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Notes: 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. Sites 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.

Notes: The particular susceptibility to insulin-dependent diabetes mellitus (IDDM) conferred by HLA-DR3,4 heterozygosity has been suggested to be an effect of transcomplementation of HLA class II molecules. To test this hypothesis of special IDDM-specific hybrid determinants and to evaluate the T-cell repertoire towards a specific antigen in IDDM patients we generated a total of 352 PPD-specific T-cell lines by the soft-agar cloning technique and studied their restriction by HLA class II molecules. Of these lines, 227 were from nine IDDM patients, of whom six were DR3,4 heterozygotes, and 125 from 10 healthy controls Forty-six T-cell lines elicited specific responses in at least two experiments and in addition to T-cell lines demonstration class-II-restricted PPD specificity, lines with an alloreactivity occurred. HLA-DQ-restricted PPD-specific T-cell line were not identified and a possible DP restriction (DPw2) was only observed with one line. These data indicate that PPD is preferentially presented to T cells in the context of HLA-DR/Dw, Presentation of PPD by hybrid molecules in IDDM patients or by IDDM-specific class II epitopes recognized by the T-cell lines was not demonstrated. By restriction fragment length polymorphism analysis using a probe for the joining region of the T-cell receptor gamma gene, T-cell lines generated by the soft-agar cloning technique were found to be oligoclonal. It is concluded that soft-agar cloning should be followed by subsequent limiting dilution in order to assure monoclonality. Different preparations of antigen-presenting cells (APC) were tested. In several cases the T-cell lines were not able to respond to PPD presenting by Epstein -Barr-virus transformed lymphoblastoid cell lines (LCL). It was demonstrated that lipopolysaccharides (LPS) of E. coli potently reduce the proliferative response of antigen-specific and alloreactive T cells when T-cell-depleted peripheral blood mononuclear cells (E− cells) were used as APC, whereas only limited inhibition was observed when LCL were used as APC in the presence of LPS. This effect of LPS is suggested to be mediated by increased prostaglandin secretion by monocytes among the E− cells since indomethacin abolished the effect of LPS. This observation may have implications for T cell cloning procedures since we have found that most commercially available culture media are heavily contaminated with endotoxin.

Notes: HLA-class III region genes may be associated with susceptibility to insulin-dependent diabetes mellitus (IDDM). In this study an Ncol polymorphism of the tumour necrosis factor beta (TNF-β) gene, which is positioned next lo the tumour necrosis factor alpha (TNF-α) gene in the HLA class 111 region, was detected by restriction fragment length polymorphism (RFLP). This polymorphism has previously been reported lo be located in the TNF-α gene. Caucasian HLA-DR3.4 heterozygous IDDM patients (n=-26) and DR-matched healthy controls (n=19). as well as randomly selected IDDM patients (n = 27) and controls (n = 25) were studied. In addition four multiplex families (49 individuals) and eight HLA-non-identical sibpairs concordant for IDDM were analysed.The TNF-β gene RFLP analysis showed fragments of 5.5 kb and 10.5 kb, which behaved as alleles. In all groups there was a haplotype assignment of the TNF-β 5.5-kb allele to BS, DR3 haplotypes, and of the TNF-β 10.5-kb allele to B15.DR4-positive haplotypes. The allelic and genotypic frequencies differed between DR3.4 IDDM patients and DR3,4 controls, and the DR3.4 control group differed significantly from the randomly selected control group (P 〈 0.0079), In HLA-DR3,4-atid DQw8-positive persons, the DR3 haplotypes carried the 10.5-kb allele ihrcL- times more frequently in IDDM patients than in controls, suggesting that the 10.5-kb allele when present on DR3 haplotyes may contribute lo susceplibility to IDDM in DR3.4 heterozygous individuals, A contributory role of the Hl.5-kballele in genetic IDDM susceptibility was supported by the sibpair analysis, in which all were TNF-/1 identical. Five were 10,5 kb homozygous. and the remaining three pairs were 5.5.10,5 kb heterozygous.Twenty-five healthy and eight newly diagnosed IDDM patients were randomly selected to study the Escherichia coli lipopolysaccharides (LPS)-purified protein derivate (tuberculin) (PPD)-, and phytohaemagglutinin (PHA)-stimulated monocyte (Mo) secretions of interleukin 1 bela (IL-1/J)and TNb-α in relation lo the Ncol TNF-/f gene polymorphism. The LPS- and PHA stimulated Mo IL-l/f and TNF-a: secretions were significantly lower for the TNF-β 5.5.10,5 kb heterozygous individuals than for TNF-β 10.5 kb homozygous individuals. Furthermore, the Mo IL-1β and TNF-a secretions of IDDM patients were significantly higher than the Mo secretions of TNF-β genolype-matched healthy controls.This study suggests an association between the 10.5 kb TNF-β allele and IDDM, and demonstrates an association between monokine responses and TNF-β genotypes. These observations may have implications for understanding the pathogenesis of HLA-associated autoimmune diseases including IDDM.

Notes: Interleukin 1β(IL-1β) and tumour necrosis factor alpha (TNF-α) may be pathogenetically important in insulin-dependent diabetes mellitus (IDDM), which is associated with genes of the HLA region. Since a regulatory role of HLA region genes on monokine production may exist, we looked for an association between the monokine and prostaglandin E2 (PGE2) responses of monocytes (Mo) from 20 healthy males (18–50 years) with HLA-DR types relevant for IDDM susceptibility and resistance (DR 1,2, DR 1,3, DR 1,4, DR 3,4). Monokine assays were established and evaluated and the secretions of IL-1β, TNF-α, and PGE2 measured in Mo cultures (2 h, 6 h, 20 h) prepared by endotoxin-free techniques and stimulated by low-dose E. coli lipopolysaccharides (LPS). There were no significant associations between Mo responses and HLA-DR phenotype. Likewise, Mo from DR2 (n=5) and DR4 (n= 5) homozygous healthy males demonstrated no significant differences in monokine and PGE2 responses of Mo.In the HLA class III region a diallelic TNF-β gene Ncol polymorphism consisting of alleles of 5.5 kb and 10.5 kb was recently described and associated with susceptibility to autoimmune diseases including IDDM. We report that IL-1β and TNF-α responses of Mo from TNF-β 10.5 kb homozygous healthy individuals were significantly higher than for TNF-β 5.5/10.5 kb heterozygotes.IL-1β and TNF-α responses of Mo from males (18–35 years) with newly diagnosed (n= 10) and long-standing IDDM (n= 10) and from age- and HLA-DR-matched healthy males (n= 10) were studied. LPS, gamma interferon (IFN), and TNF-α-stimulated Mo cultures were investigated. No significant differences were found between Mo responses of IDDM patients and controls. IFN (1000 U/ml) in the presence of LPS significantly potentiated LPS-stimulated Mo TNF-α secretion and reduced the levels of IL-β immunoreactivity in Mo lysates. IFN and TNF-α did not have any effects on LPS-stimulated Mo secretion of IL-1 β immunoreactivity.We conclude that Mo IL-1β and TNF-α production is normal in patients with recent-onset and long-standing IDDM. The interindividual differences in monokine responses may be accounted for by the diallelic human TNF-β gene polymorphism rather than by HLA class II genes. This observation may be important for understanding the association of certain H LA haplotypes with autoimmune phenomena and disease.

Notes: The effects of dietary supplementation with ω-3-polyunsaturated fatty acids (ω-3-PUFA) on the proliferative response of PBMC and on the secretion of monokines and arachidonic acid metabolites from PBMC and monocytes (Mo) from healthy subjects and patients with recent-onset insulin-dependent diabetes mellitus (IDDM) were examined. Three groups of eight to nine healthy individuals were randomized to either 2.0 g/day or 4.0 g/day of ω-3-PUFA devoid of vitamins A and D, or an isocaloric amount of placebo. Furthermore, eight patients with recent-onset IDDM received 4.0 g/day of ω-3-PUFA. IL-lβ production and TNF-α secretion was determined before and after 7 weeks of treatment, and 10 weeks after withdrawal of treatment. Significant increases in platelet and PBMC membrane eicosapentaenoic acid was found in ω-3-PUFA-treated individuals. ω-3-PUFA treatment significantly reduced the content of IL-Ib in lysates of PBMC, but did not affect PBMC or Mo secretion of IL-1β, TNF-α or prostaglandin E2 (PGE2) or PBMC leukotriene B4 (LTB4) secretion in healthy subjects or in IDDM patients. A significant inhibition of the PHA-stimulated. but not the spontaneous or PPD-stimulated, proliferative response of PBMC was observed in healthy and diabetic subjects treated with (o-3-PUFA. No correlation was found between PHA-stimulated PBMC proliferation and PBMC secretion of TNF-α and IL-1β. There were no significant differences in the spontaneous or the PPD-or PHA-stimulated proliferative responses of PBMC between diabetic and healthy individuals at entry. We conclude that although dietary supplementation with 4.0 g/day of ω-3-PUFA inhibits the proliferation of PBMC and reduces IL-I immunoreactivity in PBMC and Mo, it does not alter monokine, PGE2 or LTB4, secretion in healthy or IDDM subjects.

Notes: The secretions of interleukin 1 (IL-1), tumour necrosis factor α (TNF), and prostaglandin E2 (PGE2) of low-dose E. coli lipopolysaccharide (LPSJ-stimulated human monocytes (Mø) were investigated in an endotoxin (ET)-free milieu (〈1.6 pg LPS/ml). Human Mø cultures from nine healthy men were stimulated with 0, 12.5–500, and 250,000 pg LPS/ml as measured by a very sensitive Limulus test. The IL-1 activity was tested by the mouse costimulatory thymocyte (LAF) assay, which was thoroughly standardized and characterized (interassay variation 22–24%, intra-assay variation 3–7%). Spontaneous Mø secretions of IL-1, TNF, and PGE2 were negligible, but 12.5 pg LPS/ml significantly stimulated the secretions of these Mø products and the monokine responses to 500 and 250,000 pg LPS/ml were almost in the same range. It was demonstrated that the secretions of IL-1-TNF and TNF-PGE2 were strongly correlated. Pronounced interindividual differences in LPS responsiveness were demonstrated, and two low-responders, one of whom was HLA-DR 1,2-positive, were identified. Three first-degree relatives of the DR1.2-positive low-responder had similar low responses. Furthermore. Mø cultures were prepared weekly for 4 weeks from four HLA-DR different men and the only DR2.2 homozygous individual had low monokine responses. In conclusion, stable interindividual differences in in vitro monokine and PGE2 secretions of LPS-stimulated Mø were demonstrated. It is suggested that HLA-DR2-positive individuals may be low responders.

Notes: The histamine-releasing capability of lipopolysaccharides (LPS) was examined in human leukocyte suspensions. LPS alone did not release histamine, but was found to enhance the histamine release caused by anti-IgE. Also the IgE-mediated histamine release caused by specific antigens (allergens or bacteria) in sensitized individuals was enhanced by LPS. The potentiating effect of LPS was observed in grass pollen and dog dander allergic patients as well as in patients sensitized to E. coli or Staph, aureus bacteria. No potentiation was obtained by exposure to unspecific allergens or bacteria to which the persons were not sensitized. Bacteria can release histamine by immunological or nonimmunological mechanisms, and only the immunological histamine release was found to be potentiated by LPS. It is speculated that endotoxins reinforce release of histamine caused by allergens in allergic patients or by bacteria in persons sensitized to these microorganisms.

Notes: Haemophilus influenza and its extracellular produets (EP) ditl not release histamine from basophil leukocytes in cell suspensions from normal individuals, patients with chronic bronchitis or patients allergic to either house dust mite, grass pollen, eat dander or to their own bacteria. However, the EP was found to enhance their basophil histamine release. IgE-mediated histamine release was examined by stimulation of the cells with anti-IgE or the specific allergens, and non-immunological histamine release by stimulating the cells with the calcium ionophore A23187 or Staphylococcus aureus. In all the experiments EP caused a significant increase in the histamine release. When H. influenzae endotoxins were removed from the EP, the potentiating effect of EP was completely abolished, whereas heating (80°C. 30 min) or treatment of EP with proteinase did not influence the potentiating effect. These results indicate that H. influenzae endotoxin potentiates histamine release caused by IgE-mediated reactions or by non-immunological mechanisms.

Notes: The in vitro study of monocytes (Mo) poses several problems. Minor contamination with endotoxin (ET) of media ami utensils as well as adherence to glass or plastic surfaces may activate the cells and cause pronounced production of monokines. Many commercially liquid culture media were found to contain ET in concentrations above 25x10−12 g/ml. A simple system for the removal of ET from media and solutions was established by use of a comniercially available Polymyxin B Septiarose gel, To measure the lipopolysaccharide (LFS) binding capacity of the gel. known concentrations of LPS were added to culture media, which were passed through a column consisting or the Polymyxin B Sepharose gel. The content of ET and added LPS in media was measured hy the Limulus amoehocyte lysatc (LAL) test before and after passage of the column. The LPS-binding capacity of the gel was approximately 2.4 × 10 g/l0 ml. The biological activity of contaminating ET and added LPS in media, before and alter passage of the column, was also characterized by the capacity of the media to induce interleukin l (IL-1) secretion in human Mo cultures. The content of IL-l in Mo culture supernalants was determined by the mouse thymocyte costimulatory(LAF) assay. By comparison of the activity of ET in these different biological systems, it was demonstrated that 15-20 × 10−12 g/ml of ET stimulate human Mo cultures lo IL-1 secretion.