Antibodies, Monoclonal↗
Incorporation of LPS in liposomes diminishes its ability to induce tumoricidal activity and tumor necrosis factor secretion in murine macrophages.
We investigated the effect of lipopolysaccharide (LPS) incorporated into phospholipid vesicles (liposomes) on the induction of macrophage-mediated tumor cytotoxicity and tumor necrosis factor (TNF) secretion. The incorporation of Salmonella minnesota rough (Re)-LPS into multilamellar or small unilamellar vesicles (liposomes) resulted in an 100- to 1,000-fold reduction in its potency to activate both the macrophage cell line RAW 264.7 and murine thioglycolate elicited peritoneal macrophages to become cytotoxic for L929 and P815 tumor cells. Liposomal LPS was also a 100- to 1,000-fold less potent inducer of TNF secretion from RAW 264.7 cells. Cytokines secreted by the activated macrophages contributed to the cytotoxic effect on the L929 cells but not the P815 cell line. Human recombinant TNF was not cytotoxic for either cell line but was cytostatic for the L929 cell line. Morphological examination of the cells after uptake of fluorescent, free, and liposomal LPS revealed that both forms were internalized by the endocytic pathway. This, together with the considerably reduced potency of liposomal LPS to induce tumor cytotoxicity and TNF secretion, suggests that the interaction of the hydrophobic part of the lipid A moiety of LPS with the macrophage plasma membrane is needed to optimally activate these cells. Incorporation of LPS into liposomes effectively abrogates this interaction.
Animals↗
The role of tumor necrosis factor and interleukin 1 in the immunoinflammatory response.
Monocytes and tissue macrophages produce at least two groups of protein mediators of inflammation, interleukin 1 (IL-1) and tumor necrosis factor (TNF). Recent studies have emphasized that TNF and IL-1 modulate the inflammatory function of endothelial cells, leukocytes, and fibroblasts. Although these cytokines share a number of biologic properties, they have quite distinct gene and protein structures. It is our purpose to focus on the role of these mediators in inflammation.
Amino Acid Sequence↗
Tumor necrosis factor causes increased pulmonary permeability and edema. Comparison to septic acute lung injury.
Tumor necrosis factor alpha (TNF), a monokine produced by mononuclear cells in response to bacterial endotoxin (LPS), creates a syndrome similar to septic shock in animal models. To study whether TNF could induce acute lung injury similar to that seen in gram-negative sepsis, we injected recombinant human TNF (rHuTNF alpha) into guinea pigs and monitored arterial blood gases, leukocyte counts, and left atrial (Pla), pulmonary artery (Ppa), and mean arterial pressures (MAP) serially for 8 h. Pulmonary histopathology was assessed microscopically, and cell counts and 125I-labeled albumin (125I-albumin) in bronchoalveolar lavage (BAL) fluid and lung wet/dry weight ratios were determined. Five groups of animals were studied; the 2 TNF groups received high (1.4 X 10(6) U/kg) or low (1.0 X 10(6) U/kg) doses of rHuTNF alpha, the sepsis group received 2 X 10(9) Escherichia coli/kg intravenously, and the control group received saline. An LPS control group receiving 40 ng/kg E. coli LPS was also included because the rHuTNF alpha contained a small amount of LPS as a contaminant. Pulmonary permeability was assessed by studying the Pla and the BAL fluid/plasma 125I-albumin ratio (permeability index). The permeability index was significantly increased in the high-dose TNF (0.0408 +/- 0.0041, p less than 0.05) and sepsis groups (0.0466 +/- 0.0068, p less than 0.01) relative to controls (0.0215 +/- 0.0028). The wet/dry lung weight ratios were also significantly increased in the high-dose TNF (6.07 +/- 0.29, p less than 0.05) and sepsis groups (6.22 +/- 0.30, p less than 0.05) relative to the control group (5.18 +/- 0.20).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Granulocyte depletion prevents tumor necrosis factor-mediated acute lung injury in guinea pigs.
To examine the role of polymorphonuclear neutrophils (PMN) and other granulocytes in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we compared the permeability edema and pulmonary histopathology in normal (granulocyte sufficient) guinea pigs and in granulocytopenic guinea pigs treated with TNF. Circulating granulocytes were depleted with cyclophosphamide. Two groups of normal animals were treated with either saline (PMN+/Control) or 1.4 x 10(6) U/kg recombinant human TNF (PMN+/TNF). Three granulocytopenic groups were treated with either saline (PMN-/Control), TNF (PMN-/TNF), or intravenous infusion of 2 x 10(9) E. coli strain J96 (PMN-/Sepsis). We measured the amount of 125I-labeled albumin in bronchoalveolar lavage (BAL) fluid and whole lung tissue and the wet/dry lung weight ratio to assess pulmonary transvascular protein flux and edema. We also quantified PMN in BAL fluid and fixed lung tissue. There were no statistically significant differences in any of these parameters between the PMN+/Control, PMN-/Control, or PMN-/TNF groups, except that the PMN+/Control predictably had more PMN/alveolus than the PMN- groups. However, both the PMN+/TNF and the PMN-/Sepsis groups had increased amounts of 125I-labeled albumin in BAL fluid and lung tissue (p less than 0.01) and increased wet/dry lung weight ratios (p less than 0.05), compared to all other groups. Histopathologically, capillary congestion and moderate inflammation were seen in the PMN+/TNF group, and acute inflammation and gross alveolar hemorrhage were seen in the PMN-/Sepsis group.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
Generation of a protective human monoclonal for the treatment of gram-negative sepsis.
Using a combination of Epstein-Barr virus transformation and cell fusion to a mouse/human heteromyeloma cell line, we have generated a human monoclonal antibody, D234, that recognizes cross-reactive determinants on the lipopoly-saccharides (LPS) of Gram-negative bacteria. Direct binding to a series of rough mutant LPSs and smooth LPSs demonstrates the broad cross-reactivity of this monoclonal. D234 inhibits Re LPS-induced chemiluminescence. In a murine model of Gram-negative sepsis, D234 given after infection significantly increases survival. This antibody may have therapeutic potential for the treatment of life-threatening Gram-negative infections in humans.
Animals↗