The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant.

Systemic and mucosal immune responses were effectively induced following the subcutaneous administration of Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine in a formulation containing the active form of vitamin D3. IgA and IgG antibodies with specificity for both the protein and oligosaccharide components of the vaccine were detectable in mucosal secretions following immunization. The IgA and IgG mucosal antibodies were produced locally, and were functional as demonstrated by their diphtheria toxin neutralizing activity. Our data suggests that subcutaneous tissues can effectively serve as effective antigen presenting sites for both mucosal and systemic immune responses to antigens administered in combination with vitamin D3.

Gonococcal infection of human fallopian tube mucosa in organ culture: relationship of mucosal tissue TNF-alpha concentration to sloughing of ciliated cells.

BACKGROUND AND OBJECTIVES: An experimental model consisting of gonococcal infection of human fallopian tube mucosa in organ culture has proven useful in studying the molecular pathogenesis of acute gonococcal salpingitis and postsalpingitis sequelae. Gonococcal infection of human fallopian tube mucosa in organ culture results in the sloughing of ciliated epithelial cells from the mucosa. This damage to the mucosa can be quantified on fallopian tube pieces by an assay of the percent of the periphery that has ciliary activity (PPCA) remaining at specific time points after infection. Although assay of the PPCA has been quite valuable, it is labor-intensive, somewhat subjective, and requires that the observers have training and experience. A more practical assay for genital mucosal damage is desirable for further investigations that employ the fallopian tube experimental model. Gonococcal infection of fallopian tube mucosa in organ culture also results in the production of easily quantified tumor necrosis factor-alpha (TNF-alpha) by the mucosa. Furthermore, treatment of the organ cultures with recombinant human TNF-alpha (rHuTNFalpha) alone also causes sloughing of ciliated cells from the mucosa. These findings strongly suggest that TNF-alpha is a mediator of the mucosal damage that attends gonococcal infection. GOALS OF THE STUDY: To determine: (1) whether the PPCA values and the TNF-alpha concentrations in fallopian tube mucosal tissues correlate closely enough to allow prediction of the PPCA from a measurement of the mucosal tissue TNF-alpha concentration; and (2) whether the correlation of the TNF-alpha mucosal tissue concentration with the sloughing of ciliated cells (measured by the PPCA) supports the hypothesis that induction of TNF-alpha by gonococcal infection, with resultant sloughing of ciliated cells, is likely to be a major pathogenic mechanism of gonococcal salpingitis and might mediate postsalpingitis infertility and ectopic pregnancy. STUDY DESIGN: A metaanalysis was performed on studies from three research groups (two laboratories in the United States and one in the United Kingdom, using identical techniques for quantifying the PPCA, TNF-alpha, or both. RESULTS: There was a close and statistically significant correlation between the TNF-alpha mucosal tissue concentration and the proportion of ciliated cells lost from the mucosa as measured by the PPCA (r = 0.95, p < 0.001). Therefore, as the mucosal tissue concentration of endogenous TNF-alpha increased, the loss of ciliated cells from the epithelium increased proportionately. CONCLUSIONS: During gonococcal infection of human fallopian tube mucosa in organ culture, the mucosal tissue concentration of TNF-alpha can be used to predict the PPCA, and therefore, the extent of mucosal damage. This finding should facilitate studies of the molecular pathogenesis of infectious diseases involving human genital mucosa. Further, the close correlation of mucosal TNF-alpha concentration with genital mucosal damage, evaluated by the PPCA, supports the hypothesis that induction of the proinflammatory cytokine, TNF-alpha, by gonococcal infection, with resultant inflammation and sloughing of ciliated cells, is an important pathogenic mechanism of gonococcal salpingitis and may mediate postsalpingitis infertility and ectopic pregnancy as well.

Induction of common mucosal immunity by hormonally immunomodulated peripheral immunization.

The study described in this report demonstrates that peripheral lymph nodes draining nonmucosal tissues can effectively serve as induction sites for the establishment of common mucosal immunity if the microenvironmental conditions are altered to mimic those normally present within mucosa-associated lymphoid tissues (e.g., Peyer's patches). Lymph node lymphocytes exposed in situ to the immunomodulatory influences of the hormone 1 alpha, 25-dihydroxy vitamin D 3 were found to produce less gamma interferon and interleukin-2 (IL-2) and far more IL-4, IL-5 and IL-10 than lymphocytes from control animals. When couples with vaccination with hepatitis B surface antigen (HBsAg), the hormone, immunomodulated switch from a peripheral lymph node phenotype to a Peyer's patch-like pattern promoted the induction of both a systemic and a common mucosal immune response. This was determined by the observed increased concentrations of serum anti-HBsAg antibody and by finding that anti-HBsAg secretory antibodies were detectable in urogenital, lachrymal, fecal and oral secretions only in the hormone-treated animals. In addition, specific antibody-secreting cells were detectable in the lamina propria of the lungs and small intestines of the hormone-treated animals subsequent to vaccination, indicating that the homing properties of antigen-specific B cells were being affected by the treatment procedure. The humoral and mucosal immune responses were further augmented if both 1 alpha, 25-dihydroxy vitamin D 3 and dehydroepiandrosterone were used together as hormonal immunomodulators. This novel immunization technique may afford new opportunities to effectively intervene in sexually transmitted diseases and other diseases caused by mucosal pathogens.

Local induction of tumor necrosis factor as a molecular mechanism of mucosal damage by gonococci.

Tumor necrosis factor (TNF) is an endogenously produced cytokine that plays a critical role in mediating septic shock and multi-organ failure, but previous studies of the role TNF in disease have not examined its role in mucosal disease processes. In an experimental model of acute gonococcal salpingitis, gonococcal infection of human fallopian tube mucosa resulted in increased mucosal production of TNF. Recombinant human TNF-alpha damaged fallopian tube mucosa in a dose-response manner and produced epithelial damage with the same ultrastructural features as those observed in gonococcal infection. Blocking production of TNF during gonococcal infection diminished the extent of damage to fallopian tube mucosa. In addition to mediating systemic disease, such as septic shock, TNF is also produced locally, and can play a critical role in mediating mucosal disease processes, such as acute gonococcal salpingitis.

Isolation of a 30 kDa immunoglobulin binding protein from Pseudomonas maltophilia.

We have demonstrated that Pseudomonas maltophilia (ATCC No. 13637) possesses an exposed, immunologically accessible protein which binds to the Fc region of several species of immunoglobulins. Whole bacteria suspensions were incubated for 18 h with purified 125I-labelled antibodies with and without added non-labelled immunoglobulins. The suspensions were centrifuged for 30 min and the pellet containing bacteria was assessed for radioactivity. Using this crude assay, the whole organism bound 125I-labelled rabbit and mouse immunoglobulins and the purified Fc portion of human IgG. All of these labelled preparations were competitively displaced by unlabelled rabbit and mouse immunoglobulins, and Fc of human IgG, as well as human immunoglobulin subclasses. The organism was sonicated to solubilize this immunoglobulin binding protein. Using this sonicated preparation, it was shown that unlabelled Fc of IgG, unlabelled mouse and rabbit immunoglobulins, all competitively displaced 125I-labelled human Fc of IgG in a dose-response manner. A partially purified protein was prepared by Sephacryl S-300 followed by Sephadex G-100 column chromatography. This preparation was incubated with 125I-Fc gamma and with the following purified unlabelled preparations: F(ab')2 of IgG, Fc of IgG, murine monoclonal IgA, IgG1, IgG2, IgG3, and IgG4. All except F(ab')2 of IgG produced dose response competitive displacement. The molecular weight, as estimated by SDS-PAGE and Western blot, was 30,000 daltons. In Western blots, Fc gamma, murine monoclonal IgA, and human immunoglobulin subclasses, all showed affinity for the immobilized protein. Human F(ab')2 fragments did not show affinity for the protein. Radioiodinated pseudomonal Ig-binding protein showed affinity for human IgG coupled to Sepharose, and was displaced by unlabelled pseudomonal Ig-binding protein. Scatchard analysis of binding showed two binding affinities: two distinct types of Ig-binding proteins were obtained, a high affinity with Kd = 1.54 x 10(-10) and a lower affinity with Kd = 2.36 x 10(-8). This immunoglobulin binding protein may be useful in immunoglobulin purification or identification.

Isolation of a 48.5-kDa membrane protein from Pseudomonas maltophilia which exhibits immunologic cross-reaction to the beta-subunit of human chorionic gonadotropin.

In separate studies we have shown that Pseudomonas maltophilia (American Type Culture Collection 13637) possesses an immunoglobulin Fc-binding protein. We have found that this protein prevents the application of immunoassays using monoclonal antibodies to study possible production of a CG-like material by this bacteria. Employing an immunoglobulin saturation technique to block this protein as well as a zwitterion detergent membrane solubilization technique, we now report the isolation of a membrane protein from Pseudomonas maltophilia which shows immunological relationships to the beta-subunit and carboxyl tail of human pregnancy CG. This pseudomonas immunoreactive material produced dose-response curves in the following CG immunoassays: 1) a polyclonal rabbit anti-CG equilibrium assay, 2) carboxyl tail CG equilibrium assay, and 3) two CG equilibrium assays using monoclonal antibodies. The pseudomonas CG-like protein did not react in equilibrium assays for human TSH, human LH, or free alpha-subunit of CG. The pseudomonas CG-like protein was purified by affinity chromatography. The purified protein showed only 0.25% cross-reaction with pregnancy CG in the monoclonal antibody equilibrium assays. Furthermore, the purified protein showed no binding to rat testicular CG/LH receptors, but showed avid binding to the pseudomonas CG-binding protein previously described by Richert and Ryan. The pseudomonas protein showed no binding to Concanavalin-A, which avidly binds pregnancy CG. When assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, this protein had a mol wt of 48,500 daltons, which is larger than the mol wt of the unglycosylated beta-subunit of pregnancy CG. We conclude that pseudomonas contain a protein that has partial homology to the beta-subunit of pregnancy CG. This material does not bind to mammalian CG receptors, but does bind to a pseudomonas CG-binding site. The latter suggests it has a function, as yet unknown, in pseudomonas.

Effect of human chorionic gonadotropin (hCG) on Neisseria gonorrhoeae invasion of and IgA secretion by human fallopian tube mucosa.

The possible effect of human chorionic gonadotropin (hCG) on the mucosal immune response and susceptibility of the fallopian tube mucosa to invasion by Neisseria gonorrhoeae (gonococci) was investigated in the fallopian tube organ culture (FTOC) model. Immunohistochemical and radioreceptor assay techniques showed specific high affinity binding of hCG in vitro to the apices of non-ciliated fallopian tube cells (Kd approximately 10(-9) M). Continuous exposure of the FTOC mucosa to hCG during infection with gonococci resulted in a marked increase (6- to 15-fold) in IgA secretion and significantly reduced gonococcal invasion (invasion score range 0.7 to 1.75) compared to infected control tissue which was not exposed to hCG (invasion score range 2.9 to 4.95, P less than or equal to 0.01). By contrast, exposure of the mucosa to hCG during the 24 h preceding gonococcal infection followed by the removal of hCG from the system at the time of infection resulted in enhanced gonococcal invasion (invasion score range 7.95 to 9.7, P less than 0.001). We conclude that hCG can modulate the mucosal immune response and susceptibility of fallopian tube epithelium to gonococcal invasion.

Antimicrobial interference with bacterial mechanisms of pathogenicity: effect of sub-MIC azithromycin on gonococcal piliation and attachment to human epithelial cells.

The effects of subinhibitory concentrations of azithromycin (CP-62,993) on the piliation and attachment properties of Neisseria gonorrhoeae were examined. Subinhibitory concentrations of azithromycin significantly reduced the percentage of gonococci that expressed assembled pili on their surfaces by decreasing pilin subunit synthesis and substantially decreased gonococcal adherence to human mucosal cells.

The evolutionary watershed of susceptibility to gonococcal infection.

Gonococci do not cause genital infection in any convenient experimental animal, but all too easily cause genital infection in humans. To determine the 'evolutionary watershed' of gonococcal infections (the point on the evolutionary tree at which susceptibility to gonococcal infection begins) we extended previous studies of the interaction of gonococci with animal oviduct mucosa to include chimpanzees and baboons. Gonococci attached to, damaged, and invaded the oviduct (fallopian tube) mucosa of chimpanzees (which are apes) but not the oviduct mucosa of baboons (which are monkeys). Thus, the pattern of gonococcal infection in chimpanzees was identical to that in humans, whereas the pattern in baboons was like that in other animals. These studies indicate that the point in evolution at which susceptibility to gonococcal infection commences is between baboons and chimpanzees (or between monkeys and apes). Susceptibility to gonococcal disease appears to require the presence on genital epithelial cells of receptors for gonococcal ligands such as pili, receptors for gonococcal lipopolysaccharide, or both. The physiological role of these receptors may be to interact with more useful, as yet unidentified molecules.

Candidal meningitis following bacterial meningitis.

Patients with bacterial meningitis and posttraumatic and/or postsurgical access to the CSF are at risk for superinfection with Candida species. Patients who are not improving on appropriate antimicrobial chemotherapy for bacterial meningitis or are deteriorating after initial improvement should have a CSF reexamination for Candida superinfection.

Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis.

Probing the surface of Neisseria gonorrhoeae: immunoelectron microscopic studies to localize cyanogen bromide fragment 2 in gonococcal pili.

Common epitopes accessible to antibody on purified macromolecules or structurally altered gonococci may not be accessible to antibody when those macromolecules are in their native state on the surface of intact organisms. To determine the immunologic accessibility of cyanogen bromide fragment 2 (CNBr2), a portion of the gonococcal pilin molecule that is common to all gonococcal strains on the surface of viable gonococci, probes composed of specific CNBr2 antibodies linked to gold spheres were manufactured. When whole piliated gonococci were exposed to these anti-CNBr2 immunological probes and examined using transmission electron microscopy, no significant marketing of native pili was evident. These probes, however, detected CNBr2 in purified form. The epitopes encompassed within the CNBr2 portion of pili appear to be inaccessible to anti-CNBr2 probes within native gonococcal pili.

The use of neutrophils, macrophages and organ cultures to assess the penetration of human cells by antimicrobials.

Recent studies of pathogenic mechanisms of bacteria have revealed that in addition to traditional intracellular parasites such as Brucella spp. and Listeria, many other human pathogens reside inside cells during disease processes. Thus, studies of how well antimicrobials are delivered to and perform within phagocytic or endocytic vacuoles have become of increasing importance. Whereas studies of penetration of PMN neutrophils and macrophages have indicated whether antimicrobials penetrate the cytoplasm of human cells, studies in organ cultures can reveal whether antimicrobials enter phagosomes or endocytic vacuoles where the bacteria actually reside. Such information is probably more predictive of antimicrobial efficacy in naturally occurring intracellular infections than are data from studies with PMN's or macrophages.

Parasite-directed endocytosis.

Following the attachment of gonococci to human fallopian tube mucosa in organ culture, the gonococci are endocytosed by specialized low columnar epithelial cells, are transported to the base of the epithelial cells, and are subsequently exocytosed into the subepithelial tissues. This transepithelial transport process by which "invasion" of the host occurs appears to be dependent on microbial factors is designated parasite-directed endocytosis to distinguish it from host-directed endocytosis by cells such as macrophages that eventually degrade the parasites. "Invasion" of the host by a number of human pathogens--bacteria (e.g., Neisseria meningitidis, Haemophilus influenzae, and Listeria monocytogenes), viruses, or protozoa--appears to be accomplished by parasite-directed endocytosis.

Microbial invasion: a covert activity?

In contrast to nonpathogenic microorganisms that exist happily in biofilms on various organic and inorganic surfaces, many pathogenic microbes have the additional ability to invade host tissues by inducing their own endocytosis and transport across normally protective barriers. This phenomenon, designated "parasite-directed endocytosis," has been observed with a variety of surfaces (intestinal, genital, nasopharyngeal, and tracheal epithelium) as well as in endothelial cells. The mechanisms involved in invasion may involve a single factor as described for some species of Yersinia, or may require multiple factors as observed in Shigellae. For the majority of pathogens, the molecular mechanisms of invasion are not well understood (e.g., Neisseria gonorrhoeae). Because parasite-directed endocytosis is reminiscent of receptor-mediated endocytosis, it is quite possible that some pathogens engage in biologic mimicry by producing a molecule that resembles a natural host ligand, for which there is a host cell receptor. Such a masquerade may allow some microbes to enter the host's inner sanctum covertly in a manner analogous to the Trojan horse, rather than overtly by destroying the mucosa and entering host tissues directly. Whereas this hypothesis is speculative at present, bacteria that produce molecules resembling insulin, calmodulin, and chorionic gonadotropin have been described.

Immunoelectron microscopic localization of outer membrane proteins II on the surface of Neisseria gonorrhoeae.

To determine the ultrastructural distribution and immunological accessibility of proteins II (P.IIs) on the surfaces of whole gonococci, anti-P.II gold probes were developed and used in electron microscopic studies of viable P.II-expressing variants of Neisseria gonorrhoeae FA1090. Anti-P.II probes clearly marked the surfaces of organisms and their associated outer membrane blebs. The surface-exposed portion of P.II is antigenically variable. With the use of two different sizes of gold probes, it was demonstrated that individual gonococcal cells can express more than one antigenic type of P.II simultaneously.

Fungal endocarditis complicating treatment of prosthetic valve bacterial endocarditis: value of prophylactic oral nystatin.

We describe two patients in whom fungal endocarditis occurred during antibiotic therapy for prosthetic valve bacterial endocarditis. Successful management of both patients was eventually achieved with antifungal therapy and replacement of the prosthetic valves. These cases and review of the literature suggest that (1) high-dose antibacterial therapy predisposes to fungal endocarditis; (2) during prolonged antibiotic therapy in patients predisposed to endocarditis, clinicians should consider the use of oral nystatin as prophylaxis against fungemia and possible fungal endocarditis; and (3) early replacement of prosthetic valves infected with fungi is indicated because chemotherapy alone is predictably inadequate to effect a cure.

The frequency of expression of pyelonephritis-associated pili is under regulatory control.

The Escherichia coli urinary tract isolate C1212 contains two pyelonephritis-associated pili (pap) DNA sequences designated here as pap-17 and pap-21. Each of these pap sequences encodes antigenically-distinct pilin monomers, pilin-17 and pilin-21, respectively. Most individual strain C1212 cells isolated from a single bacterial colony expressed pilin-21. Only a small fraction (5%) of strain C1212 cells expressed pilin-17. Most of the latter population simultaneously expressed pilin-21, but a low percentage of cells expressed pili composed of pilin-17 alone. In contrast, almost every E. coli K-12 cell containing multicopy pap-17 expressed pilin-17 at the cell surface. These results indicated that the regulation of pilin-17 expression observed for strain C1212 was lost when pap-17 was in the multicopy state. Transfer of pap-17 to a single copy vector resulted in a pilin-17 expression frequency lower than strain C1212 (1%). Using E. coli K-12 containing single copy pap-17, we found that the frequency of pilin-17 expression increased about 15-fold when pap-21 was present in multiple copies in trans. Subcloning of pap-21 showed that a 2.2 kilobase-pair DNA sequence adjacent to, but not including, the pilin-21 structural gene was sufficient for activation of pilin-17 expression.