Normal Pooled Human Serum Research Articles

Effect of iron on cytolysin a expression in Salmonella enterica serovar Typhi

Previously, a novel protein ClyA (Cytolysin A) has been identified in Escherichia coli K-12, Salmonella enterica serovars Typhi and Paratyphi A and Shigella. Salmonella spp. synthesize substantial amounts of ClyA upon infection of the human host, although the mechanism by which ClyA is induced in vivo is unclear. Since environmental signals could control the expression of virulence determinants, ClyA expression in S. Typhi Ty2 was tested by Western blotting in the presence of normal pooled human serum (NPS). The level of ClyA expression increased in the presence of NPS in a concentration-dependent manner. RPMI 1640 medium similarly induced ClyA expression. ClyA expression was inversely proportional to the concentration of iron in RPMI medium. Therefore, we speculated that iron inhibited the expression of ClyA in S. Typhi Ty2, and free iron depletion may be one of the causes of S. Typhi-mediated induction of ClyA in vivo. Transcription from a clyA-lacZ fusion gene decreased as iron concentration increased, but not as significantly as the ClyA protein expression. It is concluded that the regulatory effect of iron on clyA expression is mainly at translational level.

Studies of the Inhibition of Serum Pseudocholinesterase Activity In Vitro by Commonly Used Drugs

We studied the effect of 17 commonly used drugs, including prescription and over-the-counter medications, on the activity of serum pseudocholinesterase (PCE) in vitro. Normal pooled human serum was incubated for 120 minutes at 37 degrees C with therapeutic serum concentrations of prescription and over-the-counter drugs, and the postincubation PCE activity was measured. Morphine, quinidine, and thioridazine depressed PCE activity by more than 5% while no or negligible effect was noted following incubation with acetaminophen, chlordiazepoxide, chlorpromazine, desipramine, doxepin, imipramine, methamphetamine, nortriptyline, phenobarbital, phenytoin, procainamide, salicylic acid, theophylline, and valproic acid. Depression of PCE activity can prolong the half-life of coadministered agents with metabolism mediated by PCE.

Production of Vibrio vulnificus hemolysin in vivo and its pathogenic significance

Hemolyin/cytolysin (VvhA) is one of the representative exotoxins produced by Vibrio vulnificus. Cytotoxic mechanism of VvhA has been extensively studied. However, there have been controversies concerning the pathogenic significance since vvhA − mutant showed no LD 50 change in mice. In this study, we investigated whether VvhA is expressed in vivo. When V. vulnificus was cultured in the presence of normal pooled human serum, substantial amount of VvhA was detected by ELISA and the transcription of vvhA was also evidently confirmed by RT-PCR and a transcriptional reporter assay. To investigate whether VvhA is expressed in vivo, mice were infected with V. vulnificus and bacterial RNAs were harvested from the mice. In vivo vvhA transcription was observed evidently by RT-PCR. We hereby propose that VvhA is substantially produced in vivo and would contribute to the pathogenesis of V. vulnificus septicemia.

Impaired polymorphonuclear neutrophil function in end-stage renal failure and its correction by continuous ambulatory peritoneal dialysis.

The polymorphonuclear neutrophil (PMN) function of 22 patients with end-stage renal failure (ESRF) was studied immediately before and 3 months after starting continuous ambulatory peritoneal dialysis (CAPD) and compared with a control group of healthy normal volunteers. The PMN functions studied were phagocytosis and killing of Staphylococcus epidermidis and respiratory burst activity. The results show that in the presence of normal pooled human serum PMN from patients with ESRF before CAPD treatment phagocytose bacteria normally, but have impaired killing. Before treatment, the PMN from patients with ESRF also showed an increase in both unstimulated and stimulated superoxide anion production. Abnormal PMN function was corrected by CAPD treatment, suggesting the involvement of a dialyzable toxin.

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm2) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm2, respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm2). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm2 at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.

Regulation of PGI 2 activity by serum proteins: serum albumin but not high density lipoprotein is the PGI 2 binding and stabilizing protein in human blood

Although previous studies have shown that serum albumin binds PGI 2 and protects it from rapid degradation, it remains debatable whether it is physiologically important due to its low binding affinity for PGI 2. We were intrigued by the observations of Yui et al. (J. Clin. Invest. 82 (1988) 803–807) which suggested that apo A-I of the high density lipoprotein (HDL) is the “serum PGI 2 stabilizing factor”. To clarify this, we carried out experiments to determine the binding kinetics and parameters of HDL and albumin purified from normal pooled human serum. Despite the use of multiple binding assays, we could not detect any binding activity in HDL 2, HDL 3 or nascent HDL preparations, nor could we demonstrate any PGI 2 protecting activity by these molecules. By contrast, purified albumin exhibited essentially identical binding parameters as the native serum from which the albumin was purified. The binding activity of various albumin preparations was not due to the contamination of apo A-I. Computer simulation analysis also failed to provide evidence to support the notion that HDL bound and prolonged PGI 2 activity. To determine whether physiological concentrations of albumin influence PGI 2 binding to platelet receptors, we measured PGI 2 binding to platelet membrane in the absence and presence of albumin. Albumin at 40 mg/ml increased the K D of PGI 2 binding to the receptors by 2–3-fold. These findings indicate that albumin plays a major role in protecting PGI 2 activity and regulating its availability for platelet PGI 2 receptors.

Serum Factor Responsible for Thymidine Uptake by Lymphocytes Is Independent of Somatomedin-C

ABSTRACT Lymphocytes have specific receptors for somatomedin-C (Sm-C) and GH. To test whether these cells are sensitive to the growth-promoting properties of these growth factors, we studied serum (with low or normal Sm-C and GH levels) from children having various statural disorders by means of the lymphocyte proliferation assay. Peripheral lymphocytes from each subject were cultured in media containing either autologous serum (AS) or normal pooled human serum (HS). The mitogenic response (MR) was assessed by determining the amount of tritiated thymidine incorporated into cellular DNA. The MR was studied both in phytohemagglutinin-stimulated and in unstimulated lymphocytes. MR was expressed as thymidine uptake (CPM) in the presence of AS divided by CPM in the presence of HS. The MR in serum from (1) 27 control subjects was 1.01 ± 0.03 (SEM); (2) 14 children with short stature and normal serum GH-RIA and Sm-C levels was 0.98 ± 0.04; and (3) 12 children with GH deficiency showed two widely different responses. In four patients (subgroup 1), the MR was 0.05 ± 0.02 and in eight (subgroup 2) the MR was 1.11 ± 0.06. Eleven patients were studied after GH therapy (three from subgroup 1), and the MR was 1.12 ± 0.03. Neither Sm-C levels nor linear growth rate correlated well with the growth-promoting property of the serum when tested on lymphocytes. In seven additional patients with GHD, the MR when studied at varying concentrations of serum (0.1–5.0%), showed results identical to subgroup 2. Addition of purified Sm-C to cultures did not enhance the MR of serum from GHD patients prior to GH therapy. GH-dependent factors other than Sm-C may have a role in in vitro lymphocyte proliferation; in its current form, however, this bioassay is not sensitive enough to be useful in the diagnosis of children with GH deficiency.

The role of surface polysaccharide in determining the resistance of Serratia marcescens to serum killing.

Two O14:H12 strains of Serratia marcescens with different sensitivities to killing by normal pooled human serum were investigated. Complement binding, studied by measuring hydrophobicity and using rocket immunoelectrophoresis with anti-human C3, showed the sensitive cells (S1220) rapidly bound and fixed complement whereas the resistant cells (4444-60) bound less C3b. The strains had identical membrane protein composition. Crossed immunoelectrophoresis suggested that in S1220 cells the polysaccharide material including LPS was less antigenic and present in smaller amounts than in 4444-60 cells. This was confirmed by examining extracted polysaccharide material chemically and by SDS-PAGE. The resistant strain had 33% more phenol-extractable polysaccharide material than the sensitive strain, possibly comprising LPS with longer O antigen chain lengths, or a microcapsule of O antigen polysaccharide. Extra polysaccharide material on the surface of the resistant strain prevents complement components binding and reaching the hydrophobic membrane where lytic lesions occur.

In vitro effects of hyperlipoproteinemic sera on human endothelium: Inhibition of endothelial cell migration by familial hypercholesterolemic sera

Since hyperlipoproteinemia is associated with an increased risk of atherosclerosis we have evaluated the effects of sera of different hyperlipoproteinemic clinical patterns on human endothelial cells in vitro. Cultured human umbilical vein endothelial cells were treated with sera from 2 patients homozygous for familial hypercholesterolemia and 4 patients heterozygous for that disorder. Familial hypercholesterolemic sera inhibited endothelial cell migration by 50% during a 72 hour incubation (p <0.0001) compared to normal pooled human serum or single donor AB serum when measured by an agarose gel technique. The inhibition of migration was not observed when cells were treated with familial combined hyperlipidemic sera (4 patients) or familial hypertriglyceridemic sera (5 patients). Endothelial cell detachment in vitro was not induced by any of the classical patterns of hyperlipoproteinemic sera tested. The development of atherosclerosis in familial hypercholesterolemia may be in part related to an impairment of endothelial repair.

Cryoproteinemia: An immunologic phenomenon?: Electrophoretic analysis of serum proteins of a patient with cold allergy

1. 1. The electrophoretic distribution of the serum proteins in cryoproteinemia prior to and following separation of the cryoprecipitate, and after the administration of cortisone, is analyzed and compared to normal pooled human serum. 2. 2. The electrophoretic curve of “purified” cryoprotein in saline solution is illustrated. Cryoprotein, in the case described in this study, is represented electrophoretically by a “T” component which migrates between the beta and gamma globulins. 3. 3. This component was absent after treatment with cortisone for six days. Concomitantly, no cryoprotein was obtained from the serum and the patient no longer reacted abnormally to cold. 4. 4. The similarities between cryoprotein and immune bodies are discussed. This study seems to illustrate the suppressive effects of an adrenocortical hormone on serum globulins and on a “T” component with immune body properties.