Suppression of smooth muscle cell proliferation after experimental PTFE arterial grafting: a role for polyclonal anti-basic fibroblast growth factor (bFGF) antibody

The reciprocal regulation of T-helper cell (Th) subsets is widely documented in various animal models of infectious diseases. In this study IFN-gamma/IL-4 double knockout (DKO) mice were used to analyse the role of Th subsets in mucosal immune responses. We found that the DKO mice had normal IgA differentiation but impaired induction of specific gut mucosal antibody responses after oral immunization using cholera toxin adjuvant. Both Th1 and Th2 responses were reduced compared with wild-type mice. Despite the absence of both IFN-gamma and IL-4 in the DKO mice the overall results were similar to previous observations in IFN-gamma receptor-knockout (IFN-gammaR-/-) mice and did not suggest a strict cross-regulation of the two Th subsets in the gut mucosa. To further examine the role of IFN-gamma in mucosal immunity we compared two different mouse strains lacking IFN-gamma, i.e. IFN-gamma-/- (C57BL/6) and IFN-gammaR-/- mice (129/Sv). We found that IFN-gammaR-/- mice exhibited reduced mucosal antibody responses and decreased Th1 and Th2 activity after oral immunization, while IFN-gamma-/- mice had intact antibody responses and increased Th2 responses. Thus, genetic differences were found to critically affect the development of a specific gut mucosal immune response. An enhanced Th2 activity in the Peyer's patches following oral immunization was associated with an ability to mount strong intestinal IgA immunity.     

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily

It is known that lpr mice develop systemic lymphadenopathy and lupus erythematosus-like autoimmune disease that are associated with the accumulation of CD4- CD8- (double-negative; DN) CD3+ B220+ abnormal T cells as well as normal mature CD4+ or CD8+ single-positive (SP) CD3+ T cells. In order to clarify the role of B cells in the lymphoproliferation and autoimmunity of lpr mice, we created B-cell-deficient C57BL/6 (B6) lpr mice (B6lpr/lpr microMT/microMT) by crossing B6lpr/lpr mice with B6 microMT/microMT mice in which the B-cell development was arrested at pre-B stage owing to a targeted disruption of the immunoglobulin mu heavy-chain gene locus. In the B-cell-deficient B6-lpr mice, both lymphadenopathy and splenomegaly were markedly suppressed. Although the accumulation of both CD3+ B220- SP normal T cells and CD3+ B220+ DN abnormal T cells was inhibited in the B-cell-deficient lpr mice, the decrease in numbers of CD3+ B220- SP normal T cells occurred more strikingly than that of the CD3+ B220+ DN abnormal T cells. Glomerulonephritis did not develop in the B-cell-deficient lpr mice over 40 weeks. The present results indicate that the B cells thus play a crucial role in the extensive proliferation of normal CD3+ B220- mature SP T cells rather than the accumulation of abnormal DN T cells.     

Comparison of ceftriaxone, amikacin, and ciprofloxacin in treatment of experimental Yersinia enterocolitica O9 infection in mice

Ceftriaxone and ciprofloxacin were effective in the treatment of Yersinia enterocolitica O9 intestinal infection in mice. Amikacin was less effective. The impact of these drugs on indigenous bacteria from the intestinal microbiota was studied.     

Rotavirus-specific intestinal immune response in mice assessed by enzyme-linked immunospot assay and intestinal fragment culture

Primate rotavirus strain RRV and bovine strain WC3 or reassortants made between these animal viruses and human rotaviruses have been administered to infants as candidate vaccines. We compared RRV and WC3 in a murine model of oral infection. We determined the relative capacities of these viruses to induce a virus-specific humoral immune response by intestinal lymphocytes as tested by enzyme-linked immunospot assay, intestinal fragment culture, and enzyme-linked immunosorbent assay of intestinal contents. We found that inoculation of mice with RRV induced higher frequencies of virus-specific immunoglobulin A (IgA)-secreting cells in the lamina propria, greater quantities of virus-specific IgA in intestinal fragment cultures, and greater quantities of virus-specific IgA in intestinal secretions than did inoculation with WC3 or inactivated RRV (iRRV). The induction of an IgA response in serum was predictive of an IgA response among intestinal lymphocytes after inoculation with RRV but not WC3. In addition, large quantities of IgG, IgA, and IgM not specific for rotavirus were produced in fragment cultures from mice inoculated with RRV but not in cultures from mice inoculated with WC3 or iRRV. Possible mechanisms of RRV-induced polyclonal stimulation of intestinal B cells are discussed.     

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes

Although oral electrolyte solutions (OES) replenish salts and water lost during diarrhea, present formulations do not address disturbances of the normal intestinal microbiota. Therefore, we evaluated the efficacy of an OES with and without fructooligosaccharide (FOS) for treatment of pigs with acute secretory diarrhea induced by cholera toxin. Before, during, and after diarrhea, bacteriologic evaluation was made of contents collected from the mid small intestine, cecum, and distal colon and mucosa scraped from the mid small intestine. Diarrhea caused significant declines in total bacterial counts of contents from all three regions, with less of an impact on bacteria associated with the mucosa. Although total bacterial counts recovered within 24 hr, regardless of treatment, densities of Enterobacteriaceae were higher in pigs treated with OES whereas those receiving FOS had more lactobacilli. Our results show that secretory diarrhea disturbs the normal densities and relative species abundance of the microbiota, with the influences more pronounced for contents relative to the mucosa, and that adding FOS to OES accelerates the recovery of bacteria perceived as beneficial while potentially slowing the recovery of pathogenic forms.     

Secretory immunity in HIV infection

Secretory IgA plays a crucial role in the defense of pathogens at mucosal surfaces. As CD4+ T cells are lost early in the mucosa of human immunodeficiency virus (HIV)-infected patients and as CD4+ T cells play an essential role in the regulation of specific IgA responses to pathogenic agents at mucosal sides, it could be expected that this first line of defense is impaired in HIV-infected patients. Therefore, several studies were undertaken to characterize the humoral immune response at mucosal surfaces. However, the results obtained so far are in part contradictory. For intestinal IgA, reduced, increased and no changes compared to controls were described. The different results may be due to different methods applied. In most studies an abnormal predominance of HIV-specific IgG over IgA response was found in the intestine of HIV-infected patients. Studies on cytomegalovirus-specific intestinal antibodies indicate a complete lack of a specific intestinal IgA response. However, in cryptosporidiosis of HIV-infected patients, diarrhea persists despite a secretory IgA response indicating that other factors are also important for the clearance of this pathogen.     

Secretory and serum IgA in children with protein-calorie malnutrition

The local immune system of children suffering protein-calorie malnutrition (PCM) was investigated by analyzing the amount of immunoglobulins in the nasal washing on admission, repeatedly during 84 days of hospital therapy, and on follow-up, one to two years later. Although measured concentrations of total protein, IgG, and albumin in nasal washings were reduced in children with PCM, only secretory IgA concentrations were significantly lower (P less than .01) in PCM compared to normal children. Mean secretory IgA concentrations were significantly reduced on admission through hospital day 70 and returned to near normal thereafter. At one to two years after hospital discharge, mean concentrations of secretory IgA in nasal secretions were within normal limits. The concentrations of secretory IgA in nasal washings were lowest at a time when serum IgA was markedly elevated; serum IgA concentrations fell to normal values during dietary treatment. The possible role of secretory IgA deficiency in PCM and infection is discussed.     

Oral immunization with recombinant Norwalk virus-like particles induces a systemic and mucosal immune response in mice

Recombinant Norwalk virus-like particles (rNV VLPs) produced in insect cells were evaluated as an oral immunogen in CD1 and BALB/c mice by monitoring rNV-specific serum total and subclass immunoglobulin G (IgG) and intestinal IgA responses. Dose and kinetics of response were evaluated in the presence and absence of the mucosal adjuvant cholera toxin (CT). rNV-specific serum IgG and intestinal IgA were detected in the absence of CT, and the number of responders was not significantly different from that of mice administered VLPs with CT at most doses. The use of CT was associated with induction of higher levels of IgG in serum; this effect was greater at higher doses of VLPs. IgG in serum was detected in the majority of animals by 9 days postimmunization (dpi), and intestinal IgA responses were detected by 24 dpi. In the absence of CT, IgG2b was the dominant IgG subclass response in both mouse strains. Thus, nonreplicating rNV VLPs are immunogenic when administered orally in the absence of any delivery system or mucosal adjuvant. These studies demonstrate that rNV VLPs are an excellent model to study the oral delivery of antigen, and they are a potential mucosal vaccine for NV infections.     

Urease-specific monoclonal antibodies prevent Helicobacter felis infection in mice

Experiments were performed to determine the antigenic specificity of a monoclonal antibody (immunoglobulin A [IgA] 71) previously demonstrated to neutralize the ability of Helicobacter felis to colonize mice. Immunoprecipitation of radiolabeled H. felis outer membrane proteins with IgA 71 revealed specificity for a 62-kDa protein. Another of our monoclonal antibodies, IgG 40, precipitated a protein of similar molecular weight. IgA 71 but not IgG 40 also precipitated purified recombinant H. pylori urease. The antigenic specificity of both antibodies was confirmed to be urease by the ability of each to select Escherichia coli clones expressing the H. felis urease genes. The two antibodies were shown to bind nonoverlapping epitopes in a competition enzyme-linked immunosorbent assay. Both IgA 71 and IgG 40 could effectively neutralize H. felis infectivity by incubating the bacteria with the antibodies prior to oral administration to naive mice. The mechanism of protection does not appear to be inhibition of urease activity, as IgA 71 does not inhibit the conversion of urea to ammonia by H. pylori urease in vitro. These results support a protective role for the secretory humoral immune response in Helicobacter immunity and provide further evidence that the urease enzyme can serve as a protective antigen.     

Small intestine in lymphocytic and collagenous colitis: mucosal morphology, permeability, and secretory immunity to gliadin

There is a recognised association between the "microscopic" forms of colitis and coeliac disease. There are a variety of subtle small intestinal changes in patients with "latent" gluten sensitivity, namely high intraepithelial lymphocyte (IEL) counts, abnormal mucosal permeability, and high levels of secretory IgA and IgM antibody to gliadin. These changes have hitherto not been investigated in microscopic colitis. Nine patients (four collagenous, five lymphocytic colitis) with normal villous architecture were studied. Small intestinal biopsies were obtained by Crosby capsule; small intestinal fluid was aspirated via the capsule. IEL counts were expressed per 100 epithelial cells, and intestinal IgA and IgM antigliadin antibody levels were measured by ELISA. Small intestinal permeability was measured by the lactulose:mannitol differential sugar permeability test. IEL counts were normal in all cases, median 17, range 7-30. Intestinal antigliadin antibodies were measured in six cases and were significantly elevated in two patients (both IgA and IgM). Intestinal permeability was measured in eight cases and was abnormal in two and borderline in one. These abnormalities did not overlap: four of nine patients had evidence of abnormal small intestinal function. Subclinical small intestinal disease is common in the two main forms of microscopic colitis.     

Studies on translocation of immunoglobulins across intestinal epithelium. II. Immunoelectron-microscopic localization of immunoglobulins and secretory component in human intestinal mucosa

To define mechanisms involved in the transport of immunoglobulins into intestinal fluids, we localized IgM, IgA, IgG, and secretory component (SC) in human intestinal mucosa by the peroxidase-labeled antibody technique. At the light microscopic level, immunocytes containing IgA, IgM, or IgG were found in the lamina propria. IgA, IgM, and SC were prominent in the epithelium of gland crypts; IgG was limited to a few cells at tips of villi. At the electron-microscopic level, SC was localized to perinuclear spaces, endoplasmic reticulum, saccules associated with Golgi complexes, cytoplasmic vesicles, and lateral and basal plasma membranes of columnar epithelial cells. IgA and IgM, but not IgG, also were localized to plasma membranes and cytoplasmic vesicles of these cells. Neither the immunoglobulins nor SC was found within other types of epithelial cells (Paneth, goblet, endocrine). The findings provide evidence that (1) the site of SC synthesis in intestinal epithelium is secretory columnar cells, principally those in gland crypts; (2) the polymeric immunoglobulins IgM and IgA are translocated through such SC-containing cells by a process that involves formation of cytoplasmic vesicles; (3) IgM and IgA could combine with SC during transcellular transport (likely sites are lateral or basal plasma membranes or supranuclear cytoplasm); (4) the monomeric immunoglobulin IgG does not share the transepithelial cell route involved in IgM and IgA transport.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

In vitro synthesized immunoglobulin A from nu/+ and reconstituted nu/nu mice against a dominant surface antigen of Giardia lamblia

Nu/+ mice (ZU.ICR-strain) experimentally infected with Giardia lamblia (clone GS/M-83-H7) cleared the infection by day 45 postinfection (p.i.). Athymic nu/nu mice were reconstituted with immune Peyer's patch lymphocytes obtained from self-healed nu/+ littermates and thus acquired the potential to decrease their intestinal parasite mass. Intestinal B-cells from self-healed nu/+ mice as well as from immune-reconstituted athymic nude mice synthesized in vitro parasite-specific immunoglobulin A (IgA). This IgA was subsequently analyzed by immunoblotting, showing a predominant reaction with the major surface antigen (a 72,000-Da polypeptide) characterizing the Giardia clone in question. The hypothesis on the causative role of intestinal IgA and immune lymphocytes in the control of G. lamblia infection thus deserves further attention.     

Secretory immune response in patients with intestinal amoebiasis

The secretory immune response in saliva from intestinal amoebiasis patients against antigens obtained from Entamoeba histolytica membranes was studied. Western blot analysis indicated that patient saliva contains secretory IgA antibodies against antigens with molecular masses ranging from 170 to 24 kDa, some of which were also recognized by saliva from healthy subjects. However, antigens of 170, 125, 46 and 37 kDa are recognized more frequently (> 90%) by the secretory IgA from patients with intestinal amoebiasis than by that from healthy subjects (< 10%).     

Interaction with secretory component stimulates effector functions of human eosinophils but not of neutrophils

Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component bound to IgA mediates transepithelial transport of IgA and confers increased stability on the resultant secretory IgA; however, the effect of secretory component on the biologic activity of IgA is unknown. Here, we report that secretory IgA and secretory component preferentially activate human eosinophils. When eosinophils were stimulated with immobilized secretory IgA, degranulation and superoxide production were two- to threefold greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to secretory IgA and serum IgA. Flow cytometric analysis showed that eosinophils bound to purified secretory component. The binding of 125I-labeled secretory component was inhibited by unlabeled secretory component or secretory IgA but not by serum IgA. Superoxide production by eosinophils stimulated with cytokines or IgG was enhanced synergistically by immobilized secretory component; secretory component showed no effect on neutrophil activation. Finally, anti-CD18 mAb abolished eosinophil superoxide production stimulated with secretory IgA or secretory component but not with serum IgA, suggesting a crucial role for beta2 integrins in eosinophil interactions with secretory IgA or secretory component. Thus, secretory component plays important roles in activating eosinophil functions but not neutrophil functions. This preferential interaction between secretory component and eosinophils may provide a novel mechanism to regulate mucosal tissue inflammation.     

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.     

Primary prevention of heart disease and stroke: a simplified approach to estimating risk of events and making drug treatment decisions

We evaluated the ability of mice made genetically deficient for B cells to resolve a primary infection and to develop protective immunity against vaginal challenge with a human isolate of Chlamydia trachomatis bacteria. The B-cell-deficient microMT mice cleared a primary ascending infection with similar or faster kinetics compared with wild-type mice. The presence of chlamydial inclusion bodies and the degree of inflammation in the upper genital tract was comparable and showed similar kinetics in microMT as in wild-type mice. Following resolution of the primary infection the mice were challenged by 100 ID50 of live bacteria and the level of protection and the extent of local inflammation was assessed. Strikingly, all microMT mice, as well as most of the wild-type mice, demonstrated complete immune protection with no bacterial shedding. While high titres of chlamydia-specific antibodies were stimulated locally and systemically in wild-type mice, no antibodies were detected in microMT mice. However, in both strains, immunohistochemical analysis of the upper genital tract demonstrated the presence of large numbers of CD4+ T cells and increased levels of interferon-gamma (IFN-gamma)-producing cells. The results unequivocally demonstrate that antibodies are not required for full protection to develop against ascending infection with a high dose of C. trachomatis in the female genital tract. Our study confirms the notion that cell-mediated immunity, in particular that owing to CD4+ T helper I (Th1)-type cells, is critical for host resistance against C. trachomatis in mice.     

Continued differentiation during B lymphopoiesis requires signals in addition to cell survival

During B lymphopoiesis, cells undergo successive rounds of division and growth arrest coupled to intermittent selection on the basis of Ig expression. It is unresolved whether differentiation requires specific signaling or is merely the consequence of sustained cell survival. Transgenic expression of the cell death antagonist, Bcl-2, promoted accumulation of B lymphoid cells in mice deficient in antigen receptor rearrangement (scid or rag-1-/-) and in mice lacking the IgM transmembrane domain (microMT). Continued differentiation occurred, however, only in the bcl-2/scid and bcl-2/microMT mice. The appearance of B lineage cells expressing CD21, CD22 and CD23 was associated with DHJH rearrangements which encode a truncated C mu-containing protein called D mu in bcl-2/scid mice and with expression of Ig heavy chain classes other than IgM in the bcl-2/ microMT mice. In neither case, however, were proliferating cells observed in the more mature B lineage compartments in the bone marrow. Thus, continued B cell development requires signaling via Ig heavy chain-containing receptors and is not simply a consequence of blocking apoptosis.     

Divergence between biochemical and cytogenetic differences in three species of the Callicebus moloch group

Four viruses were compared for their ability to induce an intestinal antibody response in piglets. Antibodies were not detected in response to oral vaccination with either fowlpox virus or a baculovirus (BV). Simultaneous oral dosing and parenteral inoculation with high concentrations of BV in an oil emulsion adjuvant induced high levels of circulating virus neutralising (VN) antibodies, and also low levels of intestinal antibodies when booster doses of virus were given. In response to oral vaccination with swinepox virus (SPV), low levels of circulating and intestinal VN antibodies, and higher titres of antibodies reactive in an enzyme immunoassay, including intestinal antibodies of the IgA class, were detected. Oral vaccination with porcine adenovirus type 3 (PAV-3) stimulated both circulating and intestinal VN antibodies, and IgA antibodies were demonstrated in the intestinal contents. It was concluded that SPV and PAV-3 might be suitable vectors for the expression of genes encoding the protective antigens of porcine enteric viruses.     

gamma/delta T cell-deficient mice have impaired mucosal immunoglobulin A responses

Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCRdelta-/-) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCRdelta-/- mice. The TCRdelta-/- mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toxoid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toxoid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody-producing cells were present in the intestinal lamina propria and Peyer's patches of TCRdelta-/- mice compared with the orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta cells in mucosal immunity.