T cell cytokine polarity as a determinant of immunoglobulin A (IgA) glycosylation and the severity of experimental IgA nephropathy.

Immunoglobulin A (IgA) glycosylation, recognized as an important pathogenic factor in IgA nephropathy (IgAN), is apparently controlled by the polarity of T helper (Th) cytokine responses. To examine the role of cytokine polarity in IgAN, inbred mice were immunized by intraperitoneal priming with inactivated Sendai virus (SeV) emulsified in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), which promote Th1- or Th2-immune response, respectively, and then boosted identically twice orally with aqueous suspensions of inactivated virus. Next, some mice were challenged intranasally with infectious SeV. Mice primed with CFA or IFA had equal reductions in nasal viral titre relative to non-immune controls, and equally increased serum levels of SeV-specific IgA antibody. Mice primed with CFA showed higher SeV-specific IgG than those with IFA. Splenocytes from mice primed with IFA produced copious amounts of interleukin (IL)-4 and IL-5, but little interferon-gamma and IL-2; those primed with CFA had reciprocal cytokine recall responses. Total serum IgA and especially SeV-specific IgA from mice primed with IFA showed a selective defect in sialylation and galactosylation. Although the frequency and intensity of glomerular deposits and haematuria did not differ, glomerulonephritis in mice primed with IFA and challenged with infectious virus was more severe than in those given CFA, as judged by serum creatinine level. We conclude that the polarity of T cell cytokines controls the pattern of IgA glycosylation and exerts direct or indirect effects on functional glomerular responses to immune complex deposition.

Explantation and subculture of epithelial cells from human uterine ectocervix.

Human ectocervical explant cultures were grown in medium with D-valine substituted for L-valine. Pure epithelial cell monolayers were obtained with both dialyzed and undialyzed fetal calf serum. Epithelial cell explant colonies grown in D-valine medium supplemented with undialyzed serum could routinely be subcultured if plated at a density of 1.5 x 10(4) cells/sq cm or higher. Such cultures could be passaged at least three times and could yield up to 21 population doublings per culture lifetime. Contaminating fibroblastic colonies were never detected in these cultures, which were free of surface-associated fibronectin as revealed by immunofluorescent tests. Both primary and passaged epithelial colonies retained many characteristic morphological features of ectocervical epithelium when examined by light and electron microscopy. Such cultures may be of use in investigating the action of viral and chemical carcinogenic agents upon epithelial cells in vitro.

Oral immunization with Sendai virus in mice.

We have shown that in mice cholera toxin can be an effective adjuvant for gastrointestinal immune responses against a virus. The adjuvant properties can be increased and even dissociated from the toxic properties if virus and toxoid are covalently linked. Finally, oral immunization with these preparations of cholera toxin/toxoid and Sendai virus can be used to prime for respiratory immune responses to Sendai virus in which protection from infection correlates with IgA in the upper and with IgG in the lower respiratory tract.

Host response and vaccine development to Helicobacter pylori infection.

Studies in both humans and animals demonstrate that H. pylori is capable of illiciting an innate response that in part is regulated by the genetic makeup of the host. These innate responses includes stimulating immune effector mechanisms at the cellular and biochemical level resulting in the influx of neutrophils into the lamina propria and have even been shown to modify gastric acid secretion. The availability of good animal models of chronic Helicobacter infection has also allowed investigators to begin to examine how the adaptive host immune response prevents and/or exacerbates Helicobacter-induced gastroduodenal disease. The experimental H. felis/mouse model has been utilized by a number of laboratories to investigate mechanisms of host defense against chronic Helicobacter infection. This model and the more recently developed H. pylori rodent model has not only allowed investigators to confirm the feasibility of immunotherapy to prevent and/or cure Helicobacter infection but also to begin to examine how the host immune response prevents and/or exacerbates Helicobacter-induced gastroduodenal disease. Based on these studies a hypothesis is emerging that suggests that protection and/or cure from Helicobacter infection is mediated primarily by an upregulated cellular immune response which may act via an antibody independent mechanism. Paradoxically, following natural infection with H. pylori, a component of the cellular immune response also promotes chronic gastric inflammation without clearance of the organism. The recent development of reliable and reproducible H. pylori/rodent models of disease and the availability of numerous inbred strains, transgenic and knockout animals, will allow investigators to continue to explore the role the host cellular and humoral immune response plays in promoting or preventing this infection.

Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract.

The protective efficacy of anti-Sendai virus IgA was compared to that of IgG after topical application of monoclonal antibodies (MAb) to the respiratory tract of mice. BALB/c mice were passively intranasally immunized with 50 microliters ascites containing equivalent ELISA titers of MAb 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Lung viral titers were determined by plaque assay 3 days following challenge. In most instances IgA MAb afforded equivalent protection to IgG MAb in that there was no significant difference in virus recovery from the lungs of animals treated with either IgA or IgG MAb, including subclasses of IgG. When IgA MAb was fractionated into monomers and oligomers, there was no inherent advantage to the oligomeric form with respect to passive protection against viral challenge. The data indicate that IgA and IgG antibodies are equally efficacious in protecting the airways from viral infection. The experiments suggest that the advantage of IgA for protecting mucosal surfaces, such as the respiratory tract, relates to the presence of a specialized mechanism for transporting oligomeric IgA across epithelial surfaces. The results also support the rationale for active mucosal immunization protocols designed to generate an IgA response.

IgA and mucosal defense.

The traditional role of IgA antibodies in mucosal defense has been considered as providing an immune barrier to keep exogenous substances, including microbial pathogens, from penetrating the mucosa. In this way infections can be prevented. More recently, studies in vitro and in vivo are providing evidence to suggest that IgA may have additional roles in mucosal defense. For example, during their passage through the lining epithelial cells of mucous membranes en route to the secretions, IgA antibodies may have an opportunity to neutralize intracellular pathogens like viruses. Also, IgA antibodies in the mucosal lamina propria have opportunities to complex with antigens and excrete them through the adjacent mucosal epithelium, again by the same route to the secretions that is taken by free IgA. These latter functions could aid in recovery from infection.

Animal models for gastric Helicobacter immunology and vaccine studies.

Over the last decade animal models have been used extensively to investigate disease processes and therapy for Helicobacter pylori infections. The H. pylori animal models which have been used in pathogenesis and vaccine studies include the gnotobiotic pig, non-human primates, cats, dogs, and several species of rodents including mice, rats, gerbils and guinea pigs. H. felis infection of mice and H. mustelae infection of ferrets have also been used. Recently, investigators have begun using transgenic mice and gene-targeted 'knock-out' mice to investigate Helicobacter infections. Each of these animal models has distinct advantages and disadvantages which are discussed in this minireview. The choice of an animal model is dictated by factors such as cost and an understanding of how each model will or will not allow fulfillment of experimental objectives.

Vaccine strategies for prevention of Helicobacter pylori infection.

OBJECTIVE: To examine the level and duration of the humoral immune response to Helicobacter felis following oral immunization or infection. DESIGN AND METHODS: Germ-free mice were orally immunized with sonicated H. felis plus cholera toxin five times over 6 weeks. One week after immunization was completed, immunized and control non-immunized mice received an oral challenge of live H. felis organisms. The animals were killed at 3-week intervals and serum, gastric washings, intestinal washings and gastric biopsies were obtained. H. felis infection was confirmed by a positive urease test or culture of the gastric biopsy. Serum gastric and intestinal antibody titers were determined by enzyme-linked immunosorbent assay. CONCLUSION: Infection and immunization against H. felis produces a specific humoral response. The humoral response in infection alone is significantly smaller than that of immunized animals until 6 weeks after infection. The humoral response following oral immunization persists for at least 18 weeks without further stimulation. The presence of an H. felis-specific antibody immune response before infection may be needed to protect animals from acute Helicobacter infection.

Local and systemic antibody responses in humans with Helicobacter pylori infection.

Immunization can prevent or cure an otherwise chronic helicobacter infection in several animal models despite the chronic nature of natural helicobacter infections. Differences in the antigenic specificity of the antibodies may contribute to the protection observed in these experimental animals. The goal of the present study was to compare the local and systemic antibody responses of humans with chronic Helicobacter pylori infection with those of an individual with spontaneous resolution of infection to find an immunological correlate of protection. Spontaneous resolution of infection was accompanied by a change in immunoblot profiles. Whereas a broad range of H pylori antigens was recognized in chronically infected patients (including the patient who ultimately cleared the infection spontaneously), resolution of infection in the absence of therapeutic agents resulted in the recognition of only several immunodominant antigens. The most dominant antigen was approximately 66 kDa in molecular mass. Immunoblot analysis demonstrated that these antibodies were specific for the structural subunits of the urease enzyme. These studies suggest that the success of antihelicobacter immunization may be due to the ability of vaccination to induce an immune response against antigens that are normally not immunodominant during the course of infection.

Oral immunization against Helicobacter pylori.

Helicobacter pylori, which has been associated with gastritis and duodenal ulcers, commonly chronically infects adults. Eradication of this microorganism, which is difficult to achieve, results in normalization of gastritis and marked reduction in the relapse rate of duodenal ulcers. Since eradication is difficult to achieve, prevention of initial colonization of the gastrointestinal tract may be a viable alternative for abrogation of H. pylori-associated gastroduodenal disease. To test the feasibility of this approach, mice and ferrets were orally immunized with killed H. pylori. Immunization induced immunoglobulin A and G anti-H. pylori antibodies in both gastrointestinal secretions and sera of mice. These responses were enhanced when cholera toxin was included in the immunization protocol as a mucosal adjuvant. In ferrets, addition of cholera toxin resulted in significant enhancement of anti-H. pylori antibody levels in sera and intestines. Thus, oral immunization with killed H. pylori may be feasible approach to protect hosts from this infection and the accompanying gastroduodenal disease.

In vitro mouse cytomegalovirus infection of mouse tracheal epithelial cells requires the presence of other cell types.

Recently, methods have been developed to culture dissociated tracheal epithelial (TE) cells. Attempts were made to infect these epithelial cells with mouse cytomegalovirus (MCMV) to see if the dissociated epithelial cells share characteristics of infection with MCMV-infected tracheal organ cultures. When isolated TE cells were incubated with MCMV at multiplicities between 0.10 and 2.0, no infection or minimal infection resulted. Centrifugation of virus onto the TE cell sheets also resulted in only minimal infection. If MCMV-infected mouse embryo fibroblasts were added to cultures of TE cells, however, the TE cells subsequently became infected. TE cells could also be infected by incubating MCMV with mixed cultures of uninfected fibroblasts and TE cells. The epithelial nature of infected cells was confirmed by electron microscopy. Reconstruction experiments demonstrated that fibroblast-mediated infection of mouse TE cells with MCMV was not simply due to the large amounts of virus provided by the infected fibroblasts. It is suggested that cell-cell contact, or fusion with infected cells is required for productive infection of TE cells with MCMV.

Cholera toxin as a mucosal adjuvant: III. Antibody responses to nontarget dietary antigens are not increased.

The effect of cholera toxin, a potent mucosal adjuvant, upon the murine immune response to both soybean meal, a regular dietary antigen, and ovalbumin, a simulated dietary antigen, was examined. Mice were orally immunized with a target antigen, Sendai virus, with and without cholera toxin as a mucosal adjuvant. Both serum and intestinal IgA and IgG anti-Sendai virus antibody responses were significantly increased when cholera toxin was used. For soybean meal, the major protein source in laboratory animal chows, however, no increases in serum antibody titers were found in animals which received oral cholera toxin plus virus. In addition, in mice orally immunized with ovalbumin plus cholera toxin (and virus), both serum and intestinal IgA and IgG antibody titers against ovalbumin were increased. Oral pre-immunization with ovalbumin prior to oral ovalbumin plus cholera toxin, however, resulted in no increase in either serum or intestinal anti-ovalbumin antibody responses. Thus, the use of cholera toxin as an oral adjuvant does not increase antibody responses against dietary antigens if there has been pre-exposure to the dietary antigen.

Helicobacter animal models.

In this unit, protocols for growing Helicobacter organisms on plates or in liquid cultures are presented, followed by protocols for infecting mice with Helicobacter felis and H. pylori and for infecting ferrets with H. mustelae. Also, a procedure is described for adapting an H. pylori isolate to growth in mice. Support protocols describe methods for quantifying numbers of Helicobacter organisms, and how to create a growth curve for Helicobacter cultures. One important technique in investigating Helicobacter infection is assaying the disease processes that occur in the stomach, and a protocol is provided for preparing tissue sections for this purpose. It is also important to confirm that organisms recovered from tissue samples are, in fact, Helicobacter species, and a support protocol describes morphological and biochemical tests for this purpose. Helicobacter bacteria produce large amounts of the enzyme urease, and a support protocol describes how to perform a rapid urease test on animal-tissue biopsies. Assays of Helicobacter-specific immune responses require appropriate antigens, and preparation of both Helicobacter lysates and outer-membrane proteins are detailed for use in these assays.

Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus.

Successful oral immunization to prevent infectious diseases in the gastrointestinal tract as well as distant mucosal tissues may depend on the effectiveness of an Ag to induce gut immune responses. We and others have previously reported that cholera toxin possesses strong adjuvant effects on the gut immune response to co-administered Ag. To explore further adjuvant effects of cholera toxin, the holotoxin or its B subunit was chemically cross-linked to Sendai virus. The resulting conjugates, which were not infectious, were evaluated for their capacity to induce gut immune responses against Sendai virus after oral administration to mice. Conjugating cholera toxin to virus significantly enhanced the adjuvant activity of cholera toxin compared to simple mixing. Cholera toxin B subunit, however, did not show an adjuvant effect either by itself or conjugated with the virus. Oral administration of the Sendai virus-cholera toxin conjugate was also able to prime for protective anti-viral responses in the respiratory tract. Mice that were orally immunized with the conjugate and intra-nasally boosted with inactivated virus alone showed virus-specific IgA titers in nasal secretions that correlated with protection against direct nasal challenge with live Sendai virus. For comparison, s.c. immunization was also studied. Systemic immunization with the virus-cholera toxin conjugate induced virus-specific antibody responses in serum as well as in the respiratory tract but failed to protect the upper respiratory tract against virus challenge. Systemic immunization plus an intra-nasal boost did, however, confer a variable degree of protection to the upper respiratory tract, which correlated primarily with bronchoalveolar lavage (lung) antibody titers.

Protection of germ-free mice from infection by Helicobacter felis after active oral or passive IgA immunization.

Helicobacter pylori infection of human gastric epithelium has been associated with gastritis, ulcers and gastric cancers. In an H. felis, germ-free mouse model of infection, oral immunization with bacterial antigens plus cholera toxin resulted in elevated serum, gastric and intestinal anti-H. felis antibody titres and protection from acute infection. Mice given monoclonal IgA anti-H. felis antibody at the time of initial challenge were also protected from infection. These results demonstrate that oral vaccination may be a feasible approach for the prevention of H. pylori infection of humans.