When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulation Factor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an acceleration of fibrinolysis which was proportional to the degree of the release of alpha 2PI. These results suggest the possibility that alpha 2PI could be released from fibrin in vivo by disrupting the equilibrium of the alpha 2PI-fibrin cross-linking reaction, and that the release would result in accelerated thrombolysis.
J Mimuro, S Kimura, N Aoki
Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B beta-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at approximately 10(-8) M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionyl-leucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10(-7) M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and beta-glucuronidase), or the production of superoxide anion. These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover. The capacity of hFpB to cause PMN chemotaxis without causing concurrent release of lysosomal enzymes or the production of superoxide anion is further evidence for the complexity of PMN responses to chemotactic agents.
R M Senior, W F Skogen, G L Griffin, G D Wilner
Epidermal cell-derived thymocyte activating factor (ETAF), a cytokine produced by keratinocytes, has previously been shown to be biochemically and functionally very similar, if not identical, to interleukin 1 (IL-1). Both ETAF and IL-1 have been demonstrated to be chemotactic for neutrophils and mononuclear cells in vitro. In order to demonstrate that this activity has physiological relevance we have used a simple in vivo model. The present study demonstrates that injection of high-titer ETAF or purified recombinant murine IL-1 into the mouse footpad results in an influx of neutrophils into the site with peak accumulation at 4 h. Footpad swelling also occurs with a time course roughly paralleling that of the neutrophil accumulation. Injection of control proteins failed to reproduce this phenomenon. Margination of neutrophils within blood vessels was seen within 1 h of injection of ETAF or IL-1, followed by entry into the stroma by 4 h. This suggests that chemotactic activity and not merely increased adherence or inhibition of migration is occurring. 5-10 d of daily, subcutaneous injection of ETAF on the mouse flank resulted in an infiltrate of neutrophils, and to a lesser degree, mononuclear cells in association with epidermal hyperplasia, subcutaneous fibrosis, and focal muscle necrosis in the panniculus carnosus. These findings were not seen in control sites injected with media. These findings provide direct in vivo experimental evidence suggesting a physiologic role for ETAF/IL-1 in local inflammation.
R D Granstein, R Margolis, S B Mizel, D N Sauder
The molecular mechanism of volatile anesthetic action remains unknown. Attempts to elucidate this mechanism have been complicated by the absence of models in which changes in neuronal cellular properties can be correlated with changes in whole animal anesthetic effect. In this study we describe a model where diet-induced alterations in rat brain fatty acid composition are correlated with alterations in volatile anesthetic potency. Rats maintained on a fat-free diet showed significant depletion of arachidonic acid (20:4 omega 6; 5,8,11,14-eicosatetraenoic acid) and docosahexaenoic acid (22:6 omega 3; 4,7,10,13,16,19,-docosahexaenoic acid) in brain, and a corresponding increase in Mead acid (20: 3 omega 9; 5,8,11-eicosatrienoic acid). These fat-deprived rats were significantly more sensitive to all volatile anesthetics tested than were age-controlled rats on a normal diet. Parenteral supplementation of the fat-deprived animals with linolenic acid (18: 3 omega 3, 9,12,15-octadecatrienoic acid) completely reconstituted the docosahexaenoic acid content of brain without affecting anesthetic sensitivity. In contrast, supplementation of the fat-deprived rats with linoleic acid (18: omega 6; 9,12-octadecadienoic acid) caused a dramatic decrease in anesthetic sensitivity, but only a small change in whole brain arachidonate content. Further analysis revealed that linoleate supplementation of fat-deprived animals resulted in a preferential normalization of the arachidonate content of brain phosphatidylinositol as compared with other brain phosphoglycerides. These results demonstrate for the first time a correlation between changes in membrane composition and anesthetic effect, and indicate that the precise fatty acid composition (perhaps in specific phospholipids) of brain is important in the mechanism of volatile anesthetic action.
A S Evers, W J Elliott, J B Lefkowith, P Needleman
In order to determine whether the A cell may be directly suppressed by glucose in the absence of insulin, canine pancreata were perfused in vitro, both antegrade, through the arterial system and retrograde, through the venous system. Studies of the islet microvasculature have suggested that blood flows from the B cell core to the mantle; thus, the A cell may be tonically inhibited by intra-islet insulin. Retrograde perfusion may then be expected to prevent insulin from reaching the A cell, releasing it from inhibition. Retrograde perfusion with 88 mg/dl glucose markedly increased both insulin and glucagon secretion relative to antegrade levels. In a series of experiments, glucose concentrations were changed from 88 to 200 mg/dl. An antegrade glucose change resulted in increased insulin (134+/-21%; P less than 0.0025) and decreased glucagon (-26+/-9%, P less than 0.025) secretion. A retrograde glucose increase resulted in increased secretion of both insulin (91+/-15%; P less than 0.0005) and glucagon (23+/-9%; P less than 0.0125). To confirm that retrograde perfusion deprived the A cell of endogenous core derived, vascularly delivered insulin, possibly resulting in increased insulin sensitivity, 0.3 mU/ml exogenous porcine insulin was infused. Antegrade, 0.3 mU/ml insulin, had no effect on glucagon secretion (P less than 0.250), while retrograde infusion of 0.3 mU/ml insulin significantly inhibited glucagon secretion (-31 + 8%; P less than 0.0005). The results of our study support the concept that the direction of blood flow and of flow-dependent intra-islet hormone interactions are from the islet B cell core to the mantle. It was further concluded that the normal A cell may not be suppressed by glucose in the absence of insulin.
J I Stagner, E Samols
Pancreatic polypeptide and neuropeptide Y share 50% amino acid homology (18 out of 36 residues), suggesting that they may have common ancestral origins. cDNA clones complementary to human mRNAs encoding pancreatic polypeptide and neuropeptide Y were used to detect specific human genomic DNA sequences in human-mouse somatic cell hybrid lines. The pancreatic polypeptide gene (PPY) segregated with human chromosome 17, while the neuropeptide Y gene (NPY) segregated with human chromosome 7. Examination of cell hybrids with chromosomal rearrangements assigned PPY to the p11.1-qter region and NPY to the pter-q22 region of their respective chromosomes.
T Takeuchi, D L Gumucio, T Yamada, M H Meisler, C D Minth, J E Dixon, R E Eddy, T B Shows
Seropositive rheumatoid arthritis (RA) in adult and juvenile patients is associated with the serologic marker HLA-DR4. This association is incomplete; about one-third of the patients lack the disease-associated HLA-DR4 haplotype. The main biological function of class II molecules is to restrict the recognition of antigen by T lymphocytes. We therefore tested the hypothesis that patients with seropositive RA share T cell recognition sites for an unknown antigen and that such T cell "epitopes" are not identified by conventional serologic typing. We generated alloreactive human T cell clones by stimulating peripheral blood lymphocytes of normal donors against a lymphoblastoid cell line from a juvenile patient with seropositive RA. A panel of clones that recognized only HLA-Dw14 cells on a panel of homozygous typing cells was used to analyze class II molecules of adult patients with seropositive RA. By inhibition studies using monoclonal antibodies, the epitopes recognized by the different clones could be further characterized and assigned either to DR- or to DQ-encoded cell surface products. By using four different clones, it was possible to identify Dw14-associated T cell epitopes on all seropositive rheumatoid patients tested who typed HLA-DR4-positive and also on all eight DR4-negative patients tested. Approximately one-half of nonrheumatoid DR4-positive donors carried one or more determinants recognized by these clones; the expression of these allodeterminants in DR4-negative nonrheumatoid patients was rare (less than 10%). Thus, alloreactive human T cell clones are powerful tools to define T cell recognition sites on class II molecules that are not identified by conventional typing. Using T cell clones with specificities for determinants expressed on Dw14 homozygous typing lines, we were able to demonstrate shared epitopes on cells of all patients tested with seropositive RA irrespective of their HLA-D or HLA-DR type. These data suggest that major histocompatibility complex class II antigens of RA patients might be much more homogeneous than demonstrated by the incomplete HLA-DR4 association.
J Goronzy, C M Weyand, C G Fathman
The turnover of apolipoprotein B (apo B) in very low density, intermediate density, and low density lipoproteins (VLDL, IDL, and LDL) and in the light and heavy fractions of LDL was determined in seven patients with hyperapobetalipoproteinemia (hyperapo B), six normolipidemic subjects, and five patients with heterozygous familial hypercholesterolemia (FH). After receiving an injection of 125I-VLDL, hyperapo B patients were found to have a higher rate of synthesis of VLDL-apo B than controls (40.1 vs. 21.5 mg/kg per d, P less than 0.05) but a reduced fractional catabolic rate (FCR) (0.230 vs. 0.366/h, P less than 0.01). After receiving an injection of 131I-LDL, hyperapo B patients had higher rates of LDL-apo B synthesis than controls (23.1 vs. 13.0 mg/kg per d, P less than 0.001), as did FH patients (22.7 mg/kg per d). The FCR of LDL was similar in hyperapo B patients and controls (0.386 vs. 0.366/d) but was markedly decreased in FH patients (0.192/d). Most subjects exhibited precursor-product relationships between VLDL and IDL, and all did between IDL and light LDL; an analogous relationship between light and heavy LDL was evident in most hyperapo B patients and controls but not in FH patients. Simultaneous injection of differentially labeled LDL fractions and deconvolution analysis showed increased light LDL synthesis with normal conversion into heavy LDL in hyperapo B, whereas in FH conversion of light LDL was reduced and there was independent synthesis of heavy LDL. These data show that the increased concentration of LDL-apo B in hyperapo B is solely due to increased LDL synthesis, which is secondary to increased VLDL synthesis; in contrast, in FH there is both an increase in synthesis of LDL (which is partly VLDL-independent) and reduced catabolism.
B Teng, A D Sniderman, A K Soutar, G R Thompson
Fetal liver and thymus transplantation can be successfully employed for the treatment of severe combined immunodeficiency disease. In virtually all cases, donor and recipient cells are HLA mismatched. In a patient suffering from a severe combined immunodeficiency disease, full immunological reconstitution was obtained after fetal liver and thymus transplantation. HLA typing revealed that the patient's T cells were of donor origin, while the B cells and monocytes were of host origin. Despite this complete HLA mismatch, the patient was found to mount a subnormal to normal antibody response in vivo. This finding is in contrast with the concept that antigen recognition by T cells is major histocompatibility complex (MHC) restricted. To define the mechanism responsible for this in vivo antibody response, antibody production by peripheral blood mononuclear cells from the patient was tested in vitro after in vivo booster. The in vitro anti-tetanus toxoid antibody production was similar to that of the control group. In addition, specific proliferative responses to tetanus toxoid were obtained. Immunoglobulin allotype determination showed that antibodies were synthetized by host B cells. The results of the present study indicate that transplanted T lymphocytes and recipient cells cooperate despite complete HLA mismatch.
M G Roncarolo, J L Touraine, J Banchereau