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Biomedical subjects

A Fomsgaard

Publications and source records attributed to A Fomsgaard.

At least 73 records · Page 4Linked to original sources
PubMed1990

Molecular clones of SIVsm and SIVagm: experimental infection of macaques and African green monkeys.

We have investigated the ability of biologically-active proviral molecular clones of SIVsm and SIVagm to infect rhesus macaques, pig-tail macaques, and African green monkeys. Two clones of SIVsm were individually inoculated into four rhesus and four pig-tail macaques. All eight macaques became infected, and two have experienced a significant decline in absolute numbers of circulating CD4+ cells. None of three African green monkeys were infected by an SIVsm molecular clone. However, one of four African green monkeys did become infected by SIVsm after receiving lymphocytes directly from an SIVsm-infected rhesus macaque. A molecular clone of SIVagm infected three of four macaques and three of three African green monkeys. None of the three infected macaques had a significant decline in circulating CD4+ cells. Interestingly, infection of pig-tail macaques (but not rhesus macaques) with uncloned SIVagm induced a significant drop in circulating CD4+ cells. These data suggest that molecular clones of SIVsm and SIVagm can be used in experimental models of AIDS for the evaluation of viral gene functions and for the study of in vivo genetic variation.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Molecular characterization of simian lentiviruses from east African green monkeys.

Asymptomatic infection with simian lentiviruses (also called simian immunodeficiency viruses, or SIV) is common among feral African green monkeys. To characterize the range of SIV genetic diversity among infected African green monkeys, we have determined nucleotide sequences from complete or partial molecular clones of four distinct SIVagm isolates from Kenya and Ethiopia. The nucleotide and amino acid variability we observed among the SIVagm isolates was greater than the variability within any other group of primate lentiviruses. These data suggest that: a) African green monkeys have been infected with simian lentiviruses for many years; and b) novel and uncharacterized primate lentiviruses may exist in the feral African green monkey population in other parts of Africa.

Amino Acid Sequence↗
PubMed1989

Determination of the components of immune complexes made in vitro with antigens derived from Pseudomonas aeruginosa.

A method for identification of the components of immune complexes would increase our understanding of the pathogenesis of chronic diseases such as Pseudomonas aeruginosa lung infection in cystic fibrosis. Capillary tube, gel diffusion and turbidimetric methods of determining immune complex formation were investigated with antigens from P. aeruginosa and the homologous rabbit antisera. Visible complexes were formed in the first two methods with flagella antigens. Purified lipopolysaccharide from P. aeruginosa would not form visible precipitates and a rapid and economical turbidimetric method was developed with 96-well microtiter plates. Larger quantities of immune complexes were formed in vitro with antigen/antibody ratios determined by the above methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (Western blotting) were evaluated and found to be useful in determining the antigen and antibody components of these immune complexes.

Animals↗
PubMed1989

Immunochemical and biological reactivity of human anti-lipopolysaccharide IgG obtained by screening of blood donors.

An anti-lipopolysaccharide (LPS) preparation for the intravenous treatment of septic endotoxic shock was prepared by purifying immunoglobulin G (IgG) from pooled serum from Danish blood donors. The sera were selected by the use of an enzyme-linked immunosorbent assay (ELISA) to screen blood donors for high concentrations of antibodies to a mixture of LPS from 11 different Gram-negative bacteria. ELISA was also used for indirect quantification of IgG antibodies to lipid A, and to rough LPS from Escherichia coli Ra and Salmonella minnesota R60 (Ra). The concentration of human antibodies to the LPS mixture correlated with the concentration of antibodies to the E. coli and S. minnesota rough LPS and to lipid A. The specificity of sera with high concentrations of anti-LPS IgG was investigated by immunoblotting. Sera from individual donors reacted with LPS from different bacteria and recognized different sites on the LPS molecules. The range of specificities to different LPS was increased by the pooling of selected sera. The IgG fraction from the high titre donor pool neutralized biological activities of LPS such as activation of the Limulus amoebocyte lysate reaction and induction of tumour necrosis factor secretion from human monocytes.

Absorption↗
PubMed1989

IgG autoantibodies against interleukin 1 alpha in sera of normal individuals.

A pool of human sera from healthy blood donors was found to interfere competitively with the binding of 125I-labelled human recombinant interleukin 1 alpha (rIL-1 alpha) to the murine T-cell line EL4. The interference was reversible at the cellular level, and direct binding of the ligand to serum factors was therefore investigated. After preincubation of [125I]rIL-1 alpha with pooled serum, the 125I activity eluted in two peaks from a Sephadex G-75 column. The first was located in the void volume. The second eluted together with monomer rIL-1 alpha. An almost complete displacement of the high molecular weight 125I fraction was achieved with an excess of unlabelled rIL-1 alpha but not with rIL-1 beta. The serum factors binding to [125I]rIL-1 alpha were located in the molecular weight range 100,000-200,000, judged by fractionation on a Sephacryl S-400 column, and the factors were bound to immobilized protein A. Furthermore, [125I]rIL-1 alpha preincubated with serum co-precipitated with a specific rabbit anti-human IgG antibody. Screening of 29 sera from normal individuals showed similar effects in three cases. We conclude that approximately 10% of normal human sera contains detectable IgG autoantibodies to IL-1 alpha.

Adult↗
PubMed1989

Auto-antibodies to tumour necrosis factor alpha in healthy humans and patients with inflammatory diseases and gram-negative bacterial infections.

A semi-quantitative immunoblotting method was developed to screen for serum auto-antibodies against tumour necrosis factor alpha (TNF alpha). Forty nitrocellulose strips containing identical amounts of human recombinant TNF alpha (rTNF alpha) were prepared for each set-up, and the anti-TNF alpha antibody immunoreactivities were scored according to the density of the resulting colour reaction. A significant number of sera from apparently healthy donors contained detectable auto-antibodies to TNF alpha (40%), while the strongest reaction was observed in 8%. A higher prevalence of anti-TNF alpha antibodies was found in sera from patients with Gram-negative bacterial septicaemia (66%), cystic fibrosis with chronic Pseudomonas aeruginosa lung infection (72%), and various rheumatic diseases (61%). The antibodies in sera from these patients belonged primarily to the IgG and IgM classes, the latter exhibiting the strongest response. Longitudinally collected serum samples from patients in septic endotoxin shock revealed that the anti-TNF alpha antibodies were induced initially during septicaemia, reaching maximum reactivities within the first week and returning to low or undetectable levels on days 9-20.

Adolescent↗
PubMed1989

Protective properties of a human IgG preparation rich in antibodies to a wide spectrum of lipopolysaccharides.

Naturally occurring human IgG, rich in antibodies to different lipopolysaccharides was investigated for possible protective effects against lethal endotoxin shock and lethal gram-negative infection in mice. The IgG preparation was obtained from pooled serum of selected blood donors with high concentrations of antibodies to 11 different LPS as measured by ELISA. The human IgG (5 mg/mouse) protected C3H/TifF mice against an otherwise lethal infection with Salmonella typhimurium. The human IgG also inhibited the lethality induced by purified LPS in D-galactosamine sensitized C57B1/6 mice. The protection was dependent on the IgG dose given. However, protection was not obtained against all the LPS preparations tested. Absorption of the IgG with different LPS, showed the protection to be caused by serotype-specific anti-LPS antibodies. Protection against a given LPS was not related directly to the corresponding anti-LPS titer as measured by ELISA and passive hemolysis. The interpretation of these results is discussed.

Animals↗
PubMed1989

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.

Antibodies, Bacterial↗
PubMed1989

Preliminary study on treatment of septic shock patients with antilipopolysaccharide IgG from blood donors.

A novel intravenous therapy consisting of polyvalent IgG antibodies to lipopolysaccharide (LPS, endotoxin) obtained from screening of blood donors was used for treatment of patients with profound septic endotoxin shock. Investigation of the anti-LPS IgG pharmacokinetics in the 10 patients revealed time related changes in the plasma concentrations of anti-LPS IgG, endotoxin, tumour necrosis factor (TNF) and the clinical parameters. A decrease in serum concentrations of IgG and IgM antibodies to LPS was observed prior to the immunotherapy as well as in a clinical example of lethal septicemia without anti-LPS immunotherapy. Increasing serum concentrations of anti-LPS IgG during antibody infusion was followed by a decrease in the concentration of endotoxin and TNF. In survivors an IgM and IgG anti-LPS antibody response developed. Using clinical parameters and APACHE II clinical severity scores to measure the clinical condition, a beneficial effect was observed within 24 h corresponding to a decrease in the calculated expected mortality rate from more than 80% to about 50%. Five patients (55%) expired during the study. One patient died in the early septic shock phase. One patient expired due to superimposed hemorrhagic shock. Three immunosuppressed patients died 1-2 weeks after initial recovery, 1 with fungal sepsis and 2 patients due to pseudomonas infection.

Adult↗
PubMed1988

Association between HLA-DR2 and production of tumour necrosis factor alpha and interleukin 1 by mononuclear cells activated by lipopolysaccharide.

The production of tumour necrosis factor (TNF) and interleukin 1 (IL-1) by lipopolysaccharide-activated mononuclear cells from 39 healthy donors was studied in vitro by bioassay and ELISA. The donors were typed for HLA-A, -B, -C, -DR, and -DP antigens. There was no detectable production of TNF beta (lymphotoxin). The intracellular levels of bioactive TNF alpha were minimal or undetectable in all cases. Cells from HLA-DR2+ individuals secreted significantly lower amounts of TNF alpha than cells from HLA-DR2- donors [2 ng/ml (1.5-4.4) and 7.5 ng/ml (3.9-8.3) respectively (medians 25-75%); P less than 0.01]. The difference disappeared if the cells were preactivated for 2 days with 1000 U/ml of recombinant gamma interferon (rIFN-gamma). In some individuals, the TNF alpha response increased considerably after IFN-gamma priming, in particular in those possessing the HLA-DR2 antigen. In contrast, there was no detectable difference in the production of IL-1 beta between the donors, and the IL-1 beta response decreased significantly after rIFN-gamma priming in HLA-DR2+ individuals [2.3 ng/ml (1.1-8.4) versus 7.2 ng/ml (5-7.9); P less than 0.05] and in HLA-DR2- individuals [3 ng/ml (1.1-5.3) versus 5.7 ng/ml (3.9-7.5); P less than 0.01]. There was no correlation between the TNF alpha and IL-1 responses and any of the other HLA-DR, -DP, or -B antigens. There was a significant positive correlation between the levels of TNF alpha measured by ELISA and by cytotoxicity assay. However, the TNF alpha-containing supernatants from 9 out of 37 individuals appeared to contain inhibitor(s) of the biological activity of TNF alpha. The presence of inhibitor(s) was not associated with any HLA antigens.

Adult↗
PubMed1988

Detection of tumour necrosis factor from lipopolysaccharide-stimulated human mononuclear cells by enzyme-linked immunosorbent assay and cytotoxicity bioassay.

Tumour necrosis factor (TNF) or cachectin is an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF (rTNF)/100 microliter. There was no interference of medium, serum, plasma, spinal fluid, or urine and no cross-reaction with natural or recombinant IL-1-alpha, IL-1-beta, IL-2, IFN-gamma, or lymphotoxin (TNF-beta). Recovery of TNF added to the media was 85-123% (n = 22). The relative standard deviations within and between assays were 7% and 8%, respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30 microliter or 12.5 pg/30 microliter of rTNF. IL-1-alpha and IL-1-beta slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50-300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100-1000 ng/ml). In contrast, using 0.01-100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine. Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.

Cytotoxicity Tests, Immunologic↗
PubMed1988

Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Antibodies to Pseudomonas aeruginosa from 10 cystic fibrosis patients with chronic P. aeruginosa lung infections were quantitatively and qualitatively analyzed. The development of specific antibodies in patient serum was evaluated in a longitudinal study (1972 to 1987). The concentrations and specificities of immunoglobulin G (IgG) and IgM antibodies to purified lipopolysaccharides (LPS) from clinical isolates of P. aeruginosa and to a variety of other gram-negative bacteria were studied by immunoblotting and enzyme-linked immunosorbent assay techniques. Results were compared with the number of immunoprecipitates to P. aeruginosa whole-cell extracts detected by crossed immunoelectrophoresis. IgG, but not IgM, anti-Pseudomonas LPS concentrations increased significantly at the onset of chronic infection and continued to increase during the course of the infection. There was a good positive correlation between the concentration of IgG anti-Pseudomonas LPS antibodies and the number of crossed-immunoelectrophoresis precipitins. The increases in IgG anti-LPS antibody concentrations were much higher to Pseudomonas LPS than to other LPSs. Binding studies demonstrated an increase in binding of IgG anti-Pseudomonas LPS during infection, whereas the binding of other anti-LPS antibodies decreased. Immunoblotting studies confirmed that antibodies reacted strongly with Pseudomonas LPS and weakly with Escherichia coli core-lipid A. The specificity of the reaction with Pseudomonas LPS increased with the duration of infection. It is concluded that anti-LPS response in cystic fibrosis patients during chronic P. aeruginosa infection demonstrates a marked increase in IgG anti-Pseudomonas LPS antibody concentration, specificity, and affinity. The anti-LPS enzyme-linked immunosorbent assay is proposed as a routine test to diagnose and to follow the course of chronic P. aeruginosa lung infection in patients with cystic fibrosis.

Adolescent↗
PubMed1988

Comparative immunochemistry of lipopolysaccharides from typable and polyagglutinable Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis.

Lipopolysaccharide (LPS) was extracted and purified from three Pseudomonas aeruginosa strains isolated from the infected lungs of patients with cystic fibrosis. Two of the strains could be typed by O-specific antibody (O:3 and O:9), and the third was polyagglutinable (O:3/9). The separated LPS was characterized by chemical and serological methods. The main neutral sugar constituents (glucose, rhamnose, and heptose) were found in various proportions in the three strains, whereas the amounts of glucosamine, galactosamine, ketodeoxyoctonate, and phosphate were more constant. Ester-bound C12, C16, 3-OH-C10, and 2-OH-C12, together with amide-bound 3-OH-C12, fatty acids were present in equimolar proportions in all three strains. Considerable amounts of LPS were liberated in the culture supernatant of the O:3 bacteria but not in those from the other two strains. This free LPS was shown to be immunologically identical to the cell-bound LPS and the extracted LPS. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, O:3 and O:9 LPS showed a ladder pattern characteristic of smooth LPS, while O:3/9 LPS appeared rough. Rabbit antisera used for O-typing were found by enzyme-linked immunosorbent assay to contain anti-LPS antibodies that reacted strongly with homologous LPS, moderately with O:3/9 LPS, and slightly with heterologous LPS. Immunoblotting showed that common antigenic determinants in the core-lipid A part were involved in the observed cross-reaction. The polyagglutinability of P. aeruginosa may be explained by the antibodies to these common determinants that arose from the partial absence of O polysaccharides.

Antigens, Bacterial↗
PubMed1987

ELISA for human IgG and IgM anti-lipopolysaccharide antibodies with indirect standardization.

A new attempt to standardize ELISAs for the quantitation of human IgM and IgG anti-lipopolysaccharide antibodies (anti-LPS), without the use of a specific standard material, is described. Sandwich ELISAs for total IgG and IgM were combined with indirect ELISAs for anti-LPS IgG and IgM antibodies on a 96-well microtest plate using identical assay conditions. The concentration of specific IgG or IgM anti-LPS was read on the respective standard curve for total IgG or IgM. The results were corrected for residual immunoreactivity remaining unbound in the wells after one sample incubation in the combined assays. The quantitative results of IgG anti-LPS correlated well with results obtained using an ELISA with direct standardization (r = 0.969). 28 mg/l of IgM anti-LPS was found as median value among 121 blood donors using the described ELISA principle. Binding studies demonstrated a lower apparent affinity of donor anti-LPS IgM than anti-IgG.

Enzyme-Linked Immunosorbent Assay↗