Jean-Pierre Vaerman

Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative study.

Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG sub-classes (59) or Gm allotypes (4) have been evaluated for reactivity and specificity in eight laboratories employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities McAb have been produced that can be satisfactorily applied in most methodologies employed and have potential as reference reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated demonstrating apparent assay restriction and whilst performing well in some assays proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable variability rather than capricious behaviour by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the physical and chemical procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.

Stimulation of secretory IgA and secretory component of immunoglobulins in small intestine of rats treated with Saccharomyces boulardii.

Saccharomyces boulardii (S.b.) is largely used in Western European countries for the treatment of acute infectious enteritis and antibiotic-induced gastrointestinal disorders. To study the mechanisms of the protective effect of S.b. against enteral pathogen infection, we assessed the response of the intestinal secretion of secretory IgA (s-IgA) and of the secretory component of immunoglobulins (SC) to oral administration of high doses (0.5 mg/g body weight, three times per day) of S.b. cells in growing rats. S.b. cells (biological activity: 2.8× 109 viable cells/100 mg) were administered daily by gastric intubation to weanling rats from day 14 until day 22 postpartum. Control groups received either 0.9% saline or ovalbumin following the same schedule. Expressed per milligram of cell protein, SC content was significantly increased in crypt cells isolated from the jejunum (48.5% vs saline controls, P< 0.05) as it was in the duodenal fluid (62.8% vs saline controls, P<0.01) of rats treated with S.b. Oral treatment with S.b. had no effect on the secretion of SC by the liver. In the duodenal fluid of rats treated with S.b. cells, the mean concentration of s-IgA was increased by 56.9% (P<0.01) over the concentration of s-IgA measured in saline controls. Compared to control rats treated from day 14 until day 22 postpartum with an antigenic load of ovalbumin equivalent to the total protein load provided by Sb cells (0.05 mg protein/g body weight, three times per day), S.b.-treated rats also exhibited a significantly higher intestinal concentration of SC (69% in villus cells, P<0.025 and 80% in crypt cells, P<0.01 These changes in intestinal SC and s-IgA concentration appeared not to be due to an increase in enterocyte turnover rate, since the mucosal mass parameters and the incorporation rate of [3H]thymidine into DNA measured in the jejunum, ileum, and colon remained unchanged in S.b.- treated rats. Our findings suggest that one of the mechanisms by which S.b. exerts its immunoprotective effect in the gastrointestinal tract is a stimulation of the intestinal secretion of s-IgA and of the secretory component of immunoglobulins.

Selective Transport of Polymeric Immunoglobulin-a in Bile - Quantitative Relationships of Monomeric and Polymeric Immunoglobulin-a, Immunoglobulin-m, and Other Proteins in Serum, Bile, and Saliva

In 17 adults, serum, hepatic bile, and saliva samples were analyzed for their sedimentation profile of IgA and secretory component (SC), and for their concentrations of albumin, orosomucoid, transferrin, IgG, IgA, alpha 2-macroglobulin (alpha 2M), IgM, and SC. Polymeric IgA(p-IgA) averaged 13% (50-700 micrograms/ml) of total IgA in serum, 70% (43-88%) in bile, and 93% (74-98%) in saliva. Most of the p-IgA in bile sedimented with SC, which also occurred free (8-44%), and with IgM. In bile, albumin (155-1,485 micrograms/ml) was the predominant protein, followed by IgG (32-480 micrograms/ml), and total IgA (37-209 micrograms/ml). In saliva, p-IgA (72-902 micrograms/ml) predominated, followed by albumin (16-385 micrograms/ml) and IgG (9-178 micrograms/ml). Secretion-to-serum albumin-relative concentration ratios (S/S-ARCR = 1 for albumin) in bile averaged 22 for p-IgA, 1.91 for IgM, 1.28 for monomeric IgA (m-IgA), 0.70 for IgG, and 0.57 for alpha 2M, indicating for p-IgA, IgM, and to a lesser extent for m-IgA, a selective excretion into bile. In saliva, a 16-fold greater selective excretion of p-IgA (mean S/S-ARCR = 354) was found. Labeled m- and p-IgA were injected intravenously into five patients. Specific activities indicated that for p-IgA 50% was serum derived in bile, as compared with 2% in saliva, and to 85% for m-IgA in bile. In the patient with the highest excretion of 125I-p-IgA in bile, only 2.8% of the injected dose was recovered in bile within 24 h after injection. Compared with rats and rabbits, the serum-to-bile transport of p-IgA in humans is much smaller.

Glutaradehyde-activated aminohexyl-derivative of sepharose 4B as a new versatile immunoabsorbent

Abstract 6-Aminohexyl-Sepharose 4B can be activated with glutaraldehyde to give a stable intermediate Sepharose 4B derivative which after removal of excess glutaraldehyde, covalently binds proteins during a second step, thereby furnishing Sepharose-protein conjugates of high protein content. Such conjugates constitute effective immunosorbents, having a 13-atom long spacing arm between the Sepharose and the protein. Their preparation is safe, easy, reproducible and particularly fast.

Changes in size, subclass, and metabolic properties of serum immunoglobulin A in liver diseases and in other diseases with high serum immunoglobulin A.

We have studied the relative contributions of monomeric (m-) and polymeric IgA (p-IgA) and of IgA1 and IgA2 to total serum IgA in healthy adults and patients with liver disease (LD) or with other diseases and high serum IgA. Serum concentration of total secretory component (SC) was also determined. In addition, fractional catabolic rates (FCR) and synthetic rates for both m- and p-IgA were measured in nine controls and nine cirrhotics. Our results support four main conclusions: (a) In healthy adults, intravascular p-IgA contributes to only 4-22% (mean 12%) of serum IgA, because its FCR and synthetic rate are approximately two times higher and four times smaller, respectively, than those of intravascular m-IgA. (b) in LD, biliary obstruction does not result in a significant increase in serum p-IgA unlike in rats and rabbits, indicating that in humans the SC-dependent biliary transport of p-IgA plays a much less significant role in selective removal of p-IgA from plasma than in rats and rabbits. (c) In contrast to biliary obstruction, parenchymal LD results in a significant and preferential increase in serum p-IgA, which in cirrhotics correlates with a selective reduction of the p-IgA-FCR. This supports a role for the human liver in selective removal of p-IgA from plasma, but another mechanism than the SC-dependent biliary transport should be considered. (d) Total SC, p-IgA, and IgA2 in serum are unlinked parameters, not necessarily reflecting mucosal events. A marked increase in serum SC occurs almost selectively in LD. Although a shift to IgA2 is suggested in Crohns disease and alcoholic cirrhosis, a shift to IgA1 frequently associated to a shift to p-IgA occurs in chronic active LD, primary Sicca, and connective tissue diseases.

Species-specific binding of human secretory component to SpsA protein of Streptococcus pneumoniae via a hexapeptide motif

SpsA, a pneumococcal surface protein belonging to the family of choline‐binding proteins, interacts specifically with secretory immunglobulin A (SIgA) via the secretory component (SC). SIgA and free SC from mouse, rat, rabbit and guinea‐pig failed to interact with SpsA indicating species‐specific binding to human SIgA and SC. SpsA is the only pneumococcal receptor molecule for SIgA and SC as confirmed by complete loss of SIgA and SC binding to a spsA mutant. Analysis of recombinant SpsA fusion proteins showed that the binding domain is located in the N‐terminal region of SpsA. By the use of different truncated N‐terminal SpsA fusion proteins, the minimum binding domain was shown to be composed of 112 amino acids (residues 172–283). The sequence of this 112‐amino‐acids domain was used to spot synthesize 34 overlapping peptides, consisting of 15 amino acids each, with an offset of three amino acids on a cellulose membrane. One of the peptides reacted specifically with both SIgA and SC. By using a second membrane with immobilized synthetic peptides of decreasing length containing parts of the identified 15‐amino‐acid motif a hexapeptide, YRNYPT was identified as the binding motif for SC and SIgA. SpsA proteins with a size smaller than the assay‐positive domain of 112 amino acids were able to inhibit the interaction of SIgA and pneumococci provided they contained the binding motif. The results indicated that the hexapeptide YRNYPT located in SpsA of pneumococcal strain type 1 (ATCC 33400) between amino acids 198 and 203 is involved in SIgA and SC binding. Because synthetic peptides containing only parts of the hexapeptide also assayed positive, these results further suggest that at least the amino acids YPT of the identified hexapeptide are critical for binding to SC and SIgA. Amino acid substitutions in the identified putative binding motif abolished SC‐/SIgA‐binding activity of the mutated SpsA protein, confirming the functional activity of this hexapeptide and the critical role of the amino acids YPT in SC and SIgA binding. Identification of this motif, which is highly conserved in SpsA protein among different serotypes, might contribute towards a new peptide based vaccine strategy.

Secretion of immunoglobulins and plasma proteins from the jejunal mucosa. Transport rate and origin of polymeric immunoglobulin A.

Parameters of secretion of IgA and several other plasma proteins from the jejunal mucosa were investigated in 11 individuals who had a normal distribution of Ig-containing cells in the lamina propria and in one patient who was totally deficient in jejunal IgA and IgM plasmacytes. Jejunal samples were collected during segmental gut perfusion. The following results were obtained: (a) The secretion of polymeric IgA (p-IgA, mean equals 217 micrograms/40 cm per min) exceeded those of albumin (132 micrograms), IgG (35 micrograms), and monomeric IgA (m-IgA, 15 micrograms, or 6.4% of total IgA). About 35% of IgA was IgA2 in the jejunal secretion, compared with approximately 23% in serum. This closely corresponds to the 35 and 24% of IgA2 plasmocytes in jejunal mucosa and peripheral lymph nodes, respectively. (b) For each protein, a relative coefficient of excretion (RCE) was calculated (jejunum to serum concentration ratio expressed relative to that of albumin). RCEs of 1.41 for orosomucoid, 1.0 for albumin, 0.83 for IgG, and 0.74 for IgE and, in the deficient patient, of 0.64 for m-IgA and 0.016 for IgM were obtained. This was inversely related to the molecular weight of these proteins and indicated their predominantly passive transport into the jejunum. However, in normal individuals, the RCE of transferrin (approximately 1.11 greater than 1, P greater than 0.05), alpha 2-macro globulin (approximately 0.77), m-IgA (approximately 1.98), and p-IgA (approximately 218) exceeded the value expected from simple seepage from plasma, thus pointing to an additional role of either local gut synthesis and/or active transepithelial transport. (c) Approximately 98% of p-IgA, approximately 99% of IgM, and approximately 68% of m-IgA in jejunal secretions were derived from local production in the gut wall, as determined by 125I-p-IgA specific activities and/or by comparison between the RCE values of the deficient patient to the values of controls. Therefore, the jejunal production of p-IgA (approximately 312 mg/d per 40 cm vs. approximately 54 mg/d from bile) contributes the majority of upper intestinal IgA in humans. The active transport of plasma p-IgA across the intestinal mucosa (approximately 0.08 mg/40 cm per kg per d) contributes less than 2% of the total amount of p-IgA (4.5 mg/kg per d) that is cleared daily from plasma.

Subclasses of Human Immunoglobulin A Based on Differences in the Alpha Polypeptide Chains

Antiserum from goats immunized with heavy polypeptide chains from a γA-type myeloma globulin was absorbed with serum from patients with selective absence of immunoglobulin A (γA). The resulting reagents could be used for the classification of 58 γA-myeloma proteins into two distinct antigenic types, respectively called subclasses He and Le. These differences were shown to be related to the heavy (alpha) polypeptide chains and independent of the integrity of interchain disulfide bridges. The γA-immunoglobulin from normal serum appears to consist, for the most part, of molecules with Le subclass specificity.

Lung mucosal immunity: immunoglobulin‐A revisited

Mucosal defence mechanisms are critical in preventing colonization of the respiratory tract by pathogens and penetration of antigens through the epithelial barrier. Recent research has now illustrated the active contribution of the respiratory epithelium to the exclusion of microbes and particles, but also to the control of the inflammatory and immune responses in the airways and in the alveoli. Epithelial cells also mediate the active transport of polymeric immunoglobulin-A from the lamina propria to the airway lumen through the polymeric immunoglobulin receptor. The role of IgA in the defence of mucosal surfaces has now expanded from a limited role of scavenger of exogenous material to a broader protective function with potential applications in immunotherapy. In addition, the recent identification of receptors for IgA on the surface of blood leukocytes and alveolar macrophages provides an additional mechanism of interaction between the cellular and humoral immune systems at the level of the respiratory tract.We read through the Review by PILETTE et al. [1] with extreme interest and would like to add a contribution to the subject of airways immunity in chronic obstructive pulmonary disease (COPD). For a long time, we have witnessed, at least in Italy, the predominance of the notion that all COPD patients have a certain degree of immune deficiency as a basic pathogenetic mechanism, since they experience recurrent bronchitis exacerbations. Scientific evidence of true general or local immune defects in COPD is, in our opinion, inconclusive, although we fully agree on the point made by the authors that all the studies in the literature share methodological limitations, both in sampling and analysis techniques and in selection of patients. In our experience, different immune components can appear either similar to controls or increased (as a likely consequence of repeated stimulation by exacerbations or chronic bacterial colonization of the airways), or decreased in a specific group of COPD patients. Indeed, we recently reported decreased numbers of CD3 and CD8 lymphocytes in the bronchial biopsies of severe COPD patients, associated with an increase of neutrophils and macrophages [2]. With particular regard to immunoglobulin (Ig)-A, we found only an insignificant increase in patients with mild COPD who had never smoked [3], and a high increase in severe but clinically stable COPD patients with chronic tracheostomy and a high level of bacterial colonization [4]. The experience with tracheostomized COPD patients is very interesting because they provide a model of bacteria/host interaction in which the role of immunity can be evaluated prospectively. The presence of high levels of IgA in bronchial aspirates could, in part, justify the relatively low rate of lower respiratory tract infections in these patients after discharge from hospital [5]. We appreciated the long and accurate list of defence mechanisms in the respiratory tract reported by the authors, which clearly shows that specific immune functions are only a part of the airways protection system. Indirect proof that immunoglobulins are only a part, albeit an important part, of mucosal immunity, comes from studies on the stimulation of mucosaassociated lymphoid tissues with oral vaccines or bacterial extracts, which have been demonstrated to reduce the impact of bronchitis exacerbations, at least in mild-to-moderate chronic obstructive pulmonary disease [6]. The mechanisms of action were reflected in an increase of immunoglobulin-A in the airways9 fluids, but more solid evidence, from a biological point of view, was the activation of alveolar macrophages [7]. In our opinion, the potential of oral bacterial extracts to stimulate immunoglobulin-A production in the airways should be further investigated. In conclusion, we would once again like to stress that the impairment of defence systems in the airways is not simply a question of specific immunity, and that a clinically relevant imbalance in chronic obstructive pulmonary disease must be evaluated within a complex defence network concept, rather than in a simple cause/effect perspective.

Relevance of biliary IgA antibodies in rat intestinal immunity.

Two intraperitoneal administrations of foreign red cells in Freunds complete adjuvant, or two intragastric intubations of erythrocytes, given to rats at 15 days interval, both elicit the appearance of specific antibodies in bile and serum. When bile was compared to serum, the selective predominance of IgA antibodies in this secretion was observed for both the intraperitoneally and intragastrically immunized groups, being more pronounced for the orally immunized group when related to IgG or IgM antibodies. The IgA content of upper intestinal washings was roughly ten‐fold smaller in rats with bile duct cannulation than in sham‐operated controls. Altogether, the data demonstrate that bile IgA may significantly contribute to the secretory IgA system of the gut.