Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.

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.

A B ST R A CT We have studied the relative contributions of monomeric (m-) and polymeric IgA (p-IgA) and of IgAl 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 mIgA. (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 Address reprint requests to Dr. Delacroix, ICP-MEXP, Brussels, Belgium.

A B S T R A C T 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, a2-macroglobulin (a2M), IgM, and SC. Polymeric IgA(p-IgA) averaged 13% (50-700 ltg/ 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 ug/ml) was the predominant protein, followed by IgG (32-480 gg/ml), and total IgA (37-209 ,g/ml). In saliva, p-IgA (72-902 Mig/ml) predominated, followed by albumin (16-385 ug/ml) and IgG (9-178 Mg/ml). Secretion-toserum 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 a2M, 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, Dr. Delacroix is an Aspirant for the Fond National de la Recherche Scientifique, Brussels, Belgium. His present address is UCL-ICP-MEXP,

This study investigated whether differences in fecal and serum antitoxin A antibody levels may account for the duration of Clostridium difficile-associated diarrhea (CDAD) and the occurrence of relapses. By an enzyme linked-immunosorbent assay, we tested 40 patients with CDAD including 25 patients without immunodeficiency and 15 patients receiving antineoplastic drugs. Two hundred eighty serum samples and 80 normal stool samples were investigated as controls. In nonimmunocompromised patients, serum immunoglobulin (IgG) and fecal IgA antitoxin A antibody titers were significantly higher in patients who suffered a single episode (n = 21) than in those with relapsing CDAD (n = 4) whose titers were at control levels. Of these 25 patients, eight suffered from diarrhea which lasted for more than 2 weeks. These patients had significantly lower serum-and feces-specific antibody levels than the others who presented symptoms of shorter duration. In cytostatic-treated patients, antitoxin A antibody levels were similar to controls, but relapses occurred in a single case. These data suggest an association between a defective humoral response to toxin A and a more severe form of C. difficile infection. They also indicate that other host-related factors control the severity of CDAD and remain to be elucidated.

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.

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