THE FIBRINOLYTIC PATHWAY OF HUMAN PLASMA : ISOLATION AND CHARACTERIZATION OF THE PLASMINOGEN PROACTIVATOR

The conversion of the plasminogen proactivator to plasminogen activator by activated Hageman factor or its fragments has been recognized as an essential step in the conversion of plasminogen to plasmin. The plasminogen proactivator has been completely separated from prekallikrein and pre-PTA, two other proenzyme substrates of activated Hageman factor or its fragments. Plasminogen proactivator, free of any contaminating proteins as assessed by disc gel electrophoresis or isoelectric focusing, revealed a single band with an isoelectric point of 8.9 corresponding in position to the Hageman factor activatable material eluted from replicate unstained gels. After conversion of plasminogen proactivator by Hageman factor fragments to the plasminogen activator, the active site of the plasminogen activator is not inhibited by C1INH and is thus readily distinguished from that of kallikrein or PTA. The plasminogen activator is susceptible to inactivation by DFP while the plasminogen proactivator is not, as has been the case for esterases having a serine in the active site. Its interaction with plasminogen is inhibited by ε-aminocaproic acid.     

Low Density Lipoprotein: A METABOLIC PATHWAY FOR RETURN OF CHOLESTEROL TO THE SPLANCHNIC BED

The mechanism(s) by which cholesterol returns to the splanchnic bed from peripheral tissues are not well understood. To study this phenomenon in fasting man, lipoproteins were isolated from plasma obtained from hepatic vein and aorta. Cholesterol content of each lipoprotein class was determined and arteriovenous (AV) differences could be calculated for each patient. The results in the first 24 patients indicated splanchnic secretion of very low density lipoprotein cholesterol (mean AV difference - 3 mg/100 ml, P < 0.01), but not significant AV difference for total cholesterol, high density lipoprotein cholesterol, or low density lipoprotein (LDL) B protein. In contrast, for LDL (d 1.006-1.063 g/ml), there was significant uptake of cholesterol across the AV bed +8 mg/100 ml, P < 0.0002). In a further 15 patients, similar samples were obtained and intermediate density lipoprotein isolated at d 1.006-1.019 g/ml and LDL at 1.019-1.063 g/ml. The AV difference previously noted could now be localized to the 1.019-1.063 cholesterol ester moiety (+8 mg/100 ml, P < 0.0005). In the final 14 patients, the LDL cholesterol AV difference was again confirmed and shown to be unrelated to heparin. As well, there was secretion of triglyceride in the hepatic vein LDL. These quantitative data obtained in man raise the possibility that LDL rather than high density lipoprotein transports cholesterol ester to the splanchnic bed.     

ALGEBRAIC POTENTIAL OF THE HILL EQUATION AS AN ALTERNATIVE TOOL FOR PLOTTING DOSE (OR TIME)/EFFECTS RELATIONSHIPS IN TOXICOLOGY: A THEORETICAL STUDY

Use of the Hill equation in plotting the results of toxicological experiments offers the following advantages: 1. In dose/effect relationships, the maximum response RM can be accurately determined by means of a described new noniterative algebraic method, from both hyperbolic and sigmoidal responses expressed in natural (nontransformed) units. The Hill coefficient (n) and the dose giving 50% (f50) or X% (fX) of RM, as well as their SD, are accurately deduced. 2. In time-course experiments with sigmoidal shape, an additional set of parameters, readily available from the former basic 3, makes it possible to avoid arbitrary choices, such as the time at which a percentage or mortality is considered. The slopes of the tangents at the inflexion point (maximum rate of response) and at half-maximum effect, then the coordinates of these points and the horizontal intercept of the tangent at inflexion point (as an index of initial lag-phase) will give increasing and decreasing functions of doses, respectively, depending on RM, f50, and (n). 3. The processes described first allow the classical parameters LD50 (or LDX) and LT50 (or LTX: X%--lethality time) to be calculated with high algebraic accuracy, in addition to RM and (n), both of which are of great interest in the general case of ligand-receptor interactions. Thus, the deduced set of additional indexes, not readily available from classical "Probits-type" transformations, eliminates the mortgage of subjective operations.     

THE DIFFERENTIATION PATHWAY OF T LYMPHOCYTES : EVIDENCE FOR TWO DIFFERENTIATED CELL TYPES

Two experimental models have been used to study T-cell differentiation. The first, a graft-vs.-host reaction, was induced by injecting thymocytes or cortisone-resistant thymocytes into lethally irradiated allogeneic mice. The second was tumor graft rejection in allogeneic hosts. Ultrastructural studies at various time intervals revealed two differentiated T-cell types. One of these (the "pale" cell) is probably high cytotoxic as measured in the chromium-release assay, the other (the "dark" cell) may be an "amplifier" cell, helping in the differentiation of cytotoxic cells.     

The Inhibition of Polymorphonuclear Leukocyte Cytotoxicity by Dapsone: A POSSIBLE MECHANISM IN THE TREATMENT OF DERMATITIS HERPETIFORMIS

The effect of the sulfone compound 4,4'-diaminodiphenyl sulfone (dapsone) on normal human polymorphonuclear leukocytes (PMNL) has been investigated in vitro. The drug has a dramatically beneficial effect in dermatitis herpetiformis in which the PMNL and immune complexes has been stressed to be of importance for the development of the skin lesions. Pruritus disappears and the inflammatory eruptions clear within a few days of starting therapy. The effect of dapsone has been evaluated on the different stages of phagocytosis. Using dapsone concentrations (1-30 mug/ml) comparable with those found after therapeutic doses, we have found that the drug interferes primarily with the myeloperoxidase (MPO)-H(2)O(2)-halide-mediated cytotoxic system in the PMNL. No effect was observed on random locomotion, chemotaxis, phagocytic ingestion, oxidative metabolism, or the release of lysosomal enzymes. Kinetic studies in a cell-free system with purified MPO revealed a competitive type of inhibition using varying concentrations of NaI. Furthermore, the inhibition resulted in reduced candidicidal activity during phagocytosis of Candida albicans, and reduced cytotoxicity to adjacent mammalian cells measured as the (51)Cr release from virus-induced lymphoma cells. Because the MPO-H(2)O(2)-halide system not only fulfills the antimicrobial activity but is suggested to be a modulator of the inflammatory reaction as well, the action of dapsone in dermatitis herpetiformis may in part be explained by its effect on this system.     

MECHANISM OF ACTIVATION OF THE BONE MARROW-DERIVED LYMPHOCYTE : III. A DISTINCTION BETWEEN A MACROPHAGE-PRODUCED TRIGGERING SIGNAL AND THE AMPLIFYING EFFECT ON TRIGGERED B LYMPHOCYTES OF ALLOGENEIC INTERACTIONS

Peritoneal exudate cells from nu/nu mice stimulated with proteose peptone broth, but in general not from unstimulated mice, permitted cultures of spleen cells from congenitally athymic (nu/nu) mice to respond to the thymus-dependent antigen fowl gamma globulin (FγG). Supernatants of cultures of peritoneal cells were also effective, the activity being sensitive to trypsin. Since nu/nu mice were effective sources of the peritoneal cells it would not seem obligatory for the thymus-derived (T) cell to be involved in the triggering of the bone marrow-derived (B) cell by a thymus-dependent antigen FγG. It is proposed that the B cell is triggered at the macrophage surface where it encounters two signals (a) the antigen and (b) a protein secreted by the activated macrophage. In vivo the T cell may have a role in B-cell triggering, either in activating the macrophage or in aiding in presentation of antigen on the macrophage surface. Thymus-independent antigens are proposed to induce an IgM response because they are able to provide "signal two" either by direct interaction with the B cell or via irritation or activation of the macrophage. The stimulatory effect of T cells activated by an allogeneic interaction was used as a model of one influence of the T cell on the development of an antibody response. The presence in cultures of nu/nu spleen of an allogeneic interaction had no effect on the inability of these cells to respond to FγG. However when a source of the postulated second signal such as the supernatant of a macrophage culture was present, an allogeneic interaction had a powerful amplifying effect on the anti-FγG response. In contrast the response of nu/nu spleen cultures to heterologous erythrocytes was greatly enhanced by the presence of an allogeneic interaction. It is suggested that since there was a definite basal response to the heterologous erythrocytes added alone, the enhancement represented not an activation of more B cells but rather an amplification of this basal response. Thus the anti-FγG response in cultures of nu/nu spleen differentiates between factors such as those released by activated macrophages that are involved in B-cell triggering and factors released by activated T cells that amplify the numbers of antibody-forming cells resulting from a B cell already triggered.     

ENDOTOXEMIA AND ADRENAL HEMORRHAGE : A MECHANISM FOR THE WATERHOUSE-FRIDERICHSEN SYNDROME

An experimental model that produces adrenal cortical hemorrhage with endotoxin has been described. When stimulated by thorotrast, endotoxin, or its tropic hormone (ACTH), the adrenal cortex is susceptible to the development of a hemorrhagic reaction during endotoxemia. The hemorrhagic reaction resembles that described in the Waterhouse-Friderichsen syndrome. A pathophysiologic mechanism for the occurrence of adrenal hemorrhage occurring during acute sepsis is presented. Increased metabolic activity associated with the production of corticosteroids seems to make the adrenal cortex susceptible to endotoxin-induced hemorrhage. Adrenal hemorrhage observed during sepsis, as in the Waterhouse-Friderichsen syndrome, may be attributable to endotoxemia occurring during or shortly after stimulation of the adrenal cortex by infection. Significant differences between adrenal cortical hemorrhage and the Shwartzman phenomenon are described.     

STUDIES ON THE MECHANISM OF ENDOGENOUS PYROGEN PRODUCTION : III. Human Blood Monocytes

The characteristics of pyrogen production and release by human blood monocytes were investigated. A dose-response assay of monocyte pyrogen in rabbits indicated a linear relationship of temperature elevation to dose of pyrogen at lower doses. Monocytes did not contain pyrogen when first obtained, nor did they release it spontaneously even after 5 days of incubation in vitro. Pyrogen production was apparent 4 h after stimulation by endotoxin or phagocytosis, and continued for 24 h or more. Puromycin, an inhibitor of protein synthesis, prevented both initiation and continuation of pyrogen production and release. Pyrogen-containing supernates retained most pyrogenic activity during overnight incubation even in the presence of activated cells. Lymphocytes appeared to play no role in either initiation or continuation of pyrogen production in these studies.     

A NEUTROPHIL-DEPENDENT PATHWAY FOR THE GENERATION OF A NEUTRAL PEPTIDE MEDIATOR : PARTIAL CHARACTERIZATION OF COMPONENTS AND CONTROL BYα-1-ANTITRYPSIN

A biologically active neutral peptide mediator is cleaved from a plasma protein substrate by an α-1-antitrypsin-inhibitable serine protease apparently residing on the membrane of the human neutrophil. The peptide mediator has an approximate mol wt of 1,000, and is distinguished from the kinin peptides by a neutral isoelectric point, susceptibility to inactivation by trypsin as well as chymotrypsin and activity on the isolated, atropinized, and antihistamine-treated guinea pig ileum with relatively little action on the estrous rat uterus. The neutrophil protease is fully inhibitable by DFP, trypsin inhibitors from lima or soy bean, and α-1-antitrypsin and is associated with the high mol wt fragments of the neutrophil and not the nuclear, lysosomal, or cytoplasmic subcellular fraction. The substrate has an approximate mol wt of 90,000 and is chromatographically separable from kininogen. The exquisite sensitivity of the neutrophil protease to α-1-antitrypsin was established both by inhibition with highly purified α-1-antitrypsin and by the inability of the protease to generate detectable neutral peptide in a homozygous (ZZ) α-1-antitrypsin-deficient patient without heat inactivation of the residual inhibitor. On the other hand, plasma from a (null) α-1-antitrypsin-deficient patient supported neutral peptide generation and revealed an additional factor which inactivated neutral peptide.     

机械伤害刺激激活丘脑-皮层神经网络的多通道记录研究

目的:在清醒大鼠脑内同步记录内、外侧痛觉传导通路神经元的伤害性活动,在丘脑-皮层水平观察各脑区的编码模式以及脑区之间的功能联系.方法:麻醉状态下在动物丘脑和皮层的四个区域单侧植入微电极阵列,在清醒状态下给予伤害和非伤害性机械刺激,同步记录多个神经元的单位电活动,并利用滑行窗口分析、交互相关分析、聚类分析以及判别分析等运算方法进行信息提取.结果:在7只大鼠上共记录到180个神经元的单位电活动.伤害和非伤害机械刺激均引起以兴奋为主的反应.与非伤害刺激相比,伤害性刺激使各脑区神经元反应比例显著增加,平均反应强度显著增加,两条通路上脑区之间的相关性亦显著增加.另外,从神经元群体编码的角度看,内侧通路对伤害与非伤害机械刺激的分辨能力稍优于外侧通路.结论:中枢对伤害性机械刺激的编码有赖于多个脑区的共同参与,内、外侧系统对刺激的编码能力不同,可能与刺激本身的特性有关.     

MECHANISM OF ACTION OF MIGRATION INHIBITORY FACTOR (MIF) : I. EVIDENCE FOR A RECEPTOR FOR MIF PRESENT ON THE PERITONEAL MACROPHAGE BUT NOT ON THE ALVEOLAR MACROPHAGE

The initial interaction between migration inhibitory factor (MIF) and the guinea pig alveolar and peritoneal macrophage was studied. MIF-containing supernatants were generated from sensitized lymph node lymphocytes obtained from guinea pigs immunized with bovine gamma globulin in complete Freund's adjuvant. MIF-containing supernatants were markedly inhibitory for the migration of the peritoneal macrophage but had no effect on the alveolar macrophage. A linear relationship was observed between per cent inhibition of migration and serial twofold dilution of supernatant. Reexpressed in arbitrary MIF units, this relationship reflects a dose-response relationship with saturation characteristics. Pulse exposure of peritoneal macrophages to MIF resulted in adsorption of MIF onto both viable and nonviable cells with corresponding depletion of supernatant MIF. The alveolar macrophage did not adsorb MIF. Pulse adsorption of MIF onto the peritoneal macrophage is dependent on time, temperature, and cell number. Pretreatment of the cells with proteolytic enzyme prevents the adsorption of MIF while leaving migration unaffected. These observations support the existence of a specific cell surface receptor for MIF. The existence of such a receptor provides selectivity of immune modulation of macrophage populations by lymphocytes in delayed hypersensitivity reactions.     

QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES : VI. IDIOTYPIC SPECIFICITY AS A POTENTIAL GENETIC MARKER FOR THE VARIABLE REGIONS OF MOUSE IMMUNOGLOBULIN POLYPEPTIDE CHAINS

Antisera were prepared in rabbits against anti-p-azobenzoate antibodies of an A/J and a BALB/c mouse and anti-p-azophenylarsonate antibodies of an A/J mouse. After appropriate absorption the antisera reacted with the anti-hapten antibody of the donor mouse but, by sensitive quantitative tests, not at all with other components of the hyperimmune serum or with preimmune serum of the donor mouse. The absorbed antiserum therefore appeared to be specific for idiotypic determinants. Nearly all idiotypic specificities identified in the serum of the donor were also present in the serum of other mice of the same strain, immunized against the same hapten group, but not in mice immunized with a different hapten. In each case the antibodies of the donor mouse reacted most effectively on a weight basis with antiidiotypic antiserum. Cross-reactions were observed among different strains of mice but homologous anti-bodies reacted most effectively with antiidiotypic antisera. C57/BL and DBA antisera contained very low concentrations of specificities present in the A/J and BALB/c antibody populations; antibodies of A/J and BALB/c antisera are more closely related to one another. The results indicate that idiotypic specificity may provide a genetic marker for the variable regions of immunoglobulin polypeptide chains.     

ANTIBODY-MEDIATED ACTIVATION OF A DEFECTIVE β-D-GALACTOSIDASE : II. IMMUNOLOGICAL RELATIONSHIP BETWEEN THE NORMAL AND THE DEFECTIVE ENZYME

Two closely related protein antigens were used to study immunogenic competition. Namely, normal β-D-galactosidase of Escherichia coli (Z) and a genetically defective β-D-galactosidase (AMEF) which seems to differ from the normal in one amino acid substitution. A unique characteristic of this pair of antigens is that, although they are indistinguishable in precipitation and absorption tests with antibodies, the enzymatic activity of AMEF is specifically increased several-hundredfold in the presence of antibodies directed against Z. The following results show that Z and AMEF also differ in their immunogenic ability: (a) antibodies directed against Z activated AMEF; antibodies directed against AMEF did not activate, but competed specifically with activating antibodies. (b) Animals immunized with AMEF failed to produce activating antibodies when they were subsequently challenged with Z, although the presence of some cells primed to produce activating antibodies could be demonstrated by adoptive transfer. (c) Animals preimmunized with Z were stimulated in their production of activating antibodies by AMEF challenge, although not as efficiently as with Z. A model explaining these observations by competition for the immunogenic site among antigen-sensitive cells carrying cross-reacting receptors is presented.     

STUDIES ON IMMUNE HUMAN HEMOLYSIS : II. THE DONATH-LANDSTEINER REACTION AS A MODEL SYSTEM FOR STUDYING THE MECHANISM OF ACTION OF COMPLEMENT AND THE ROLE OF C'1 AND C'1 ESTERASE

1. The inactivation, of C'1 blocks the completion of the cold phase of the Donath-Landsteiner reaction; inactivation of the other components of complement does not interfere with the cold phase of the reaction. 2. C'2, C'3, and C'4 are required for the completion of the hemolytic reaction. C'4 reacts in either the cold or warm phase, but C'2 and C'3 must react in the warm phase. 3. Partially purified C'1 or C'1 esterase can be substituted for whole serum in the cold phase, although neither reagent contains any of the other components of complement 4. Partially purified serum inhibitor of C'1 esterase blocks the effect of C'1 esterase in the cold phase, and reverses the effect of complement, C'1 or C'1 esterase when incubated with "activated" cells after the cold phase. 5. C'1 esterase activity can be eluted from "activated" erythrocytes with Na₃EDTA. 6. The components of human complement in this human hemolytic reaction act in the order C'1, C'4, C'2, C'3. Ca⁺⁺ is required for the reaction with C'1, and Mg⁺⁺ is required for the reaction with C'2. 7. Accordingly, a functional role of C'1 esterase in a human disease state is demonstrated, and a human model is established for the study of the mechanism of action of complement.