Several allergic reactions under conditions of functioning of an inhibitor of antibody activity in the body]

Allergization under conditions of functioning an inhibitory factor in the organism was studied by the reaction of leukocytolysis with Tsuverkalov's dysenterin and in the reproduction of hypersensitivity of the immediate type on the Schultz-Dale's model. It was shown that in functioning an inhibitory factor in the organism the degree of leukocytolysis reaction with the dysentery allergen decreased in the immunized animals in parallel with the fall in the serum antibody activity. A depression of the activity of macromolecular sensitizing antibodies was also observed.     

Effect of serum fractions on the incorporation of tritiated thymidine in activated lymphocytes: II. Comparison between cord blood and the blood of newborn infants

After serum Sephacryl 300 gel filtration, the major circulating forms of TA in serum are macromolecular. However different patterns are found in cord blood and in the blood of newborns at 24 hr of life. The low values found in cord blood are not due to inhibiting factor(s). TA in serum fractions does not parallel immunoreactive Sm-C levels. The pattern of immunoreactive Sm-C are similar in both cord blood and newborn blood.     

Nature of the inhibitory serum factor in mice injected with isologous antibodies conjugated with cellulose

The formation of antigen-specific serum inhibitory factor was induced by injection of covalently bound to cellulose syngeneic antibodies to sheep red blood cells into (CBA X C57BL/6)F1 mice. This factor was absorbed by cellulose immunosorbents immobilized with antibodies against sheep red blood cells and with rabbit antibodies against mouse gamma-globulin and was not absorbed by immunosorbents immobilized with immunoglobulins of intact mice or immunoglobulin containing antibodies against rat red blood cells. These data, and evidence obtained by the authors previously, indicate that inhibitory factor of the serum is likely to be due to idiotypic antibodies.     

The Hurler syndrome: treatment of cultured Hurler fibroblasts with normal human serum.

Prolonged replacement of fetal calf serum by normal human serum for the enrichment of medium during tissue culture of Hurler fibroblasts resulted in increased acid mucopolysaccharides in the cells and in the medium. The predominant intracellular mucopolysaccharide had the characteristics of dermatan sulfate when Hurler cells were treated with either serum. Normal human serum contains a nonspecific coreective factor capable of augmenting the loss of 35SO4-AMPS from Hurler cells, but not from normal cells. Fetal calf serum and Hurler serum have similar corrective factor activity for labeled Hurler cells. The corrective factor activity of all three sera was recovered from reconstituted dialyzed ammonium sulfate precipitates. The corrective factor of normal human serum did not increase degradation of mucopolysaccharide, but increased secretion of macromolecular and large oligosaccharide components. Failure of the corrective factor of normal human serum to effectively decrease the dermatan sulfate content of Hurler cells during prolonged exposure may be a quantitative phenomenon due partly to the brief duration of corrective factor activity and partly to increased synthesis of mucopolysaccharide.     

Spontaneous production of a suppressor factor by a human macrophage-like cell line U937. II. Suppression of antigen- and mitogen-induced blastogenesis, IL 2 production and IL 2 receptor expression in T lymphocytes

U937, a human macrophage-like cell line, spontaneously produces a factor which inhibited blastogenic responses of human blood T lymphocytes stimulated with tuberculin-purified protein derivative (PPD) or phytohemagglutinin (PHA). We investigated the mechanism of suppressor action of the U937 factor. The U937 suppressor factor inhibited interleukin 2 (IL 2) production by human blood T lymphocytes stimulated with PPD or PHA. IL 1 did not overcome the inhibitory action of the U937 factor on PPD-induced IL 2 production by human blood T lymphocytes. The U937 factor also inhibited the production of IL 2 by a human leukemic cell line, JURKAT, stimulated with PHA. The U937 suppressor factor interfered with the expression of Tac antigen (IL 2 receptor) on PPD- or PHA-stimulated blood T lymphocytes. The inhibitory activity of the U937 factor on Tac expression was not affected by the addition of IL 2 or a crude lymphokine-containing T cell supernatant. Tac expression was more sensitive than IL 2 production to inhibition by U937-conditioned medium. The U937 suppressor factor was precipitable by 33 to 67% saturated ammonium sulfate and was inactivated at pH 2 or pH 11. Sephacryl S-200 Gel filtration analysis of U937 culture supernatants revealed that the inhibitory activities for blastogenesis, IL 2 production, and Tac expression co-purified in fractions with an apparent m.w. between 67,000 and 130,000. These data indicate that U937 spontaneously produces a macromolecular suppressive factor with major locus of action on the production of IL 2 and the expression of the IL 2 receptor.     

A loop of coagulation factor VIIa influencing macromolecular substrate specificity

Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).     

The correlation between plasminogen activator activity and thymidine incorporation in mouse bone marrow-derived macrophages : Opposing actions of colony-stimulating factor, phorbol myristate acetate, dexamethasone and prostaglandin E

Plasminogen activator activity and [3H]thymidine incorporation were studied in mouse bone marrow-derived macrophages. The two activities correlated closely in the presence of stimulatory (colony-stimulating factor, phorbol myristate acetate, PMA) and inhibitory (dexamethasone, prostaglandin E1) signals. The actions of dexamethasone and prostaglandin E1 could be overcome by either stimulatory agent, so that the net effect was an alteration in sensitivity of the macrophages to colony-stimulating factor, or PMA. The sensitivity of bone marrow-derived macrophages to CSF-1 was also reduced by the addition of small numbers of CSF-1 unresponsive peritoneal macrophages. Plasminogen activator induction was not a sufficient signal for [3H]thymidine incorporation which requires an additional macromolecular serum component. The serum component was found not to be plasminogen.     

INHIBITORY EFFECT OF RABBIT SERUM FACTOR ON IN VITRO OOCYTE MATURATION OF THE TELEOST FISH,ORYZIAS LATIPES*

The present study indicates that a factor in rabbit serum inhibits the in vitro steroid- and gonadotropin-induced maturation of oocytes of the teleost fish, Oryzias latipes. Such inhibitory activity could not be recognized in the serum of this fish or in the fluids from mammalian follicles. Passage of the serum inhibitor through a cellulose membrane indicated that it has a molecular weight of less than 3,500. The inhibitory activity on steroid-induced oocyte maturation was not destroyed by heating, by repeated freezing and thawing or by treatment with proteolytic enzymes, lipase or amylases. However, its activity could be removed by extraction with charcoal but not with ethyl ether or toluene. The inhibitory action of the heat-stable and dialyzable serum factor was reversible. The factor appears to exert its inhibitory effect upon the oocyte itself in an early step of maturation, rather than upon the follicle cells.     

Effect of serum on prostaglandin production during the co-culture of human thyroid cells and peripheral blood mononuclear cells

Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co-culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process. Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH₄) ₂SO₄ precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion-exchange chomatography of human serum showed the inhibitor to co-migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum. Prostaglandins are potent modulators of immune-cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.     

Impaired monocyte function in liver cirrhosis.

Monocyte function in patients with cirrhosis of the liver was measured by phagocytosis and killing of Candida pseudotropicalis and C albicans. Both variables were significantly decreased in the patients compared with controls. Control monocytes exposed for two hours to patients'' serum showed a significant decrease in intracellular killing compared with control monocytes incubated in autologous serum. This suggests the presence of an inhibiting factor in the patients'' serum. This inhibitory factor passed through a dialysis membrane that permitted the passage of molecules of less than 12 000 daltons. Treating monocytes from patients with trypsin significantly increased phagocytosis, indicating that the possible inhibitory factor was attached to the monocyte surface. Metabolism of monocytes during phagocytosis as determined by chemoluminescence was significantly lower in monocytes from patients compared with controls, indicating metabolic impairment. Monocytes are a component of the monocyte-macrophage system, which includes Kupffer''s cells. Impairment of the function of these cells, which have a pivotal role in clearing portal blood, might well be extremely important in the development of chronic liver disease.     

丁香苷抗炎镇痛作用及部分机制研究

研究丁香苷抗炎镇痛作用及部分机制。以阿司匹林作阳性对照药,观察丁香苷对二甲苯致小鼠耳廓肿胀、醋酸致小鼠毛细血管通透性增加、角叉菜胶致大鼠足趾肿胀、棉球致大鼠肉芽肿的抗炎作用;对小鼠热板试验、醋酸扭体试验的镇痛作用;同时测定角叉菜胶致大鼠炎足炎性渗出物中的PGE2、MDA和血清中的NO、SOD,初步探讨丁香苷抗炎镇痛的部分机制。结果表明,丁香苷对急慢性炎症反应有明显抑制作用,能明显降低角叉菜胶致炎足炎性渗出物中PGE2、MDA和血清中NO含量,明显增加血清中SOD的活性。因此,丁香苷具有较强的抗炎镇痛作用,其机制可能与抑制PGE2、NO等炎症介质生成、增强自由基清除能力有关。     

Macromolecules mediate prostacyclin release from human umbilical artery

Previous reports regarding the modulation of prostaglandin release from tissues by serum components did not identify these components. We have found that inhibition of prostacyclin release from human umbilical artery by human serum is attributable to serum macromolecules. We demonstrate that such inhibitory activity depends on macromolecular size and may result from macromolecule/cell surface interactions.     

Characterization of a lymphokine produced by human T cells which inhibits collagen synthesis

Human lymphocytes, isolated from peripheral blood, were cultured for 48 hr in a defined medium containing 10 mg/ml bovine serum albumin and phytohemagglutinin. A lymphokine which inhibits collagen synthesis by cultured human dermal fibroblasts was purified from the lymphocyte incubation medium by successive steps of ammonium sulfate precipitation, gel filtration chromatography, and isoelectric focusing. Good recovery of this collagen synthesis inhibitory factor (CSIF) was obtained and a factor with an approximate molecular weight of 55,000 and a pI of 6.2 was isolated. The purification of the factor should permit further studies on its mechanism of action.     

Rat Serum Factors Inhibiting the G1-S Transition In Hepatocytes

An attempt was made to detect the serum factors inhibiting the G₁-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G₁-S inhibitory factor. the same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10³. the active period in the cell cycle of both the LMW and HMW factors was the same: the late G₁ phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. an enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.     

Effects of heterologous sera on fertilized rabbit ova

Undiluted blood serum of various species was used to culture two-celled rabbit ova for 24 hours. It was found that there is an ovocidal factor present in the serum of man, sheep, cattle, goat, and fowl. The factor is lethal rather than inhibitory; exposure to it for 10 minutes will cause the death of the ova. This factor is unstable, thermolabile (destroyed at 55 degrees C. in 30 minutes), and of large molecular size. The strength or concentration of this factor varies according to the origin of the serum, increasing in the order man, sheep, cattle, goat, fowl. The blood serum of rabbit, horse, dog, guinea pig, rat, and pig contains no ovocidal factor against rabbit ova. The ovocidal factor differs from the spermicidal factor, which is present in all the sera of the different species studied with rabbit spermatozoa. Immunization of the guinea pig against rabbit ova is possible. Normal development of young rabbits was obtained by transplantation of ova cultured in the heated or normal serum of other species after 24 hours.     

Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule II. Evidence that complements is involved

The strong inhibitory effect of intraluminally perfused serum on the isotonic absorption by the rat kidney proximal tubule is abolished when serum is preincubated with inulin, zymosan, cobra venom factor, hydrazine or isolated kidney brushborder membrane. Rabbit serum genetically deficient in C6 does not have an inhibitory effect but is fully reconstituted by the addition of purified rabbit C6. Treatment of serum with anti-C6 serum also abolishes its inhibitory effect. These and previously reported data are best interpreted to indicate that complement-mediated cell lysis is the mechanism for the serum-induced inhibition of isotonic absorption.     

Inhibiting alternative pathway complement activation by targeting the factor D exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.     

Binding of low-density lipoprotein to heparin-Sepharose: characterization and inhibition by serum high-molecular-weight components

In this study, the interaction of human serum low-density lipoprotein (LDL) with heparin immobilized on Sepharose was reinvestigated. Binding of isolated LDL (stabilized with human serum albumin (HSA] was compared with that of LDL in full serum. (1) Binding of isolated LDL was slightly decreased by CaCl2 and was not affected by MgCl2. In contrast, with full serum LDL binding was increased by these divalent cations. (2) In both situations, binding of LDL was saturable, but the maximum degree of binding that could be reached was much higher with isolated LDL than with LDL in full serum. This could be ascribed to an inhibitory action of a factor found in the d greater than 1.24 fraction of serum. (3) The effect of this factor was diminished in the presence of CaCl2 or MgCl2, which suggests that the stimulation of LDL binding by these cations in full serum is due to suppression of the inhibitory activity of this factor. (4) The inhibitory factor in the d greater than 1.24 fraction can be partially purified by absorption to heparin-Sepharose, followed by elution with 6 M guanidine chloride. The resulting preparation had a 30- to 50-fold higher specific activity. Attempts to purify the factor further resulted in loss of activity. (5) The activity is decreased upon treatment with trypsin and also upon acetylation or reduction with dithiothreitol, indicating that free amino groups and S-S bridges are essential.     

Identification and characterization of a T cell growth inhibitory factor produced by K562 erythromyeloid cells

Cells of the human erythroleukemia cell line K562 constitutively secrete a factor that inhibits human T lymphocyte proliferation induced via CD3/Ti. The factor, termed K-TIF (K562-derived T cell inhibitory factor) is produced in either the presence or absence of fetal calf serum in cultures of K562 cells and can be precipitated by 70% NH4SO4. Gel filtration chromatography on Superose 12 resin by FPLC showed that the inhibitory factor has a molecular weight of approximately 30-35 kDa. A protein of this size, metabolically labeled with [35S]methionine, specifically bound human peripheral blood mononuclear cells. Chromatofocusing with Mono P by FPLC (pH gradient 7.2-5) indicates that the inhibitory factor has an isoelectric point of 6.0-6.4.     

Inhibitory and anti-inhibitory factors of rat serum active on the G1-S transition of hepatocyte cell cycle.

Rat hepatocytes are responsive to a serum factor inhibiting their progression through the cell cycle from the late G1 phase to the S phase. After fractionation of normal adult rat serum by two chromatographic steps on DEAE cellulose and sephadex gel filtration, the inhibitory activity was linked to proteins having a high electronegative charge and of apparent high molecular weight. Polyacrylamide gel electrophoretic analysis of active fraction showed that the alpha1 macroglobulin was its main component. Male and female baby rats were sensitive to the inhibitory factor from normal rats. Contrary to the normal adult rat serum the whole hepatectomized adult rat serum did not exhibit any ingibitory activity on the G1-S transition. However, two components having antagonist activities: an alpha1 globulin and a gamma globulin, were separated by chromatographic procedures from hepatectomized rat serum. (a) The alpha1 globulin showed an inhibitory activity. It had an apparent molecular weight lower than that found in normal rats. Its activity was sex related: only male baby rats were responsive. (b) The factor present in the gamma globulin fraction was found to be antagonistic to the alpha1 globulin factor. Its occurrence after hepatectomy explains the absence of inhibitory activity in the serum of hepatectomized rats.