Presence Of Normal Human Serum Research Articles

Isolation of a lipopolysaccharide mutant of Neisseria gonorrhoeae: an analysis of the antigenic and biologic difference.

Analysis of the surface constituents of a pyocin 611 131-resistant variant of strain no. JW-31 of Neisseria gonorrhoeae revealed substantial differences in the lipopolysaccharide (LPS) but not changes in the auxotype or outer-membrane proteins. Immunodiffusion and an enzyme-linked immunosorbent assay showed that the variant strain (no. JW-31R) lost both the LPS serotype and the variable antigens while retaining at least a portion of the common determinant. The use of monoclonal antibody indicated that LPSs from strain no. JW-31R and pyocin 611 131-resistant strains of other LPS serotypes lack a D-galactosaminyl-D-galactopyranosyl-D-glucose moiety. The LPS-derived polysaccharide from strain no. JW-31 binds to wheat-germ lectin in precipitin and inhibition systems, whereas the JW-31R polysaccharide exhibits a markedly reduced affinity. In the presence of normal human serum, 99% of strain no. JW-31R was killed within 20 min and strain no. JW-31 was not.

Effects of bovine parathyroid extract and [Asu1,7]-eel calcitonin on 35S incorporation into chick embryonic cartilage.

35S incorporation into embryonic chick pelvic cartilage was measured using the method of somatomedin bioassay and the effects thereupon of the bovine parathyroid extract (PTE) and the synthetic [Asu1,7]-eel calcitonin (CT) were examined. Bovine PTE potentiated 35S incorporation stimulated by normal human serum. [Asu1,7]-eel CT did not affect the sulfation activity in the presence of normal human serum. However, it showed a slight stimulation of 35S incorporation in the absence of normal human serum. No definite interaction on 35S incorporation between PTE and CT was observed. It is speculated from these results that PTH may be involved in the stimulation of the cartilage growth in concert with somatomedins in vivo.

Variability in the degree of opsonization and phagocytosis of strains of Staphylococcus aureus isolated from patients with disseminated intravascular coagulation.

Patients with septicemia due to Staphylococcus aureus occasionally manifest DIC. We studied the ability of organisms isolated from 30 patients with S. aureus (5 with DIC) to be opsonized and phagocytosed by normal human PMNs in the presence of normal human serum chelated with MgEGTA as a possible measure of alternative complement pathway activation. By this assay, nine S. aureus strains were found to be opsonized at 50% level or greater. Five of those organisms were isolated from the five patients who had clinical and laboratory manifestations of DIC, three of whom died. Patients from whom the other four activating strains were isolated were more seriously ill (by a method of grading clinical severity of disease) than the remaining 21 patients. Four of the nine patients from whom activating strains were isolated died compared to only four of the other 21 patients. The accelerated phagocytosis of staphylococcal strains from patients with DIC after incubation in MgEGTA-chelated serum suggests that surface factors on these strains interacted with serum factors differently than did strains from patients not developing DIC. However, all (except one) staphylococcal strains, from patients both with and without DIC, depleted hemolytic complement activity in MgEGTA-chelated serum; therefore a direct relationship between activation of the alternative complement pathway and opsonization seems not to exist. Thus strains of S. aureus capable of being opsonized by PMNs in human serum chelated with MgEGTA were associated with more severe infections often associated with DIC.

Naturally occurring blocking antibodies modulate immediate hypersensitivity responses in human filariasis.

Although the basophils and mast cells of patients with chronic helminth infection are sensitized with specific IgE antibody and frequently exposed to parasite antigens in vivo , these rarely manifest allergic reactions to their parasites. To investigate the regulatory mechanisms limiting immediate hypersensitivity responsiveness in such patients, we used the in vitro antigen-induced histamine- release (HR) reaction of human basophils as a correlate of in vivo allergic responses. For 13 patients with bancroftian filariasis HR responses were elicited by graded doses of microfilarial antigen in the absence of serum or in the presence of normal human serum, autologous serum, or serum from other infected patients. In all instances, sera from patients with filariasis contained a factor that specifically inhibited HR to parasite antigen. Normal sera had no such inhibitory effect, but sera from other filariasis patients inhibited as effectively as autologous serum. This HR blocking factor was heat stable (56°C x 2 hr) and nondialyzable. Its parasite antigen specificity was demonstrated by its inability to block the HR of patient cells triggered by anti-IgE anti-body and its lack of inhibitory effect on the Hr response of ragweed-sensitized cells reacted with ragweed antigen E. Fractionation of the sera by staphylococcus protein A-Sepharose chromatography showed that the blocking factor was an IgG antibody whose activity could be removed by specific immunoabsorption with filarial antigen. The levels of blocking antibody in the sera of these patients were high, comparable to those reported for atopic patients on immunotherapy regimens. These findings demonstrate that IgG blocking antibodies directed against parasite allergens are a regular component of the immune response to chronic filarial infection and suggest their potential role in vivo for specifically modulating allergic responsiveness to parasite antigens.

Immunoregulation of the proliferative response of lymphocytes by soluble E receptors

The role played by soluble human T-lymphocyte receptors for sheep erythrocytes (E) upon the proliferative response of lymphocytes was investigated. Preparations containing soluble E receptors, e.g., normal human serum dialysate (NHSD) and leukocyte dialysate containing transfer factor (TF) were added to cultures performed in the presence of normal human serum. In most of the cultures stimulated by allogeneic cells or by phytohemagglutinin (PHA), the addition of these preparations induced inhibition of the [ 3H]thymidine uptake. Previous absorption with E abolished or diminished significantly this inhibitory effect. Addition of NHSD absorbed with E restored the proliferative response in mixed leukocyte cultures performed in medium containing dialyzed normal human serum instead of NHS. The immunoregulation exerted by soluble E receptors upon the proliferative response of human lymphocytes seems to be more clearly expressed in the response to allogeneic cells than in the response to PHA.

Influx of thyroid hormones into rat liver in vivo. Differential availability of thyroxine and triiodothyronine bound by plasma proteins.

The transport of [(125)I]thyroxine (T(4)) and [(125)I]triiodothyronine (T(3)) into liver was investigated with a tissue sampling-portal vein injection technique in the anesthetized rat. The method allows the investigation of the effects of plasma proteins in human serum on the unidirectional influx of T(4) or T(3) into liver cells. The percent extraction of unidirectional clearance of T(3) and T(4) was 77+/-2% and 43+/-2%, respectively, after portal injection of a bolus of Ringer's solution. Cell membrane transport of T(4) or T(3) was nonsaturable because 50-muM concentrations of unlabeled hormone had no effect on transport. The addition of bovine albumin in concentrations of 1, 5, or 10 g/100 ml bound >98% of T(4) or T(3) in vitro, but had no significant effect on T(3) or T(4) transport in vivo. Conversely, 10% rabbit antisera specific for T(3) or T(4), completely abolished the intracellular distribution of thyroid hormone into liver. In the presence of rat serum, which contains albumin and thyroid hormone binding pre-albumin (TBPA), 18 and 81% of total plasma T(4) and T(3), respectively, were available for transport in vivo. The fraction of hormone available for transport in the presence of normal human serum, which contains albumin, TBPA, and thyroid hormone binding globulin (TBG) was 11% for T(4) and 72% for T(3). The fraction of hormone transported into liver after injection of serum obtained from pregnant or birth control pilltreated volunteers was 4% for T(4) (but this was not significantly different from zero) and 54% for T(3). THESE DATA SUGGEST: (a) The mechanism by which T(4) and T(3) traverse the liver cell membrane is probably free diffusion. (b) Albumin-bound T(4) or T(3) is freely cleared by liver, approximately 50% of TBG-bound T(3) is transported, but little, if any, of TBPA-bound T(4) or TBG-bound T(4) is cleared by liver cells. (c) Although the albumin-bound fraction of T(4) greatly exceeds the free (dialyzable) moiety, the two fractions are both inversely related to the existing TBA or TBG level; therefore, in vitro measurements of free T(4) would be expected to accurately reflect what is available for transport in vivo. Conversely, TBG-bound T(3) is readily transported in vivo; therefore, it is proposed that in vitro measurements of free T(3) do not reliably predict the fraction of T(3) available for transport into liver in vivo.

Participation of normal human immunoglobulins M, G, and A in opsonophagocytosis and intracellular killing of Bacteroides fragilis and Bacteroides thetaiotaomicron by human polymorphonuclear leukocytes.

Restoration of the ability of hypogammaglobulinemic serum to support opsonophagocytosis and intracellular killing of Bacteroides fragilis and Bacteroides thetaiotaomicron by human polymorphonuclear leukocytes was achieved by supplementation with normal human immunoglobulin M, but not with normal human immunoglobulin G. Polymorphonuclear leukocyte bactericidal activity in the presence of immunoglobulin A-deficient human serum was equivalent to that observed in the presence of normal human serum.

Immunosuppressive effect of soluble E receptors in uremic serum

The effect of serum and urine from uremic patients upon the proliferative response of lymphocytes to phytohemagglutinin and to allogeneic cells was investigated. Addition of uremic serum dialysate, to cultures performed in the presence of normal human serum, promoted a significantly higher inhibition of blastogenesis, compared with the addition of normal serum dialysate. Uremic urine and its dialysate induced degrees of depression upon the lymphocytes proliferative response equivalent to those observed with normal urine and normal urine dialysate, respectively. Absorption with sheep erythrocytes (E) removed the inhibitory effect from uremic serum and diminished significantly the inhibition induced by uremic urine. We concluded that accumulation of soluble E receptors, in uremic serum, is the main cause of its immunodepressive properties.

Acquired heterophile antigens on the surface of human cell lines.

Chicken IgM antibodies raised against sheep erythrocytes (SE) reacted with a heterophile carbohydrate antigen designated as "HC". This antigen was visualized by SE rosette formation with antibody-sensitized and formaldehyde-fixed target cells. Cells from eight lymphoblastoid lines and ten lines of diverse histogenetic origin all expressed HC on their surface to a greater or lesser degree when grown in fetal calf serum (FCS). Lymphoblastoid cell lines, HSB2 and Namalva, lost most of their HC antigen after three cell divisions when grown in medium which contained normal human serum (NHS) instead of FCS. The acquired origin of HC was demonstrated by the conversion of HSB2-HC- into HSB2-HC+ cells after incubation in the presence of FCS or fetuin for 18 h at 37 degrees C. The acquisition of HC antigen depended on the concentration of fetuin as well as the time and temperature of incubation. The presence of NHS in the culture medium reduced the degree of HC incorporation. The level of HC expressed on the cell surface was reduced by treating HSB2 cells with sodium periodate but not by proteolytic enzymes. Competitive inhibition with hog blood group substance and sheep IgG suggested similar specificity of HC on target cells which had been sensitized with either fetuin or sheep IgM (Eur. J. Immunol. 1977.7:204). The avidity of chicken antibodies to HC in either of these systems was several orders of magnitude lower than the binding to SE.

The effect of normal human serum on proteoglycan synthesis

Embryonic chick cartilages were incubated with the radio-precursors 35S, [ 3H]glucosamine, [ 3H]serine in the absence and presence of normal human serum in order to elucidate the steps in proteoglycan synthesis that are stimulated by normal serum. In the presence of serum there was similar increase in the uptake rates of all three precursors into both the entire tissue and isolated glycosaminoglycan chains which was proportional to the concentration of the serum. The effect of serum, cycloheximide, and D-xylose added separately and in various combinations was studied by following the distribution of 35S-labelled macromolecules between tissue-bound and soluble fractions. The addition of serum alone led to a marked increaseof 35S uptake in the tissue-bound fraction, whereas cycloheximide inhibited 35S uptake into the tissue-bound fraction; however 35S uptake into soluble fractions was unchanged for both additions. The introduction of D-xyclose to the incubation medium, led to a marked incrase in the soluble 35S-labelled fraction with respect to the tissue-bound fraction, both with and without serum. The combination of cycloheximide and D-xyclose caused a further increase in the proportion of soluble/tissue-bound fractions. A comparison was made of the type of proteoglycan being synthesized in the presence of serum with that formed in its absence. Serum was found to stimulate proteoglycan with a higher protein content than that found in its absence. We conclude that serum stimulated the initial steps of proteoglycan synthesis either upon core-protein synthesis and/or its xylosylation, forming a slow diffusible product which is rapidly integrated into the tissue matrix.

Cellular cholesterol ester accumulation induced by free cholesterol-rich lipid dispersions.

The influence of lipoprotein composition on free cholesterol (RC) esterification and accumulation of esterified cholesterol (EC) was studied in cells of Fu5AH rat hepatoma exposed to culture media supplemented with (FC/P) ranging from 0.6 to 2.8. In the presence of normal human serum, FC-lecithin dispersions witb a FC/P greater than one stimulated both the esterification of FC and the accumulation of cellular EC. Conversely, dispersions with FC/P values of approximately one had no effect on either FC esterification or the accumulation of EC when compared to cells grown in the absence of lipid dispersions. No stimulation of cellular response was obtained when FC-rich dispersions were added to cells in the absence of serum; however, stimulation was observed when cells were exposed to isolated human serum lipoproteins (very low density, low density, and high density). With each lipoprotein, FC-lecithin dispersions with FC/P values less than one inhibited cholesteryl ester accumulation, FC-rich dispersions stimulated, and dispersions with FC/P values of approximately one had an effect equivalent to that of isolated lipproteins alone. These studies extablish that cellular FC esterification and EC content are not influenced solely by the FC concentration of medium, but rather respond to the ratio of FC to phospholipidl These studies also suggest that serum lipoproteins participate in the transfer of FC carried by FC-rich lecithin dispersions to cells.

Effect of normal and activated human macrophages on Toxoplasma gondii.

Human macrophages derived from in vitro culture of peripheral blood monocytes were studied under a variety of conditions to determine their microbicidal capacity for the obligate intracellular protozoan, Toxoplasma gondii. The effect of macrophages on intracellular Toxoplasma was evaluated morphologically by light and phase microscopy and by autoradiography. When macrophages from dye test (DT)-negative or DT-positive individuals were infected with Toxoplasma in the presence of normal human serum, the organisms were able to multiply intracellularly with resultant destruction of the monolayer. Once organisms were intracellular, the presence of antibody-containing serum in the medium did not alter this inability of the macrophages to kill Toxoplasma. However, when Toxoplasma were incubated in the presence of heat-inactivated DT-positive serum just before infection of the monolayers, the intracellular organisms were inhibited or killed by normal macrophages. Attempts were made to activate macrophages in vitro to kill Toxoplasma. Macrophages incubated in the presence of sensitized lymphocytes and Streptokinase-Streptodornase (SK-SD) or Toxoplasma lysate antigen (TLA) were found to kill Toxoplasma when compared to macrophages incubated in the presence of lymphocytes from DT-negative individuals and TLA or lymphocytes alone. Thus, in vitro induction of resistance (both specifically and nonspecifically) in human macrophages was accomplished by culturing these cells in the presence of specifically sensitized lymphocytes and antigen. These results suggest that, as in the mouse model, activated human macrophages have the ability to inhibit or kill intracellular Toxoplasma and that these cells may be important as effector cells in cell-mediated immunity (CMI) to toxoplasmosis in man.

The Use of Sensitized Latex in the Identification of Human Bloodstains

The use of latex particles as a means of establishing the origin of unknown bloodstains is described. The particles are sensitized with anti-human serum protein immunoglobulins ; when used as a rapid slide test they agglutinate readily in the presence of normal human serum diluted 1/10,000.

DANGEROUS UNIVERSAL DONORS: II. Further Observations on In Vivo and In Vitro Behavior of Isoantibodies of Immune Type Present in Group O Blood

1. Severe hemolytic reactions were observed in 3 group A (subgroup A1) recipients transfused with group O whole blood or plasma. In one case, 10 ml. of a commerical preparation of soluble A and B factors had been added to 500 ml. of whole blood prior to the transfusion and it is believed that the reaction might have been even more serious had not this material been added.2. The anti-A antibodies in the serum of the dangerous universal donors causing the hemolytic reactions fixed complement, acted as hemolysins, were difficult to neutralize with soluble A and B factors, were capable of giving positive Coombs tests and their ability to agglutinate A cells was enhanced by the presence of normal human serum. These characteristics were similar to those observed in serum from donors known to be actively immunized against the A factor, but the stimulus for development of “immune” anti-A antibodies in the dangerous group O donors was not apparent.3. Small amounts of immune A antibody were consistently demonstrated in 12 of 100 random group O sera which, after neutralization, produced indirect Coombs tests with A1 cells and agglutinated A1 cells suspended in compatible normal human serum.4. Screening procedures for elimination of dangerous group O donors are discussed.

Studies on the blocking antibody in serum of ragweed-treated patients

Abstract 1.1. Our experiments showed that the serum of a ragweed-treated patient contained a thermostable substance which, when mixed with reaginic sera, increased its capacity to neutralize ragweed antigen beyond the titer at which it was capable of neutralizing it in the presence of normal human serum or physiologic saline. 2.2. Convalescent pneumonia, searlet fever, and measles sera were not similarly capable of inhibiting the antigen-reagin reaction and thus increasing the neutralization titer. 3.3. Convalescent measles and scarlet fever sera did not appear to be capable of inhibiting the whealing phenomenon produced by ragweed antigen in ragweed-sensitive skins.

Potentiating Influence of Urine on Sulfanilamide's Bacteriostatic Effect on E. coli in vitro.

In a previous publication1 one of us (Mellon) reported a poten-tiative effect of sulfanilamide on hemolytic streptococci when the organisms had been exposed to physiological salt solution during the process of dilution preliminary to seeding the test cultures. If this exposure was omitted and dilution in broth substituted, the remarkable bacteriostatic effect of sulfanilamide in low concentrations was not noted. In the presence of normal human serum the effect is enhanced. In other words, a sterilizing effect is obtained from the combined action of these minimal factors which exceeds many times a simple summation effect.Kenny, Johnston, and von Haebler2 have recently reported a very favorable series of clinical cases of E. coli infection of the urinary tract which were successfully treated with sulfanilamide. They showed that with oral medication of 1.5 gm. a day for 5-7 days sterilization of the urine was obtained during the period of treatment in all of 46 cases of infection with a typical E. coli. The ...

The Opsonic Index in Diphtheria

According to Wright1 diphtheria bacilli are insensible to the opsonic action of blood fluids. This conclusion is based upon experiments showing that there is as much (or, in one case, more) phagocytosis in the presence of normal human serum heated at 600 C. as in unheated. In order to study this question four strains of typical diphtheria bacilli furnished me by Dr. Hamilton were tested. With all of these strains there was no opsonin demonstrable in normal human serum after heating the serum 30 minutes at 500 C. This was found to be true also of serum from diphtheria patients when tested with strain N, the organism employed in the subsequent experiments. Re que2 also found that when washed leucocytes are mixed with normal human or dog serum heated to 58-600 for 15-30 minutes, there 1s practically no phagocytosis with diphtheria bacilli. It is possible that the strain which Wright used was subject to the spontaneous phagocytosis which has been observed to occur with occasional strains of diphtheria bacilli. As the course of the specific opsonic index in certain acute infections has been found to possess characteristic features that harmonize well with the general clinical picture, rising above normal as the symptoms decline, it was thought it would be of interest to determine if this were also true in regard to diphtheria. Both the diphtheriaand the streptococco-opsonic indices were estimated on account of the close association of streptococci with tonsilar infections. The Wright method of estimating the opsonic index has been employed for the most part. The suspensions are made from 24,