Infections often involve the mucosal surfaces of the body, which form a boundary with the outside world. This review focuses on immunoglobulin A (IgA) antibodies because IgA is the principal mucosal antibody class. IgA is synthesized by local plasma cells and has a specific polymeric immunoglobulin receptor-mediated transport mechanism for entry into the secretions. By serving as an external barrier capable of inhibiting attachment of microbes to the luminal surface of the mucosal epithelial lining, IgA antibodies form the first line of immune defense. In addition to this traditional mode of extracellular antibody function, recent evidence suggests that IgA antibodies can also function in a nontraditional fashion by neutralizing viruses intracellularly, if a virus is infecting an epithelial cell through which specific IgA antibody is passing on its way to the secretions. IgA antibodies are also envisaged as providing an internal mucosal barrier beneath the mucosal lining. Antigens intercepted by IgA antibodies here can potentially be ferried through the epithelium and thereby excreted. In addition to the polymeric immunoglobulin receptor on mucosal epithelial cells, IgA antibodies can bind to receptors on a variety of leukocytes and have been shown, in some experimental systems, to be capable of activating the alternative complement pathway, making IgA antibodies potential participants in inflammatory reactions. Although the relationship of IgA antibodies to inflammation is not entirely clear, the bias presented is that IgA is basically noninflammatory, perhaps even anti-inflammatory. According to this view, the major role of the Fc portion of IgA antibodies is to transport IgA across mucosal epithelial cells and not, as in the case of the other classes of antibody, to activate secondary phenomena of the kind that contribute to inflammation. Because of IgA's key role as an initial barrier to infection, much current research in mucosal immunology is directed toward developing new vectors and adjuvants that can provide improved approaches to mucosal vaccines. Finally, because of advances in monoclonal antibody technology, topical application of antibodies to mucosal surfaces has significant potential for preventing and treating infections.

The presence of microflora in the digestive tract promotes the development of the intestinal immune system. In this study, to evaluate the roles of two types of indigenous microbe, segmented filamentous bacteria (SFB) and clostridia, whose habitats are the small and large intestines, respectively, in this immunological development, we analyzed three kinds of gnotobiotic mice contaminated with SFB, clostridia, and both SFB and clostridia, respectively, in comparison with germfree (GF) or conventionalized (Cvd) mice associated with specific-pathogen-free flora. In the small intestine, the number of alpha beta T-cell receptor-bearing intraepithelial lymphocytes (alpha betaIEL) increased in SFB-associated mice (SFB-mice) but not in clostridium-associated mice (Clost-mice). There was no great difference in Vbeta usage among GF mice, Cvd mice, and these gnotobiotic mice, although the association with SFB decreased the proportion of Vbeta6(+) cells in CD8beta- subsets to some extent, compared to that in GF mice. The expression of major histocompatibility complex class II molecules on the epithelial cells was observed in SFB-mice but not in Clost-mice. On the other hand, in the large intestine, the ratio of the number of CD4(-) CD8(+) cells to that of CD4(+) CD8(-) cells in alpha betaIEL increased in Clost-mice but not in SFB-mice. On association with both SFB and clostridia, the numbers and phenotypes of IEL in the small and large intestines changed to become similar to those in Cvd mice. In particular, the ratio of the number of CD8alpha beta+ cells to that of CD8alpha alpha+ cells in alpha betaIEL, unusually elevated in the small intestines of SFB-mice, decreased to the level in Cvd mice on contamination with both SFB and clostridia. The number of immunoglobulin A (IgA)-producing cells in the lamina propria was more elevated in SFB-mice than in Clost-mice, not only in the ileum but also in the colon. The number of IgA-producing cells in the colons of Clost-mice was a little increased compared to that in GF mice. Taken together, SFB and clostridia promoted the development of both IEL and IgA-producing cells in the small intestine and that of only IEL in the large intestine, respectively, suggesting the occurrence of compartmentalization of the immunological responses to the indigenous bacteria between the small and large intestines.

Receptors for the Fc domain of immunoglobulins play an important role in immune defense. There are two well-defined functional classes of mammalian receptors. One class of receptors transports immunoglobulins across epithelial tissues to their main sites of action. This class includes the neonatal Fc receptor (FcRn), which transports immunoglobulin G (IgG), and the polymeric immunoglobulin receptor (pIgR), which transports immunoglobulin A (IgA) and immunoglobulin M (IgM). Another class of receptors present on the surfaces of effector cells triggers various biological responses upon binding antibody-antigen complexes. Of these, the IgG receptors (Fc gamma R) and immunoglobulin E (IgE) receptors (Fc epsilon R) are the best characterized. The biological responses elicited include antibody-dependent, cell-mediated cytotoxicity, phagocytosis, release of inflammatory mediators, and regulation of lymphocyte proliferation and differentiation. We summarize the current knowledge of the structures and functions of FcRn, pIgR, and the Fc gamma R and Fc epsilon RI proteins, concentrating on the interactions of the extracellular portions of these receptors with immunoglobulins.

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.

Mammals have two genes encoding homologues of the endoplasmic reticulum (ER) disulfide oxidase ERO1 (ER oxidoreductin 1). ERO1-beta is greatly enriched in the endocrine pancreas. We report in this study that homozygosity for a disrupting allele of Ero1lb selectively compromises oxidative folding of proinsulin and promotes glucose intolerance in mutant mice. Surprisingly, concomitant disruption of Ero1l, encoding the other ERO1 isoform, ERO1-alpha, does not exacerbate the ERO1-beta deficiency phenotype. Although immunoglobulin-producing cells normally express both isoforms of ERO1, disulfide bond formation and immunoglobulin secretion proceed at nearly normal pace in the double mutant. Moreover, although the more reducing environment of their ER protects cultured ERO1-beta knockdown Min6 cells from the toxicity of a misfolding-prone mutant Ins2(Akita), the diabetic phenotype and islet destruction promoted by Ins2(Akita) are enhanced in ERO1-beta compound mutant mice. These findings point to an unexpectedly selective function for ERO1-beta in oxidative protein folding in insulin-producing cells that is required for glucose homeostasis in vivo.

Measles virus (MV), a member of the genus Morbillivirus in the family Paramyxoviridae, is an enveloped virus with a non-segmented, negative-strand RNA genome. It has two envelope glycoproteins, the haemagglutinin (H) and fusion proteins, which are responsible for attachment and membrane fusion, respectively. Human signalling lymphocyte activation molecule (SLAM; also called CD150), a membrane glycoprotein of the immunoglobulin superfamily, acts as a cellular receptor for MV. SLAM is expressed on immature thymocytes, activated lymphocytes, macrophages and dendritic cells and regulates production of interleukin (IL)-4 and IL-13 by CD4+ T cells, as well as production of IL-12, tumour necrosis factor alpha and nitric oxide by macrophages. The distribution of SLAM is in accord with the lymphotropism and immunosuppressive nature of MV. Canine distemper virus and Rinderpest virus, other members of the genus Morbillivirus, also use canine and bovine SLAM as receptors, respectively. Laboratory-adapted MV strains may use the ubiquitously expressed CD46, a complement-regulatory molecule, as an alternative receptor through amino acid substitutions in the H protein. Furthermore, MV can infect SLAM- cells, albeit inefficiently, via the SLAM- and CD46-independent pathway, which may account for MV infection of epithelial, endothelial and neuronal cells in vivo. MV infection, however, is not determined entirely by the H protein-receptor interaction, and other MV proteins can also contribute to its efficient growth by facilitating virus replication at post-entry steps. Identification of SLAM as the principal receptor for MV has provided us with an important clue for better understanding of MV tropism and pathogenesis.

In the oral cavity, indigenous bacteria are often associated with two major oral diseases, caries and periodontal diseases. These diseases seem to appear following an imbalance in the oral resident microbiota, leading to the emergence of potentially pathogenic bacteria. To define the process involved in caries and periodontal diseases, it is necessary to understand the ecology of the oral cavity and to identify the factors responsible for the transition of the oral microbiota from a commensal to a pathogenic relationship with the host. The regulatory forces influencing the oral ecosystem can be divided into three major categories: host related, microbe related, and external factors. Among host factors, secretory immunoglobulin A (SIgA) constitutes the main specific immune defense mechanism in saliva and may play an important role in the homeostasis of the oral microbiota. Naturally occurring SIgA antibodies that are reactive against a variety of indigenous bacteria are detectable in saliva. These antibodies may control the oral microbiota by reducing the adherence of bacteria to the oral mucosa and teeth. It is thought that protection against bacterial etiologic agents of caries and periodontal diseases could be conferred by the induction of SIgA antibodies via the stimulation of the mucosal immune system. However, elucidation of the role of the SIgA immune system in controlling the oral indigenous microbiota is a prerequisite for the development of effective vaccines against these diseases. The role of SIgA antibodies in the acquisition and the regulation of the indigenous microbiota is still controversial. Our review discusses the importance of SIgA among the multiple factors that control the oral microbiota. It describes the oral ecosystems, the principal factors that may control the oral microbiota, a basic knowledge of the secretory immune system, the biological functions of SIgA, and, finally, experiments related to the role of SIgA in oral microbial ecology.

BACKGROUND

Protein-carbohydrate interactions are believed to be important in many biological processes that involve cell-cell communication. Apart from the selectins, the only well-characterized vertebrate sialic acid-dependent adhesion molecules are CD22 and sialoadhesin; CD22 is a member of the immunoglobulin superfamily that is expressed by B lymphocytes and sialoadhesin is a macrophage receptor. The recent cloning of the gene encoding sialoadhesin has shown that it is also immunoglobulin-like. Both proteins share sequence similarity with the myelin-associated glycoprotein, an adhesion molecule of oligodendrocytes and Schwann cells that has been implicated in the process of myelination, raising the important question of whether myelin-associated glycoprotein is also a sialic acid-binding protein.

RESULTS

We have investigated the binding properties of these three receptors when expressed either in monkey COS cells or as chimaeric proteins containing the Fc portion of human immunoglobulin G. We demonstrate that, like sialoadhesin and CD22, myelin-associated glycoprotein mediates cell adhesion by binding to cell-surface glycans that contain sialic acid. We have dissected the specificities of these three adhesins further: whereas sialoadhesin binds equally to the sugar moieties NeuAc alpha 2-->3Gal beta 1-->3(4)GlcNAc or NeuAc alpha 2-->3Gal beta 1-->3GalNAc, myelin-associated glycoprotein recognizes only NeuAc alpha 2-->3Gal beta 1-->3GalNAc and CD22 binds specifically to NeuAc alpha 2-->6Gal beta 1-->4GlcNAc. Furthermore, we show that the recognition of sialylated glycans on the surfaces of particular cell types leads to the selective binding of sialoadhesin to neutrophils, myelin-associated glycoprotein to neurons and CD22 to lymphocytes.

CONCLUSIONS

Our findings demonstrate that a subgroup of the immunoglobulin superfamily can mediate diverse biological processes through recognition of specific sialylated glycans on cell surfaces. We propose that this subgroup of proteins be called the sialoadhesin family.

Unfolding of proteins by forced stretching with atomic force microscopy or laser tweezer experiments complements more classical techniques using chemical denaturants or temperature. Forced unfolding is of particular interest for proteins that are under mechanical stress in their biological function. For beta-sandwich proteins (a fibronectin type III and an immunoglobulin domain), both of which appear in the muscle protein titin, the results of stretching simulations show important differences from temperature-induced unfolding, but there are common features that point to the existence of folding cores. Intermediates detected by comparing unfolding with a biasing perturbation and a constant pulling force are not evident in temperature-induced unfolding. For an alpha-helical domain (alpha-spectrin), which forms part of the cytoskeleton, there is little commonality in the pathways from unfolding induced by stretching and temperature. Comparison of the forced unfolding of the two beta-sandwich proteins and two alpha-helical proteins (the alpha-spectrin domain and an acyl-coenzyme A-binding protein) highlights important differences within and between protein classes that are related to the folding topologies and the relative stability of the various structural elements.

A deletion DNA rearrangement is associated with immunoglobulin class switching from IgM to IgG, IgA or IgE This recombination occurs in immunoglobulin switch regions, which are complex, highly repetitive regions of DNA. As switch regions become transcriptionally active just before switch recombination, analysis of the behaviour of these sequences during transcription could elucidate the mechanism of switch recombination. Here, we report that transcription of a supercoiled plasmid containing the murine IgA switch region (S alpha) leads to a loss of superhelical turns. The resulting series of less supercoiled plasmids is stabilized by RNA-DNA hybrids formed by the nascent RNA transcripts, which remain base-paired with their DNA templates.

Sonicated Borrelia burgdorferi was previously reported to possess both B-cell mitogenic and interleukin-6 (IL-6) stimulatory activities. In this report, two outer surface lipoproteins, OspA and OspB, were purified from B. burgdorferi and assessed for the presence of these functions. OspA was purified from two strains, an OspB-deficient variant of HB19 and N40, while OspB was purified from the N40 strain. All lipoprotein preparations were free of endotoxin contamination, and polymyxin B failed to inhibit responses, indicating that media contamination was not contributing to biological assays. All three preparations were able to stimulate proliferation of mononuclear cells from naive C3H/HeJ and BALB/c mice. Depletion experiments indicated that the responding cells were B lymphocytes and not T lymphocytes. Purified OspA and OspB stimulated immunoglobulin M production by splenocyte cultures from naive mice, a property also previously attributed to sonicated B. burgdorferi. OspA and OspB also stimulated the production of IL-6 and tumor necrosis factor alpha by bone marrow-derived macrophages from BALB/c and C3H/HeJ mice. Cytokine production was enhanced by the presence of gamma interferon in the cultures, indicating that the magnitude of responses to these lipoproteins may be modulated by cytokines in the microenvironment of infected tissues. Human endothelial cells produced IL-6 when incubated with OspA and OspB, indicating that non-hematopoietic lineage cells can respond to the lipoproteins. Purified OspA and OspB had approximately equal activity, with responses detected in the range of 10 ng of lipoprotein per ml to 1 microgram of lipoprotein per ml. Comparison with published dose responses for lipoproteins purified from Escherichia coli indicates that OspA and OspB purified from B. burgdorferi are much more potent. The high potency of the B. burgdorferi lipoproteins and the ability of the spirochete to invade tissues and persist argue that they could be important in the localized events contributing to the pathology of Lyme disease.

Mucosal immunity reduces the need for elimination of penetrating exogenous antigens by proinflammatory systemic immunity. The adult gut mucosa contains some 80% of the body's activated B cells-differentiated to plasmablasts and plasma cells (PCs). Most mucosal PCs produce dimeric immunoglobulin A (IgA), which, along with pentameric immunoglobulin M (IgM), can be exported by secretory epithelia expressing the polymeric immunoglobulin receptor. Immune exclusion of antigens is performed mainly by secretory IgA in cooperation with innate defenses, but, in newborns and in IgA deficiency, secretory IgM is important. In the gut, induction and regulation of mucosal immunity occurs primarily in gut-associated lymphoid tissue-particularly the Peyer's patches-and also in mesenteric lymph nodes. Terminal differentiation to PCs is accomplished in the lamina propria to which the activated memory/effector T and B cells home. Lactating mammary glands are part of the secretory immune system, and IgA antibodies in breast milk reflect antigenic stimulation of gut-associated lymphoid tissue and nasopharynx-associated lymphoid tissue such as the tonsils. Breast-milk antibodies are thus highly targeted against infectious agents and other exogenous antigens in the mother's environment, which are those likely to be encountered by the infant. Therefore breast-feeding represents an ingenious immunologic integration of mother and child.

Immunoglobulins were isolated from the serum or ascitic fluid of Lou/Wsl rats bearing plasmacytomas and labeled with 125I. When labeled IgA was injected i.v. it disappeared from the blood serum much more rapidly than IgG2 so that after 3 h less than 10% remained. This rapid disappearance of the injected IgA was not seen in rats with ligated bile ducts. In rats with cannulated bile ducts, the labeled IgA appeared rapidly in the bile so that 25% of the injected dose was recovered in 3 h; at the peak of this biliary excretion the specific radioactivity of the bile (cpm/milligram protein) was about 200 times greater than that of the blood serum. Thus much of the IgA which finds its way into the blood is rapidly and actively transported across the liver so that it enters the gut lumen via the biliary tract.

In contrast with the study of alpha beta T cells, that of gamma delta T cells is relatively recent and stems from the discovery of their rearranged genes, rather than from any knowledge of their biological function. Thus, experiments designed to characterize their specificity and function have drawn heavily on our knowledge of alpha beta T cells. During the past few years, many studies, especially with mice lacking either alpha beta or gamma delta T cells, have demonstrated that gamma delta T cells can contribute to immune competence, but they do so in a way that is distinct from alpha beta T cells. It is also evident that gamma delta T cells may not recognize antigen the same way as do alpha beta T cells. Analysis of three protein antigens-the murine MHC class II IEk, the nonclassical MHC T10/T22, and the Herpes virus glycoprotein gI-indicates that gamma delta T cell recognition does not require antigen processing and that the proteins are recognized directly. In all three cases, recognition by these T cell clones involves neither peptides bound to these proteins nor peptides derived from them. Moreover, a group of small phosphate-containing nonpeptide compounds derived from mycobacterial extracts has been found to stimulate a major population of human peripheral gamma delta T cells in a T cell receptor (TCR)-dependent manner. This indicates that gamma delta T cells can respond to ligands that are different from those of alpha beta T cells. Analysis of complementarity determining region (CDR3) length distributions of gamma and delta chains indicates that they are more similar to those of immunoglobulins than to TCR alpha and beta. This further supports the idea that gamma delta and alpha beta T cells recognize antigens differently and suggests that gamma delta T cells may be more like immunoglobulins in their recognition properties. gamma delta T cells share many cell surface proteins with alpha beta T cells and are able to secrete lymphokines and express cytolytic activities in response to antigenic stimulation. These, together with the results cited above, indicate that gamma delta T cells can mediate cellular immune functions without a requirement for antigen processing. Thus, pathogens, damaged tissues, or even B and T cells can be recognized directly, and cellular immune responses can be initiated without a requirement for antigen degradation or specialized antigen-presenting cells. This would give gamma delta T cells greater flexibility than the more classical type of alpha beta T cell-mediated cellular immunity.

Immunoglobulin heavy-chain switching is effected by a DNA recombination event that replaces the C mu gene with one of the other heavy-chain constant-region (CH) genes located 3' to the C mu gene. How the specificity of this event is controlled is unknown. However, it has been shown that IgM+ cells capable of switching to specific isotypes have the corresponding unrearranged CH genes in an accessible or active chromatin state, as demonstrated by the fact that these specific CH genes are hypomethylated and are transcriptionally active. We now report that the RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote switching to these specific CH genes. For example, we find that bacterial lipopolysaccharide, which induces the IgM+ cell line I.29 mu to switch to IgA, induces transcripts from the germ-line C alpha gene(s) in I.29 mu cells prior to switch recombination. Two preparations of T-cell lymphokines (recombinant interleukin 4 and supernatant from the T-cell line 2.19, which contains interleukins 4 and 5) that promote switching to specific isotypes by lipopolysaccharide-treated spleen cells induce transcripts from the corresponding germ-line CH genes prior to expression of the new isotypes. For example, interleukin 4, which appears to be necessary for switching to IgE in vitro and in vivo, induces within 2 days large increases in germ-line C epsilon transcripts in lipopolysaccharide-treated spleen cells and in I.29 mu cells. The most straightforward interpretation of our data is that these lymphokines direct switching to specific isotypes by activating specific CH genes, making them accessible to the putative switch recombinase.

Changes in mucosal defense have been implicated as important factors affecting infections complications in critically ill patients. To study the effects of nutrient administration on gut-associated lymphatic tissue (GALT), ICR mice were randomized to receive chow plus intravenous saline, intravenous feeding of a total parenteral nutrition (TPN) solution, or enteral feeding of the same TPN solution. In a second series of experiments, a more complex enteral diet (Nutren) was compared with chow feeding and enteral TPN. After 5 days of feeding with experimental diets, lymphocytes were harvested from the mesenteric lymph nodes (MLNs), Peyer's patches (PPs), lamina propria (LP) cells, and intraepithelial (IE) spaces of the small intestine to determine cell yields and phenotypes. Small intestinal washings, gallbladder contents, and sera were collected and analyzed for immunoglobulin A (IgA) levels. In both series of experiments, there were no significant changes within the MLNs. There were significant decreases in total cell yields from the PPs, IE spaces, and LP in animals fed with TPN solution, either enterally or parenterally, as compared with chow-fed mice. Total T cells were decreased in both TPN-fed groups in the PPs and LP, whereas total B cells were decreased in the PP, IE, and LP populations. Total cell numbers remained normal in the Nutrenfed group, except for a decrease in LP T cells. CD4+ LP cells decreased significantly with a reduction in the CD4/CD8 ratio in mice fed TPN solution either intravenously or enterally, whereas IgA recovery from small intestinal washings was significantly decreased in the same groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe the most general solution to date of the problem of matching globular protein sequences to the appropriate three-dimensional structures. The screening template, against which sequences are tested, is provided by a protein "structural fingerprint" library based on the contact map and the buried/exposed pattern of residues. Then, a lattice Monte Carlo algorithm validates or dismisses the stability of the proposed fold. Examples of known structural similarities between proteins having weakly or unrelated sequences such as the globins and phycocyanins, the eight-member alpha/beta fold of triose phosphate isomerase and even a close structural equivalence between azurin and immunoglobulins are found.

Tissue-specific homing of lymphocytes is regulated by interactions with the endothelium of specialized venules, such as the high endothelial venules (HEV) in lymph nodes and mucosal lymphoid tissues. The mucosal vascular addressin, a 58-66K glycoprotein adhesion receptor for lymphocytes, is selectively expressed on HEV of mucosal lymphoid organ and on lamina propria venules and helps direct lymphocyte traffic to these mucosal tissues. We now report the isolation of a complementary DNA that, on transfection into COS cells, encodes immunoreactive addressin that specifically binds the mucosal HEV-binding T-cell lymphoma TK1. The predicted amino-acid sequence defines the mucosal addressin as a novel immunoglobulin family member, MAdCAM-1, with two amino-terminal domains that display strong homology to previously described vascular adhesion receptors for leukocytes, ICAM-1 (ref. 6) and VCAM-1 (ref. 7). The membrane proximal domain is homologous to the third domain (C alpha 2) of another mucosa-associated immunoglobulin family member, IgA1 (refs 8, 9). In addition to the immunoglobulin domains, there is a serine/threonine-rich region which may serve as a backbone to present carbohydrate ligands to lymphocytes. MAdCAM-1 is thus a complex multidomain receptor displaying several structural motifs that may participate in lymphocyte homing interactions.

Subarachnoid hemorrhage (SAH) causes secondary brain injury due to vasospasm and inflammation. Here, we studied a rat model of mild-to-moderate SAH intended to minimize ischemia/hypoxia to examine the role of sulfonylurea receptor 1 (SUR1) in the inflammatory response induced by SAH. mRNA for Abcc8, which encodes SUR1, and SUR1 protein were abundantly upregulated in cortex adjacent to SAH, where tumor-necrosis factor-alpha (TNFalpha) and nuclear factor (NF)kappaB signaling were prominent. In vitro experiments confirmed that Abcc8 transcription is stimulated by TNFalpha. To investigate the functional consequences of SUR1 expression after SAH, we studied the effect of the potent, selective SUR1 inhibitor, glibenclamide. We examined barrier permeability (immunoglobulin G, IgG extravasation), and its correlate, the localization of the tight junction protein, zona occludens 1 (ZO-1). SAH caused a large increase in barrier permeability and disrupted the normal junctional localization of ZO-1, with glibenclamide significantly reducing both effects. In addition, SAH caused large increases in markers of inflammation, including TNFalpha and NFkappaB, and markers of cell injury or cell death, including IgG endocytosis and caspase-3 activation, with glibenclamide significantly reducing these effects. We conclude that block of SUR1 by glibenclamide may ameliorate several pathologic effects associated with inflammation that lead to cortical dysfunction after SAH.

Immunoglobulin A (IgA) is prominently secreted at mucosal surfaces and coats a fraction of the intestinal microbiota. However, the commensal bacteria bound by IgA are poorly characterized and the type of humoral immunity they elicit remains elusive. We used bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) in murine models of immunodeficiency to identify IgA-bound bacteria and elucidate mechanisms of commensal IgA targeting. We found that residence in the small intestine, rather than bacterial identity, dictated induction of specific IgA. Most commensals elicited strong T-independent (TI) responses that originated from the orphan B1b lineage and from B2 cells, but excluded natural antibacterial B1a specificities. Atypical commensals including segmented filamentous bacteria and Mucispirillum evaded TI responses but elicited T-dependent IgA. These data demonstrate exquisite targeting of distinct commensal bacteria by multiple layers of humoral immunity and reveal a specialized function of the B1b lineage in TI mucosal IgA responses.

To further characterize the IgA deposits found in glomeruli of patients with IgA nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus, renal biopsies from patients with these disorders were stained by immunofluorescence with monoclonal anti-IgA subclass reagents, anti-light chain reagents and anti-J chain. The mesangium and peripheral capillary were brightly stained for IgA1 and were negative for IgA2. IgA1 and, to a lesser extent, IgA2 were contained in tubular casts. Both kappa and lambda light chains were found in all deposits. The intensity of J chain staining correlated with the intensity of IgM and not IgA staining. Biopsies brightly stained for IgA but negative for IgM were negative for J chain. These results indicate that glomerular IgA deposits in these disorders consist predominantly of monomers of IgA1.

Primary biliary cirrhosis (PBC) is characterized by antimitochondrial antibodies (AMAs), directed to the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Notwithstanding the presence of mitochondria in virtually all nucleated cells, the destruction in PBC is limited to small intrahepatic bile ducts. The reasons for this tissue specificity remain unknown, although biliary epithelial cells (BECs) uniquely preserve the PDC-E2 epitope following apoptosis. Notably, PBC recurs in an allogeneic transplanted liver, suggesting generic rather than host PBC-specific susceptibility of BEC. We used cultured human intrahepatic BECs (HIBECs) and other well-characterized cell lines, including, HeLa, CaCo-2 cells, and nontransformed human keratinocytes and bronchial epithelial cells, to determine the integrity and specific localization of PDC-E2 during induced apoptosis. All cell lines, both before and after apoptosis, were tested with sera from patients with PBC (n = 30), other autoimmune liver and rheumatic diseases (n = 20), and healthy individuals (n = 20) as well as with a mouse monoclonal antibody against PDC-E2 and AMA with an immunoglobulin A isotype. PDC-E2 was found to localize unmodified within apoptotic blebs of HIBECs, but not within blebs of various other cell lineages studied. The fact that AMA-containing sera reacted with PDC-E2 on apoptotic BECs without a requirement for permeabilization suggests that the autoantigen is accessible to the immune system during apoptosis.

CONCLUSIONS

Our data indicate that the tissue (cholangiocyte) specificity of the autoimmune injury in PBC is a consequence of the unique characteristics of HIBECs during apoptosis and can be explained by exposure to the immune system of intact immunoreactive PDC-E2 within apoptotic blebs.

Fc receptors for immunoglobulins are found on many immune cells and trigger essential functions of the immune defence system. With the exception of the high-affinity receptor for immunoglobulin E (Fc epsilon RI), these receptors were thought to consist of single polypeptides. Fc epsilon RI is a tetrameric complex of one alpha-subunit, one beta-subunit and two gamma-subunits. Here we report the cloning of a polypeptide identical to the gamma-chains of Fc epsilon RI, from mouse macrophages that do not express this receptor. Biosynthetic labelling and gene transfer together show that these gamma-chains associate with one of the macrophage receptors (Fc gamma RIIa). The human homologue, Fc gamma RIII (CD16), from natural killer cells is also expected to associate with gamma-chains. It is possible that these gamma-chains and the homologous zeta-chains of the T-cell antigen receptor belong to a new family of related proteins which share a common role in the signal transducing pathway.

The ability of differing subunit combinations of gamma-aminobutyric acid type A (GABAA) receptors produced from murine alpha 1, beta 2, and gamma 2L subunits to form functional cell surface receptors was analyzed in both A293 cells and Xenopus oocytes using a combination of molecular, electrophysiological, biochemical, and morphological approaches. The results revealed that GABAA receptor assembly occurred within the endoplasmic reticulum and involved the interaction with the chaperone molecules immunoglobulin heavy chain binding protein and calnexin. Despite all three subunits possessing the ability to oligomerize with each other, only alpha 1 beta 2 and alpha 1 beta 2 gamma 2L subunit combinations could produce functional surface expression in a process that was not dependent on N-linked glycosylation. Single subunits and the alpha 1 gamma 2L and beta 2 gamma 2L combinations were retained within the endoplasmic reticulum. These results suggest that receptor assembly occurs by defined pathways, which may serve to limit the diversity of GABAA receptors that exist on the surface of neurons.