Estrogen is a negative regulator of lymphopoiesis and provides an experimental tool for probing relationships between lymphocyte precursors and stem cells. We found that expression of lymphocyte-associated genes and immunoglobulin (Ig) gene rearrangement occurred before CD45R acquisition. Lymphoid-restricted progenitors that were Lin(-)IL-7R alpha(+)c-kit(lo)TdT(+) (lineage marker(-), interleukin receptor 7 alpha(+), c-kit(lo) and terminal deoxynucleotidyl transferase(+)) were selectively depleted in estrogen-treated mice; within a less differentiated Lin-c-kit(hi) fraction, functional precursors of B and T, but not myeloid, cells were also selectively depleted. TdT and an Ig heavy chain transgene were detected within a hormone-regulated Lin(-)c-kit(hi)Sca-1(+)CD27(+)Flk-2(+)IL-7R alpha(-) subset of this multipotential progenitor population. Identification of these extremely early lymphoid precursors should facilitate investigation of the molecular mechanisms that control lineage-fate decisions in hematopoiesis.

The multiligand, endocytic receptors megalin and cubilin are colocalized in the renal proximal tubule. They are heavily expressed in the apical endocytic apparatus. Megalin is a 600-kDa transmembrane protein belonging to the low-density lipoprotein-receptor family. The cytoplasmic tail contains three NPXY motifs that mediate the clustering in coated pits and are possibly involved in signaling functions. Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is a 460-kDa receptor with no transmembrane domain and no known signal for endocytosis. Because the two receptors bind each other with high affinity and colocalize in several tissues, it is highly conceivable that megalin mediates internalization of cubilin and its ligands. Both receptors are important for normal tubular reabsorption of proteins, including albumin. Among the proteins normally filtered in the glomeruli, cubilin has been shown to bind albumin, immunoglobulin light chains, and apolipoprotein A-I. The variety of filtered ligands identified for megalin include vitamin-binding proteins, hormones, enzymes, apolipoprotein H, albumin, and beta(2)- and alpha(1)-microglobulin. Loss of these proteins and vitamins in the urine of megalin-deficient mice illustrates the physiological importance of this receptor.

Yeast forms of Paracoccidioides brasiliensis grown in liquid medium produced exocellular components. Immunodiffusion reactions and immunoprecipitations of 131I-radiolabeled antigenic components with sera from patients having paracoccidioidomycosis (PCM) were used to monitor the isolation of specific constituents. Components having the main antigenic activity (fCon A) were isolated by exclusion from a Bio-Gel P30 column, followed by successive binding of eluted material to a Sepharose-concanavalin A column, and elution. The product contained, from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, a minor 43,000-molecular-weight (MW) component (gp43), a polydisperse high-MW glycoconjugate, and a diffusely migrating 55,000-MW glycoprotein (gp55). Other components, including a 72,000-MW glycoprotein, were irregularly expressed. The high-MW glycoconjugate complex contained, on the basis of methylation and 13C nuclear magnetic resonance data, a branched structure of mainly mannopyranosyl units. These were nonreducing ends, 6-O-, 2-O-, and 2,6-di-O-substituted, and the specific rotation of +16 degrees indicated that the glycosidic configurations of the units were alpha and beta in a ratio of ca. 1:1 (concanavalin A binding indicated that nonreducing ends or 2-O-substituted units or both of alpha-D-mannopyranose were present). A small proportion of nonreducing end units of D-galactopyranose were also present in this polysaccharide. gp55 is a glycoprotein containing a complex carbohydrate moiety with fucose, mannose, galactose, and glucose, either as terminal nonreducing units or substituted in positions indicated by methylation data. Both PCM and normal human sera precipitated the high-MW glycoconjugate from 131I-labeled fCon A preparations, whereas gp55 was unreactive with human sera. gp43 was a specific antigenic component of P. brasiliensis culture filtrates which could be isolated in a pure form by gel filtration column chromatography (Sephadex G150) or by Sepharose-patient immunoglobulin G affinity chromatography. 131I-labeled gp43 reacted equally well with 10 PCM sera and hyperimmune rabbit serum against the band E antigen of Yarzabal at a 10(-3) dilution. At the same dilution, no reaction was detected with sera from normal individuals and from patients with other mycoses. Similarly, only PCM sera and the hyperimmune anti-E serum gave precipitin lines with gp43 in the less sensitive immunodiffusion tests. gp43 consisted of three components, with pI 6.7, 6.4 and 6.2, all of which reacted with PCM serum.

BACKGROUND

Streptococcal protein G comprises two or three domains that bind to the constant Fc region of most mammalian immunoglobulin Gs (IgGs). Protein G is functionally related to staphylococcal protein A, with which it shares neither sequence nor structural homology.

RESULTS

To understand the competitive binding of these two proteins to the Fc region, the crystal structure of a single Ig-binding domain of streptococcal protein G was determined at 3.5 A resolution in complex with the Fc fragment of human IgG and compared with the structures of protein A:Fc and protein G:Fab complexes. Protein G binds to the interface between the second and third heavy chain constant domains of Fc, which is roughly the same binding site used by protein A. Protein G comprises one alpha-helix packed onto a four-stranded beta-sheet. Residues from protein G that are involved in binding are situated within the C-terminal part of the alpha-helix, the N-terminal part of the third beta-strand and the loop region connecting these two structural elements. The identified Fc-binding region of protein G agrees well with both biochemical and NMR spectroscopic data. However, the Fc-binding helices of protein G and protein A are not superimposable.

CONCLUSIONS

Protein G and protein A have developed different strategies for binding to Fc. The protein G:Fc complex involves mainly charged and polar contacts, whereas protein A and Fc are held together through non-specific hydrophobic interactions and a few polar interactions. Several residues of Fc are involved in both the protein G:Fc and the protein A:Fc interaction, which explains the competitive binding of the two proteins. The apparent differences in their Fc-binding activities result from additional unique interactions.

Immunoglobulin A has long been referred to as the antibody of mucosal secretions. However, in the last few years there has been increased interest in serum IgA, in factors that regulate the various forms of IgA produced, and in the relationship between serum and mucosal IgA. Recent data indicate that in humans, the mucosal surfaces are neither the source nor the destination of serum IgA. By estimating the amounts of IgA produced at various sites throughout the body, we have shown that in humans more IgA is synthesized and secreted each day than IgG and IgM combined; about one third of this IgA is secreted directly into the vascular compartment and never reaches the mucosal surfaces. We also consider a possible role for serum IgA as the "discreet housekeeper," the relationship between cells that produce IgA in the serum and IgA in secretions, and factors that influence the various forms of IgA produced.

Several receptors and counter-receptor pairs on T cells and on antigen-presenting cells (APCs) deliver costimulatory signals to T cells during antigen presentation. The CD28 receptor on T cells with its ligand B7 represents one of the best characterized and most important examples of this costimulation. We show here that interleukin 12 (IL-12), a cytokine also produced by APCs (monocyte/macrophages and B cells) and active on T and natural killer cells, has a strong synergistic effect with the B7/CD28 interaction in inducing proliferation and cytokine production in both mitogen-activated and freshly isolated peripheral blood T cells. Together with anti-CD28 antibodies, IL-12 induces proliferation of T cells to levels higher than those obtained with IL-2 stimulation and it is effective at IL-12 concentrations 100- to 1,000-fold lower than effective concentrations of IL-2. The proliferative effect of anti-CD28 and IL-12 is resistant to moderate doses of cyclosporin A and is largely independent of endogenous IL-2, IL-12, in synergy with anti-CD28 or B7-transfected cells, is most effective in inducing interferon gamma (IFN-gamma) production, but production of tumor necrosis factor alpha and granulocyte/macrophage colony-stimulating factor is also observed. IL-12-induced IFN-gamma production in peripheral blood mononuclear cells is inhibited by the chimeric molecule CTLA-4 immunoglobulin, which prevents binding of CD28 to B7, suggesting that endogenous B7 on the mononuclear cells and IL-12 cooperate in inducing IFN-gamma production. IL-10 inhibits both IL-12 production and B7 expression on monocytes. These two effects are largely responsible for the ability of IL-10, acting on accessory cells, to inhibit IFN-gamma production by lymphocytes, because anti-CD28 antibodies and IL-12 can reverse the inhibitory effect of IL-10 on IFN-gamma production. Our results in vitro suggest that the synergy between B7 and IL-12, a surface antigen and a soluble product of APCs, respectively, plays a role in regulating T cell activation and immune response in the microenvironment of inflamed tissues.

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.

Keywords: COVID-19; IgA; SARS-CoV-2; T cells; antibody; intranasal; mucosal immunity; pathogenesis; protection; vaccine.

OBJECTIVE

To determine whether long-term supplementation with vitamin E enhances in vivo, clinically relevant measures of cell-mediated immunity in healthy elderly subjects.

METHODS

Randomized, double-blind, placebo-controlled intervention study.

METHODS

A total of 88 free-living, healthy subjects at least 65 years of age.

METHODS

Subjects were randomly assigned to a placebo group or to groups consuming 60, 200, or 800 mg/d of vitamin E for 235 days.

METHODS

Delayed-type hypersensitivity skin response (DTH); antibody response to hepatitis B, tetanus and diphtheria, and pneumococcal vaccines; and autoantibodies to DNA and thyroglobulin were assessed before and after supplementation.

RESULTS

Supplementation with vitamin E for 4 months improved certain clinically relevant indexes of cell-mediated immunity in healthy elderly. Subjects consuming 200 mg/d of vitamin E had a 65% increase in DTH and a 6-fold increase in antibody titer to hepatitis B compared with placebo (17% and 3-fold, respectively), 60-mg/d (41% and 3-fold, respectively), and 800-mg/d (49% and 2.5-fold, respectively) groups. The 200-mg/d group also had a significant increase in antibody titer to tetanus vaccine. Subjects in the upper tertile of serum alpha-tocopherol (vitamin E) concentration (>48.4 micromol/L [2.08 mg/dL]) after supplementation had higher antibody response to hepatitis B and DTH. Vitamin E supplementation had no effect on antibody titer to diphtheria and did not affect immunoglobulin levels or levels of T and B cells. No significant effect of vitamin E supplementation on autoantibody levels was observed.

CONCLUSIONS

Our results indicate that a level of vitamin E greater than currently recommended enhances certain clinically relevant in vivo indexes of T-cell-mediated function in healthy elderly persons. No adverse effects were observed with vitamin E supplementation.

HLA class II molecules have a crucial role in the immune response to antigens. We have isolated two new class II-like complementary DNA sequences, RING6 and RING7, which map between the HLA-DNA and -DOB loci. They are novel members of the immunoglobulin gene family which may have diverged before the duplications that gave rise to the main class II loci. The RING6 and RING7 genes seem to encode alpha- and beta-chains of a previously undiscovered class II-related protein.

Invariant natural killer T (NKT) cells are a highly conserved subset of T lymphocytes expressing a semi-invariant T cell receptor (TCR), which is restricted to CD1d and specific for the glycosphingolipid antigen alpha-galactosylceramide. Their ability to secrete a variety of cytokines, which in turn modulate the activation of cells of both innate and acquired immune responses, suggests that invariant NKT cells exert a regulatory role mainly via indirect mechanisms. A relevant question is whether invariant NKT cells can directly help B cells. We document here that human invariant NKT cells are as efficient as conventional CD4+ Th0 lymphocytes in promoting proliferation of autologous memory and naive B lymphocytes in vitro, and in inducing immunoglobulin production. Help to B cells by invariant NKT cells is CD1d-dependent and delivered also in the absence of alpha-galactosylceramide, suggesting that NKT cells recognize an endogenous ligand presented by CD1d on B cells. The two major subsets of invariant NKT cells, CD4+ and double negative (CD4-CD8-), express comparable levels of CD40 ligand and cytokines, but differ in helper functions. Indeed, both subsets induce similar levels of B cell proliferation, whereas CD4+ NKT cells induce higher levels of immunoglobulin production. These results suggest a direct role for invariant NKT cells in regulating B lymphocyte proliferation and effector functions.

Many of the cell fate decisions in precursor B cells and more mature B cells are controlled by membrane immunoglobulin (Ig)M heavy chain (mu) and the Ig alpha-Ig beta signal transducers. The role of Ig beta in regulating early B cell development was examined in mice that lack Ig beta (Ig beta-/-). These mice had a complete block in B cell development at the immature CD43+B220+ stage. Immunoglobulin heavy chain diversity (DH) and joining (JH) segments rearranged, but variable (VH) to DJH recombination and immunoglobulin messenger RNA expression were compromised. These experiments define an unexpected, early requirement for Ig(beta) to produce B cells that can complete VDJH recombination.

Traditionally, immunoglobulin A (IgA) was thought to neutralize virus by forming complexes with viral attachment proteins, blocking attachment of virions to host epithelial cells. Recently we have proposed an intracellular action for dimeric IgA, which is actively transported through epithelial cells by the polymeric immunoglobulin receptor (pIgR), in that it may be able to bind to newly synthesized viral proteins within the cell, preventing viral assembly. To this effect, we have previously demonstrated that IgA monoclonal antibodies against Sendai virus, a parainfluenza virus, colocalize with the viral hemagglutinin-neuraminidase protein within infected epithelial cells and reduce intracellular viral titers. Here we determine whether IgA can interact with influenza virus hemagglutinin (HA) protein within epithelial cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the pIgR were infected on their apical surfaces with influenza virus A/Puerto Rico/8-Mount Sinai. Polymeric IgA anti-HA, but not IgG anti-HA, delivered to the basolateral surface colocalized with HA protein within the cell by immunofluorescence. Compared with those of controls, viral titers were reduced in the supernatants and cell lysates from monolayers treated with anti-HA IgA but not with anti-HA IgG. Furthermore, the addition of anti-IgA antibodies to supernatants did not interfere with the neutralizing activity of IgA placed in the basal chamber, indicating that IgA was acting within the cell and not in the extracellular medium to interrupt viral replication. Thus, these studies provide additional support for the concept that IgA can inhibit replication of microbial pathogens intracellularly.

OBJECTIVE

Oral immunization with Helicobacter pylori urease can cure Helicobacter infection in animals. As a step toward therapeutic immunization in humans, the safety and immunogenicity of oral immunization with recombinant H. pylori urease were tested in H. pylori-infected adults.

METHODS

Twenty-six H. pylori-infected volunteers were randomized in a double-blind study to four weekly oral doses of 180, 60, or 20 mg of urease with 5 microg heat-labile enterotoxin of Escherichia coli (LT), LT alone, or placebo. Side effects and immune responses were evaluated weekly after immunization, and gastric biopsy specimens were obtained after 1 month and 6 months for histology and quantitative cultures.

RESULTS

Diarrhea was noted in 16 of 24 (66%) of the volunteers who completed the study. Antiurease serum immunoglobulin A titers increased 1. 58-fold +/- 0.37-fold and 3.66-fold +/- 1.5-fold (mean +/- SEM) after immunization with 60 and 180 mg urease, respectively, whereas no change occurred in the placebo +/- LT groups (P = 0.005). Circulating antiurease immunoglobulin A-producing cells increased in volunteers exposed to urease compared with placebo (38.9 +/- 13. 6/10(6) vs. 5.4 +/- 3.1; P = 0.018). Eradication of H. pylori infection was not observed, but urease immunization induced a significant decrease in gastric H. pylori density.

CONCLUSIONS

H. pylori urease with LT is well tolerated and immunogenic in H. pylori-infected individuals. An improved vaccine formulation may induce curative immunity.

Due to their vast surface area, the mucosal surfaces of the body represent a major site of potential attack by invading pathogens. The secretions that bathe mucosal surfaces contain significant levels of immunoglobulins (Igs), which play key roles in immune defense of these surfaces. IgA is the predominant antibody class in many external secretions and has many functional attributes, both direct and indirect, that serve to prevent infective agents such as bacteria and viruses from breaching the mucosal barrier. This review details current understanding of the structural and functional characteristics of IgA, including interaction with specific receptors (such as Fc(alpha)RI, Fc(alpha)/microR, and CD71) and presents examples of the means by which certain pathogens circumvent the protective properties of this important Ig.

BACKGROUND

Fabry disease is an X-linked inherited disorder that is caused by excessive lysosomal globotriaosylceramide (CTH) storage due to a deficiency in alpha-galactosidase A (alpha-Gal A). Two recombinant enzyme preparations have been approved as treatment modality. We studied emergence and properties of alpha-Gal A antibodies in treated patients.

METHODS

During the first 6 to 12 months of intravenous administration of recombinant enzymes (rh-alpha-Gal A) formation of antibodies was studied in 18 adult Fabry patients (two females).

RESULTS

The female patients did not develop detectable amounts of antibodies following enzyme therapy. After 6 months of treatment with either agalsidase alpha or beta, 11/16 male patients showed high titers of immunoglobulin G (IgG) antibodies that cross-react in vitro similarly with both recombinant enzymes. The anti-rh-alpha-Gal A IgG neutralizes rh-alpha-Gal A activity in vitro for 65% to 95%. During infusion with rh-alpha-Gal A, circulating enzyme-antibody complexes are formed and these complexes are taken up by leukocytes in the peripheral blood. After 6 months of treatment all IgG-negative patients showed a significant (P < 0.01) reduction of urinary CTH (1890 +/- 797 to 603 +/- 291 nmol CTH/24hr urine), compared to IgG-positive patients (mean increase from 2535 +/- 988 to 2723 +/- 1212), suggesting a negative effect of circulating antibodies on renal tubular CTH clearance.

CONCLUSIONS

Emergence of antibodies with in vivo neutralizing capacities is frequently encountered in treated Fabry disease patients. Complete cross-reactivity of these antibodies suggests that it is unlikely that switching from one to the other recombinant protein prevents the immune response and related effects. Further studies on the clinical implications of alpha-Gal A antibodies are essential.

T lymphocytes recognize cell-bound antigens in the molecular context of the self major histocompatibility complex (MHC) gene products through the surface T-cell receptor(s). The minimal component of the T-cell receptor is a heterodimer composed of alpha and beta subunits, each of relative molecular mass (Mr) approximately 45,000 (refs 1-3). Recently, complementary DNA clones encoding these subunits have been isolated and characterized along with that of a third subunit of unknown function, termed gamma (refs 4-9). These studies revealed a primary structure for each subunit that was clearly similar to that of immunoglobulin and indicated a somatic rearrangement of corresponding genes that are also immunoglobulin-like. Recently, the analysis of the sequence organization of the T-cell receptor beta-chain and T-cell-specific gamma-chain gene families has been reported. We now present an initial characterization of the murine T-cell receptor alpha-chain gene family, and conclude that although it is clearly related to the gene families encoding immunoglobulins, T-cell receptor beta-chains and also T-cell gamma-chains, it shows unique characteristics. There is only a single constant (C) region gene segment, which is an exceptionally large distance (approximately 20-40 kilobases (kb) in the cases studied here) from joining (J) gene segments. In addition, the J cluster and the variable (V) segment number seen to be very large. Finally, in the case studied here, a complete alpha-chain gene shows no somatic mutation and can be assembled directly from V alpha, J alpha and C alpha segments without inclusion of diversity (D alpha) segments.

The mucosal secretory immune system provides an important primary defence against disease, as studies of humans with mucosal humoral immunodeficiencies suggest that the absence of secretory immunoglobulin A leads to an increase in mucosal infections. However, the infection risks posed do not seem to provide the evolutionary drive to retain constitutive secretion of often 'hard won' protein, suggesting that secretory antibodies may have some other important function (or functions). This Review examines the evidence that secretory antibodies provide an important defence against infection in specific animal models and explores complementary explanations for the evolution of the secretory immune system.

The gastrointestinal mucosa is the largest reservoir of macrophages in the body. These important effector cells are derived from blood monocytes that are recruited to the lamina propria by endogenous chemoattractants in the non-inflamed mucosa and by inflammatory chemokines and bacterial products during inflammation. In the non-inflamed mucosa, newly recruited pro-inflammatory monocytes are exposed to lamina propria stromal (extracellular matrix) factors that induce phenotypic and functional differentiation into non-inflammatory macrophages. As a consequence of this differentiation, resident lamina propria macrophages are strikingly downregulated for the expression of innate response receptors, such as the receptors for lipopolysaccharide, immunoglobulin G (IgG), and IgA, and the production of pro-inflammatory cytokines, including interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-alpha. Despite downregulated pro-inflammatory function, strong phagocytic and bactericidal activities remain intact. Thus, in the non-inflamed intestinal mucosa, lamina propria macrophages are non-inflammatory but retain avid scavenger and host defense functions, a unique but ideal phenotype and functional profile for effector cells in close proximity to immunostimulatory microorganisms and products.

The amino acid sequence of major outer membrane protein II (ompA protein) from Escherichia coli K-12 has been determined. The transmembrane polypeptide consists of 325 residues, resulting in a molecular weight of 35,159. The transmembrane part of the protein is located between residues 1 and 177. In this part of the protein a predominantly lipophilic 27-residue segment exists that perhaps spans the membrane in a mostly alpha-helical conformation, or a 19-residue stretch of this segment might traverse the membrane linearly. Inside the outer membrane a sequence -Ala-Pro-Ala-Pro-Ala-Pro-Ala-Pro- exists that, analogous to the -Cys-Pro-Pro-Cys-Pro- sequence in the hinge region of immunoglobulin, could assume the conformation of a polyproline helix. Computer analysis did not reveal a clear overall pattern of internal homology in the protein; besides the -Ala-Pro- repeat, only one local area (two adjacent dodecapeptide segments) shows some repetitiveness. The same analysis did not produce evidence for internal homology in the previously determined sequence of outer membrane protein I (porin) nor was any marked resemblance detected between transmembrane proteins I and II.

The initiation of immunoglobulin-E (IgE)-mediated allergic responses requires the binding of IgE antibody to its high-affinity receptor, Fc epsilonRI. Crosslinking of Fc epsilonRI initiates an intracellular signal transduction cascade that triggers the release of mediators of the allergic response. The interaction of the crystallizable fragment (Fc) of IgE (IgE-Fc) with Fc epsilonRI is a key recognition event of this process and involves the extracellular domains of the Fc epsilonRI alpha-chain. To understand the structural basis for this interaction, we have solved the crystal structure of the human IgE-Fc-Fc epsilonRI alpha complex to 3.5-A resolution. The crystal structure reveals that one receptor binds one dimeric IgE-Fc molecule asymmetrically through interactions at two sites, each involving one C epsilon3 domain of the IgE-Fc. The interaction of one receptor with the IgE-Fc blocks the binding of a second receptor, and features of this interaction are conserved in other members of the Fc receptor family. The structure suggests new approaches to inhibiting the binding of IgE to Fc epsilonRI for the treatment of allergy and asthma.

Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins. Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells. While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors. On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells. In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C. We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria. Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA). Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H. influenzae and appear to use a export system for secretion. We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E. coli (RDEC-1). Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains. To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays. We found that EspC is not necessary for mediating EPEC-induced signal transduction in HeLa epithelial cells and does not play a role in adherence or invasion of tissue culture cells.

The interleukin-2 receptor alpha (IL-2R alpha) chain gene contains a sequence similar to the immunoglobulin (Ig) kappa (kappa) enhancer NF-kappa B binding site. This site, which is bound by the nuclear protein, NF-kappa B, is critical for Ig kappa gene expression. The major T cell nuclear factor that binds to the IL-2R alpha site in vitro appears indistinguishable from NF-kappa B. NF-kappa B binds to IL-2R alpha and kappa sequences with similar affinities; however, only the kappa site potently activates transcription from heterologous promoters. Thus, high-affinity NF-kappa B binding in vitro cannot be equated with transcriptional activation in vivo. Mutation of the NF-kappa B binding site in the context of an IL-2 R alpha promoter construct markedly diminished promoter activity in human T cell lymphotropic virus type I (HTLV-I)-transformed MT-2 cells but not in phorbol myristate acetate-stimulated Jurkat T cells.

Candida albicans ALS3 encodes a large cell-surface glycoprotein that has adhesive properties. Immunostaining of cultured C. albicans germ tubes showed that Als3p is distributed diffusely across the germ tube surface. Two-photon laser scanning microscopy of model catheter biofilms grown using a PALS3-green fluorescent protein (GFP) reporter strain showed GFP production in hyphae throughout the biofilm structure while biofilms grown using a PTPI1-GFP reporter strain showed GFP in both hyphae and yeast-form cells. Model catheter biofilms formed by an als3 Delta/als3 Delta strain were weakened structurally and had approximately half the biomass of a wild-type biofilm. Reintegration of a wild-type ALS3 allele restored biofilm mass and wild-type biofilm structure. Production of an Als3p-Ag alpha 1p fusion protein under control of the ALS3 promoter in the als3 Delta/als3 Delta strain restored some of the wild-type biofilm structural features, but not the wild-type biofilm mass. Despite its inability to restore wild-type biofilm mass, the Als3p-Ag alpha 1p fusion protein mediated adhesion of the als3 Delta/als3 Delta C. albicans strain to human buccal epithelial cells (BECs). The adhesive role of the Als3p N-terminal domain was further demonstrated by blocking adhesion of C. albicans to BECs with immunoglobulin reactive against the Als3p N-terminal sequences. Together, these data suggest that portions of Als3p that are important for biofilm formation may be different from those that are important in BEC adhesion, and that Als3p may have multiple functions in biofilm formation. Overexpression of ALS3 in an efg1 Delta/efg1 Delta strain that was deficient for filamentous growth and biofilm formation resulted in growth of elongated C. albicans cells, even under culture conditions that do not favour filamentation. In the catheter biofilm model, the ALS3 overexpression strain formed biofilm with a mass similar to that of a wild-type control. However, C. albicans cells in the biofilm had yeast-like morphology. This result uncouples the effect of cellular morphology from biofilm formation and underscores the importance of Als3p in biofilm development on silicone elastomer surfaces.