Cytokine release from the nasal mucosa and whole blood after experimental exposures to organic dusts.

The aim of this study was to assess the cytokine response after nasal exposure to organic dusts. In a double blinded, crossover study five garbage workers with occupational airway symptoms and five healthy garbage workers were intranasally exposed to endotoxin (lipopolysaccharide LPS), beta-1,3-D-glucan (GLU), Aspergillus sp., compost or the saline dilute for 15 min. Nasal cavity volume and nasal lavage (NAL) were performed at baseline and 3, 6, 11 h postexposure. NAL was analysed with differential cell counts, cysteinyl-leukotrienes, tumour necrosis factor alpha, interleukin (IL)-1beta, IL-6 and IL-8. A whole blood assay on cytokine-release was performed with LPS and GLU. NAL cytokines neutrophils, lymphocytes and albumin increased significantly at 6 h after LPS exposure. GLU induced an increase in albumin and a slight increase in IL-1beta 6-11 h post exposure. In the WBA a significant increase in all cytokines after exposure to LPS as well as GLU was found. Significantly more cells were seen in NAL of the control group 6 h post LPS exposure. In conclusion lipopolysaccharide is the most potent inducer of inflammation in the nasal mucosa whereas compost and beta-1,3-D-glucan only induce minor changes. This reaction to lipopolysaccharide is attenuated in workers with occupational airway symptoms. In whole blood assay, however, beta-1,3-D-glucan also induces cytokine release, indicating a different protective effect of the nasal mucosa towards lipopolysaccharide and beta-1,3-D-glucan.

An enzyme immunoassay for rat growth hormone: validation and application to the determination of plasma levels and in vitro release.

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.

Enzyme immunometric assay for leukotriene C4.

An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

Immunometric assay of low molecular weight haptens containing primary amino groups.

A new enzyme immunometric assay of small haptens containing primary amino groups (thyroxine, MW 777; substance P, MW 1347; endothelin, MW 2492) is described. The procedure involves different sequential steps: (1) immunocapture of the haptens (standard or sample) by monoclonal anti-hapten antibodies coated on 96-well microtiter plates; (2) cross-linking of haptens via their amino groups to the wells using homobifunctional reagents (glutaraldehyde or disuccinimidyl suberate); (3) denaturing treatments (HCl or methanol); (4) measurement of linked epitope using the same monoclonal anti-hapten antibodies labeled with acetylcholinesterase. A minimal detectable concentration in the 4-10 fmoL/mL range was observed. Each assay appeared to be 70-200 times more sensitive than conventional competitive enzyme immunoassay using the same monoclonal antibody-coated plate technology and acetylcholinesterase-hapten conjugates as enzymatic tracers. Precision and specificity were very satisfying. Good correlation was noted between this assay and the competitive assays performed for different biological samples (plasma, tissues, or supernatant cell culture).

A rapid, simple method for calculating equilibrium constants and antibody site concentrations from dilution curves alone.

A rapid new practical method for calculating both the antibody-antigen equilibrium constant and the antibody concentration from antibody dilution curve data alone is described. This method is faster than the inhibition curve method for evaluating a humoral immune response. It is particularly suitable for monitoring the immune response of an immunization program. The response is assessed as an immunization index, Abi*Ka. This index is more exact than the antibody titer obtained from dilution curves and independent of the specific activity of the labelled molecule and total activity used in the assay. The method was used to monitor the production of a monoclonal antibody to the sulphide peptide leucotriene including immunization, cloning and purification.

[Prostanoids and hemostasis]. / Prostanoïdes et hémostase.

Thromboxane A2 (TXA2) and prostacyclin (two prostanoids) are produced from arachidonic acid through the cycloxygenase pathway. The enzyme cyclooxygenase is inhibited by aspirin. Prostanoids are short-lived and thus exert their effects locally. Activated platelets synthetize TXA2 which reinforces activation of those platelets and platelets in the vicinity of the former. Complete activation however can occur in the absence of TXA2 synthesis. Prostacyclin is able to block all the platelet responses, but this would be achieved in vivo together with intense vasodilation. Primary adhesion remains unaffected, since it does not require platelet activation. There are other molecules endowed with inhibitory effects on platelets: PGD2, PGE1, adenosine, and EDRF (nitric oxide). These molecules, and prostacyclin, also have effects on other cells than platelets and smooth muscle cells: leukocytes, endothelial cells. Clinical investigations on the prostanoid system in physiology and pathology of the cardiovascular system have been hampered by analytical problems. Taking into account all these restrictions, a rational pharmacological approach is difficult, but newer molecules with dual and selective activity against TXA2 (both inhibitor of TX-synthetase and antagonist at the TXA2-receptor level) seem promising as anti-thrombotic agents.

Elimination of undesired cross-reactants by using mixtures of antibodies: experimental and theoretical evaluations of hapten radioimmunoassays.

This study shows that the specificity of radioimmunoassays can be improved by including a second antibody raised against an undesired cross-reactant. In a radioimmunoassay of prostaglandin E2 (PGE2) involving a monoclonal antibody, the cross-reactivity with 6-keto-prostaglandin E1 (6kPGE1) was decreased from 20% to 2% by including a high concentration of a polyclonal anti-6kPGE1. A similar increase in specificity was obtained in the assay of a larger hapten, luliberin (luteinizing hormone releasing hormone); the cross-reactivity of a luliberin analog was decreased 20-fold. Equations derived from the Law of Mass Action were used for the mathematical analysis and for the computer simulation of changes in assay affinity and specificity according to the quantity and quality of the mixed antibodies. The model gave values that agreed well with experimental data; it promises to be quite useful in designing specific radioimmunoassays.

Lack of prostaglandin E2-mediated monocyte suppressive activity in newborn and mothers.

An excess of adult blood adherent cells (monocytes) inhibits mitogen, antigen and allogeneic cell-induced lymphocyte proliferations. This inhibition is dependent on the number of the adherent monocytes in the cultures and is substantially reduced (by 60%) by indomethacin or anti-PGE2 antiserum. Newborn monocytes exert only a weak inhibitory effect and produce about eight times less PGE2 than adult monocytes. The production of PGE2 and the suppression can be induced by incubating newborn monocytes with mixed leucocyte culture supernatants. Both monocytes from mothers at the time of delivery and from newborn infants, usually exert a poor suppressive activity related to a low production of PGE2. We strongly suggest that the expression of the PGE2-mediated suppression by monocytes is under the control of activated short-lived suppressor lymphocytes.

Respective roles and interactions of T-lymphocyte and PGE2-mediated monocyte suppressive activities in human newborns and mothers at the time of delivery.

Recently the concept of a poorly functional humoral immune response in the newborn was proposed. Data have been presented indicating that the impaired newborn B cell maturation, as shown in vitro in a pokeweed mitogen-induced B cell maturation system, is due both to an immaturity of lymphocyte subsets and to an increased suppressive T activity. In the present work, we present evidence that there exists a predominance of a naturally occurring T lymphocyte suppressive activity in the cord blood in that the removal of the suppressive activity by irradiation allows a normal maturation of newborn B cells. Such normal maturation of newborn B cells can also be obtained using mixed cultures of adult T cells and newborn B cells. Newborn suppressor T cells belong to both EA gamma (+) and EA gamma (-) fractions, and it is not known whether these two groups do or do not belong to different subsets. The PGE2-dependent monocyte suppressive activity does not play any role in the suppression observed in newborns since newborn monocytes are poorly suppressive and since they produce a smaller amount of PGE2 than adult monocytes. Some observations suggest, on the contrary, that the suppressive T lymphocytes can regulate the level of the PGE2-dependent monocyte suppressive activity. It should be noticed that similar observations about T lymphocyte and PGE2-dependent monocyte suppressive activities have been made at the same time using mothers' cells. These observations suggest the possibility that such changes in B cell immune regulation may result from an interaction between maternal and fetal lymphoid cells.

[Immunologic study of familial lymphohistiocytosis. Eight new case reports (author's transl)]. / Etude immunologique de la lymphohistiocytose familiale. A propos de huit nouvelles observations.

We report 8 cases of familial lymphohistiocytosis collected in 6 families. Several data argue for an hyperactivation of the reticuloendothelial system (RES). An abnormal visualization of all organs was observed in a scintigraphic study after 99technetium labelled red blood cells injection. Blood monocytes contained very low peroxidase activity as detected by cytoenzymology and secreted large quantities of prostaglandin E2 (PGE2). Adherent cells isolated from blood exercised a strong suppressor effect on the proliferation of normal lymphocytes induced by phytohemaglutinin. This effect was reduced by indomethacin and therefore appears PGE2-dependent. One patient's serum exerced an inhibitory activity on antigen-induced proliferation of normal lymphocytes and on mixed leucocyte reaction. In contrast, cellular and humoral functions were not deeply impaired. The hyperactivation of the RES remains unexplained and not related to a graft versus host reaction, which could be excluded in 3 of our patients. Therapeutic attempts were not efficient, all patients dying despite steroid, vincaleucoblastin and indomethacin therapy.

Biotin-responsive immunoregulatory dysfunction in multiple carboxylase deficiency.

The immunoregulatory system has recently been shown to require prostaglandins (PG) for its activation in man. We report here an impairment of immunoregulatory function, due to defective PGE monocytic production, in a 12-month-old boy with multiple carboxylase deficiency (MCD). The abnormal immune-response was corrected in vitro by adding PGE to the medium. Moreover, PGE deficiency and immunoregulatory dysfunction responded to biotin administration in vivo. It is suggested that the PGE deficiency in MCD could result from an impaired activity of a biotin enzyme, acetyl CoA carboxylase, since the product of this enzyme reaction, malonyl CoA, is required for prostaglandin synthesis.

Viroimmunoenzymoassay for detection of anti-penicilloyl antibodies and penicilloyl residues: comparison of results obtained by radio-, viro- and enzymoimmunoassay.

A new technic named viroimmunoenzymoassay is described. The principle is the same as the classical viroimmunoassay which uses as tracer a bacteriophage bound covalently to an hapten. Immuno-specific neutralization of these modified bacteriophages by an hapten antiserum allows to detect and to test hapten antibodies or the haptens with a great sensitivity. In the new technic the visualization of the bacterial lysis is estimated by measure of the amount of beta-galactosidase which is released into the medium by Escherichia coli BB bacteria. These are previously induced by isopropyl thiogalactoside or lactose. The method is applied to the assay of penicilloyl groups bound covalently with another molecule, and its performances are compared with three other classical immunological methods: the radio-, enzymo- and viroimmunoassay.

[Purification and properties of an esterase excreted during sporulation of Bacillus subtilis]. / Purification et propriétés d'une estérase excrétée pendant la sporulation de Bacillus subtilis

After growth in Difco Nutrient Broth, several proteolytic enzymes are excreted by B. subtilis, Marburg strain, namely a metalloprotease and a seryl protease. We report here the purification and some biochemical properties of a third extracellular hydrolytic enzyme for which we propose the term of esterase. As the serylprotease, the esterase is a seryl enzyme endowed with both proteolytic and esterolytic activities. Nevertheless the esterase differs from serylprotease in many aspects. In particular, it is an acidic enzyme with a low proteolytic activity and a high esterolytic activity. Its specificity toward synthetic substrates is restricted. The enzyme is active only on esters of amino acids and particularly on those of tyrosine. With Bz Tyr O Et as substrate the esterase displays a maximum of activity between pH 7.2 and 8.1 with a Km of 1.3.10-minus 3 M at 30 degrees C. At the end of this work, two questions remain unanswered: 1) the origin of the multiplicity of the bands revealed by polyacrylamide electrophoresis in the purest fraction; 2) the nature of the physiological substrate of the enzyme.