The abolition of the protective effect of Clostridium welchii type A antiserum by ferric iron

Intravenous injection of ferric ammonium citrate equivalent to 5 mg iron/kg abolished the protective effect of Clostridium welchii antiserum in guinea-pigs infected with Cl. welchii type A. In animals not given iron there was an abrupt cessation of growth 4–5 hours after infection. In those given iron bacterial growth was continuous and the animals died in 12–24 hours. In both of the animals there was always an excess of Cl. welchii alpha antitoxin in the plasma and tissues. Injection of iron did not interfere with phagocytosis of Cl. welchii. Iron abolished the protective effect of antiserum up to 6 hours after infection. It had no effect when given 8 hours after infection or later.     

Pathogenesis and immunology of experimental gonococcal infection: role of iron in virulence.

Addition of iron componds to inocula of the relatively avirulent T3 or T4 colony types of gonococci increased their lethality for chicken embryos after intravenous inoculation but had little or no effect on the highly virulent T1 or T2 types. The toxicity of nonviable inocula, killed cells or sonicates, was not significantly affected by ecogenous iron. Addition of the iron-binding protein conalbumin reduced or delayed the lethal effect of T1, but not T3, gonococci although growth of both colony types in the allantoic cavity of the embryo was inhibited by this protwin. This effect can be attributed specifically to deprivation of iron since the iron-complexed form of conalbumin had no apparent influence on growth or virulence. The results indicate that the ability to acquire iron in vivo is a significant factor in gonococcal virulence. The virulent colony types appear to have enhanced ability to compete with the host for iron and this may be related to the presence of pili, other surface components, or the synthesis of iron-chelating compounds.     

Pathogenesis and Immunology of Experimental Gonococcal Infection: Virulence of Colony Types of Neisseria gonorrhoeae for Chicken Embryos

The virulence of gonococcal strains and colony types was evaluated in embryonated hen eggs of various ages inoculated by different routes. Striking differences in virulence of colony types were revealed by intravenous inoculation of 11-day embryos. T1 and T2 colony types were found to have high virulence for embryos, whereas T3 and T4 colony types were relatively avirulent. These observations are in accord with previous studies in volunteers. The differences in virulence were not related to differences in toxicity of killed gonococci, sonic lysates, or to the susceptibility of gonococci to cidal effects of chicken embryo blood. Rather, they appeared to involve differences in clearance of gonococci from the blood stream and subsequent multiplication of the virulent colony types. Infection with virulent colony types appears to be primarily bacteremic in this model. Preliminary experiments indicated that chicken embryos may be protected against the lethal infection by prior treatment of the inoculum with normal and immune rabbit serum. This protective effect was not associated with bactericidal activity. The chicken embryo model is potentially useful as a means of investigating attributes of virulence of gonococci and factors in immunity against gonorrhea.     

Assessment of attachment of Neisseria gonorrhoeae to HeLa cells by double radiolabeling.

Attachment of Neisseria gonorrhoeae to HeLa cells was assessed by a technique using double radioisotopic labeling. Piliated, virulent bacteria from colony type 2 attached to HeLa cells to a greater extent than nonpiliated, avirulent bacteria from colony type 4. Maximal attachment rates for bacteria from both colony types occurred during the early incubation periods at 37 degrees C, and the HeLa cells appeared saturated at 4 h. Attachment was maximum at pH 6.5 and dependent upon the multiplicity of infection. Treatment of the HeLa cells with trypsin diminished the degree of attachment, but this effect substantially disappeared by 24 h after trypsin treatment. Scanning electron microscopy revealed bacteria of colony types 2 and 4 adhered to the HeLa cell surface. Thin-section transmission electron microscopy showed that bacteria were associated with the surface of the HeLa cell but not ingested.     

The abolition of the protective effect of Pasteurella septica antiserum by iron compounds

Ferric ammonium citrate, haematin hydrochloride, soluble haematin, lysed mouse red cells and a variety of purified haemoglobins abolished the protective effect of Pasteurella septica antiserum in mice when the iron compounds were injected intraperitoneally with P. septica. Ferric ammonium citrate was less effective than haematin or lysed red cells when the dose of P. septica was reduced to less than 10⁵. The ability of lysed red cells to abolish protection was greatly reduced if given 4 hours or more after infection. P. septica grew rapidly in unimmunized normal mice. In passively immunized mice the number of viable bacteria declined rapidly after infection. In passively immunized mice given haematin or lysed red cells the growth of bacteria was identical to that seen in unprotected normal mice. Large numbers of dead P. septica or carbon particles did not interfere with passive protection.     

Interaction of Gonococci with Phagocytic Leukocytes from Men and Mice

The interaction of human and mouse phagocytic leukocytes with representative virulent (F62-T1) and avirulent (F62-T4, RD-5) strains of Neisseria gonorrhoeae was studied in vitro. Leukocyte monolayers were incubated with gonococci for 30 min at 37 C, washed repeatedly, reincubated with fresh medium, and sampled for viable bacteria at intervals. After the initial incubation period and washing, human leukocytes retained larger numbers of viable T1 than of T4. During the subsequent 120 min of incubation, the numbers of viable T1 remained approximately constant, whereas viable counts of T4 declined by about two-thirds. In contrast, mouse leukocytes under similar conditions destroyed 70% of both types of gonococci. When human bactericidal serum was applied to infected human leukocytes, it had no effect on T4 but inactivated over 50% of T1. It is concluded that T4 are phagocytized by human leukocytes and are thus exposed to internal digestion, but are protected from bactericidal serum. T1, on the other hand, either adhere to the surface of the leukocytes or remain located so that they are neither digested by the leukocytes nor protected from bactericidal serum.     

Effect of siderophores on virulence of Neisseria gonorrhoeae.

The virulence of Neisseria gonorrhoeae for chicken embryos can be modified in a predictable manner by the addition of microbial siderophores to the inoculum. "Meningobactin" and "gonobactin," siderophores isolated from iron-limited cultures of meningococci and gonococci, respectively, enhance the virulence of the relatively avirulent colony type 3 (T3) organisms, but have essentially no effect on the virulence of T1 organisms. Both of these compounds were found previously to stimulate in vitro growth of the pathogenic Neisseria spp. under conditions made iron limiting by the addition of conalbumin, the transferrin counterpart of chickens. Similarly, ferrated schizokinen and arthrobactin, both dihydroxamate siderophores which stimulated growth in iron-limited conditions in vitro, also enhanced virulence of T3 organisms, whereas desferrioxamine B mesylate (Desferal), a trihydroxamate previously shown to be inhibitory in vitro, decreased the virulence of the T1 colony form. This was due to the iron-binding function of the molecule, as the iron-saturated form, ferrioxamine B mesylate, did not affect virulence. An additional trihydroxamate siderophore, ferrichrome A, which was inactive on Neisseria spp. in either the deferri- or ferrated forms in vitro, likewise did not affect virulence in the chicken embryo model. The neisserial siderophores were more effective than the other microbial siderophores in enhancing virulence of T3 gonococci. The results add to the evidence that the ability to acquire iron is an important determinant of virulence.     

Origin of T lymphocyte colony-forming cells in cell populations depleted of sheep erythrocyte rosette forming cells.

Cell populations depleted of sheep erythrocytes (E) rosette-forming T cells (E-cells) contain cells capable of giving rise to T cell colonies. We have characterized the T cell colony-forming cell from human bone marrow, blood and tonsil E- cells using a T-cell colony assay. Depletion of CD2+, CD3+ or CD4+ cells from E- cells reduced colony formation by 70-100%. Removal of CD8+ cells did not reduce, but rather enhanced colony formation by 50% or more. The most effective reduction (100%) in colony formation was obtained with anti-CD4, indicating that CD4 is a marker of all colony-forming T cells. The CD4+ lymphocytes generated two types of colonies, types I and II, in the presence or absence, respectively, of CD8+ lymphocytes. Type I were small and compact, reached a peak on days 5-7, and contained CD4+ and CD8+ cells. Type II were large and diffuse, reached a peak on days 9-10 and contained CD4+ cells. In continuous culture of single type II colony cells, we observed a consistent increase of CD8+ cells. In one colony the combined percentage of CD4+ and CD8+ cells exceeded 100% (averaging 83% CD4+ and 72% CD8+), indicating the presence of dual markers on some cells. We suggest that colony forming T cells are CD2+ CD3+ CD4+, the CD4+ antigen being the most consistent marker of such precursor cells.     

Hen Fluorescein-Labeled Gonococcal Lipopolysaccharide Antibody in the Delayed Fluorescent Antibody Technique for the Confirmation of Neisseria gonorrhoeae

A fluorescent antibody reagent (termed anti-LPS conjugate) was prepared from sera obtained from hens immunized with gonococcal R-type lipopolysaccharide. The reagent was absorbed with Formalin-treated cells of Neisseria meningitidis. The anti-LPS conjugate gave uniform brilliant staining of Neisseria gonorrhoeae with little background fluorescence, thus making interpretation and reading of fluorescence simple. The conjugate did not significantly stain cultures of N. meningitidis, Neisseria lactamica, nonpathogenic Neisseria species, or other gram-negative bacteria. Several preparations of the conjugate provided the same specificity and reproducibility of staining. The anti-LPS conjugate was compared with Difco Laboratories fluorescent antibody conjugate for staining of N. gonorrhoeae. Both conjugates stained cells of the light and dark variants of gonococcal colony types 1 and 2, as well as cells of colony types 3 and 4. When used for the confirmation of N. gonorrhoeae, the anti-LPS and Difco conjugates stained 426 of 431 (98.8%) and 210 of 213 (98.6%) of the gonococcal cultures, respectively. Absorption of the anti-LPS conjugate with R-type lipopolysaccharide removed the staining of gonococci. However, absorption of Difco conjugate with R-type lipopolysaccharide did not remove the staining of gonococci, suggesting that the majority of fluorescein-labeled antibody present in the Difco conjugate is directed to gonococcal cell surface components other than lipopolysaccharide. The results of this study indicate that fluorescein-labeled gonococcal lipopolysaccharide antibody should be a reliable fluorescent antibody reagent for the confirmation of N. gonorrhoeae.     

Interactions of Neisseria gonorrhoeae with Human Neutrophils: Effects of Serum and Gonococcal Opacity on Phagocyte Killing and Chemiluminescence

Serum-sensitive strains of Neisseria gonorrhoeae were incubated with suspensions of normal or chronic granulomatous disease human neutrophils in the absence or presence of fresh or heat-inactivated human serum; phagocytosis, gonococcal viability, and chemiluminescence were measured. Nonpiliated opaque or transparent gonococci (colony types 3 and 4, respectively) were used for phagocytic bactericidal assays. In the presence of 2.0% fresh human serum, normal neutrophils killed >90% of types 3 and 4 gonococci by 135 min. Serum alone at this concentration was not bactericidal. In the absence of serum, type 4 gonococci were not killed, whereas type 3 gonococci were killed to the same degree as in the presence of serum. Interestingly, heat-inactivated normal serum slightly inhibited phagocytic killing of type 3 gonococci. Results almost identical to those above were obtained when 5% fresh human serum deficient in complement component 7 was substituted for 2% normal autologous serum. This indicated that the later components of complement were not involved in the observed results. To investigate the mechanisms responsible for the intracellular killing of the gonococci, we used neutrophils from patients with chronic granulomatous disease. These neutrophils are deficient in an activable NADPH oxidase and do not produce bactericidal oxygen products upon phagocytic stimulation. Neutrophils from two unrelated boys with chronic granulomatous disease killed type 3 and 4 gonococci to the same degree as did normal neutrophils. As with normal neutrophils, serum was needed for killing type 4 organisms. As expected, neutrophils from these patients showed absolutely no increased chemiluminescence in the presence of type 3 or 4 gonococci, with or without serum. The effects of serum on gonococcus-induced chemiluminescence by normal neutrophils was also investigated. For these studies, in addition to type 3 and 4 gonococci, we also used transparent colony types of lightly (type 1) and heavily (type 2) piliated organisms. Chemiluminescence induced by type 1, 2, or 3 gonococci (i.e., gonococci possessing either pili or opacity-associated proteins, but not both) was augmented only slightly by serum and then only at low ratios of gonococci to neutrophils. On the other hand, chemiluminescence induced by type 4 gonococci (i.e., gonococci possessing neither pili nor opacity-associated proteins) was substantially increased in the presence of serum. Stimulation of chemiluminescence by type 1, 2, 3, or 4 gonococci was dose dependent in the absence or presence of serum. Heat-killed type 3 gonococci induced chemiluminescence to the same degree as did viable organisms. Since the gonococci used in this research was strongly catalase positive, as are gonococci in general, and since it was killed by chronic granulomatous disease neutrophils, the results indicate that gonococci can be effectively killed within neutrophils, i.e., within phagolysosomes, by nonoxidative bactericidal mechanisms. Whereas type 3 gonococci were phagocytized and killed by neutrophils equally well with or without serum, serum was obligatory for phagocytic killing of type 4 gonococci, i.e., gonococci lacking opacity-associated proteins. In addition, either pili or opacity-associated proteins were apparently necessary for maximal stimulation of neutrophil chemiluminescence. The submaximal stimulation of chemiluminescence by gonococci lacking both pili and opacity-associated proteins, i.e., type 4 gonococci was augmented by low concentrations of nonimmune serum.     

The effect of iron (Fe3+) on the cloning efficiency of human memory T4+ lymphocytes.

The effect of iron (Fe3+) and normal human liver ferritin on the proliferative response of normal human lymphocytes to tetanus toxoid was examined. This proliferative response involved memory T4+ lymphocytes as shown by a selective depletion study. Limit dilution analysis revealed that iron, present as ferric citrate, affected the initiation of clone development, and that concentrations of ferric citrate from 30 microM to 1 nM were able to reduce significantly the cloning efficiency of precursor T cells (up to 90% reduction). The reduced cloning frequency was not due to immunological suppression. Clone size was also reduced when iron was present during culture. In contrast, the presence of normal human liver ferritin during culture (concentration range: 300 micrograms/1-10,000 micrograms/1) had no effect on lymphocyte proliferation. The data indicate that low molecular weight iron (as ferric citrate) in concentrations similar to those which have been reported in the serum of patients with iron overload diseases, can interfere with antigen-specific lymphocyte responses and this may have implications for the development of infections and neoplasia in diseases of iron-overload.     

Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli.

Two strains of Escherichia coli were inhibited by complement-inactivated cow serum and to a lesser extent by precolostral calf serum devoid of specific antibodies. They were not inhibited by undiluted colostral whey or milk but colostral whey became bacteriostatic after dialysis or dilution in Kolmer saline and addition of precolostral calf serum or lactoferrin. The inhibition in all these fluids was due to iron-binding proteins (transferrin or lactoferrin). Undiluted dialysed milk was not inhibitory because of its low content of lactoferrin but became inhibitory after addition of 1 mg/ml of lactoferrin. The lack of inhibition in undiluted whey is due to the high concentration of citrate in colostral whey (and milk) and it is suggested that citrate competes with the iron-binding proteins for iron and makes it availabe to the bacteria. Addition of bicarbonate, which is required for the binding of iron by transferrin and lactoferrin, can overcome the effect of citrate; hence, the bacteriostatic effect of cow serum and precolostral calf serum is due to the presence of both transferrin and bicarbonate as well as the low lefel of citrate.     

Role of exotoxin A and elastase in the pathogenicity of Pseudomonas aeruginosa strain PAO experimental mouse burn infection

We examined the virulence of Pseudomonas aeruginosa strain PAO and xcp (extracellular proteins deficient) and xch (extracellular proteins hyperproducing) mutants derived from strain PAO in an experimental mouse burn infection model. The results showed that xcp mutants, which produced little or no extracellular elastase and exotoxin A, were as virulent as their corresponding xcp+ strains. The xch mutants produced more elastase and exotoxin A than the wild type strain, however, they had significantly lower virulence, probably due to reduced ability of these strains to take up iron. Treatment of mice with ferric ammonium citrate had no effect on the wild type strain but enhanced mortality in mice challenged with xch mutants. Neither elastase nor exotoxin A seem to play any role in burn infections with P. aeruginosa strain PAO. However, ability for iron uptake is an important virulence factor.     

Simple method for distinguishing gonococcal colony types.

Gonococcal colony types can be distinguished by a new procedure that makes use of a dissecting microscope with a concave mirror and a fluorescent lamp. Critical adjustment of the mirror angle results in illumination similar to that obtained in the dark-field microscope. When the concave mirror is set at a certain angle, colonies of the lenticular types 1 and 2 refract the light coming through them in such a way that an edge of the microscope stage is focused in each colony. By contrast, colonies of types 3 and 4, which are relatively flat, fail to refract incident light. Although distinguishable from each other by differences in color, type 3 and 4 colonies do not display the focusing effect typical for type 1 and 2 colonies and appear uniformly illuminated. This new technique permits the rapid identification and isolation of even a single type 1 or 2 colony in a field of type 3 or 4 colonies, making it possible to obtain and maintain competent colonies (type 1 or 2) for the genetic transformation assay for Neisseria gonorrhoeae strain identification as well as for other purposes.     

Chemotaxis of human polymorphonuclear leukocytes toward Neisseria gonorrhoeae

Chemoattractive properties of Neisseria gonorrhoeae were studied by measuring leukocyte migration in agarose gel. Human serum albumin (0.5%) was present in the gel and normal human serum was excluded from all components of the assay. Viable cell populations and lysates of colonial types F62T1, F62T2 and F62T3 induced migration of polymorphonuclear leukocytes. Chemotactic activity of the lysate was not altered by heating at 100 degrees C for 10 min and was retained in the 12 100 g supernatant fraction of the heated lysate. Fractionation of the supernate by Sephadex G-100 chromatography showed that the chemotactic activity was associated primarily with an absorbance peak at 280 nm of relatively low mol. wt. The chemotactic activity of this fraction was lost after dialysis and the peak was no longer present in the Sephadex G-100 elution profile of the dialysed supernate. The gonococcal leukotaxins were sensitive to digestion by trypsin, pronase and amyloglucosidase, but insensitive to treatment with RNAase, DNAase or lipase at pH 5.7-7.1.     

Heterogeneity of human T-lymphocyte clones generated from autologous mixed-lymphocyte cultures

To assess the heterogeneity of T cells activated during the autologous mixed-lymphocyte reaction (AMLR), a cloning procedure based on the soft agar colony assay was developed. Supernatants of allogeneic MLR cultures were used as a source of interleukin 2 (IL-2) to generate two types of colonies: upper and lower colonies. Both types of colonies were expanded in long-term cultures using supernatants of phytohemagglutinin (PHA)-activated lymphocyte cultures. Cloned cells underwent secondary proliferation when stimulated by autologous monocytes, although certain clones also responded to autologous B cells. Most autoactivated clones expressed the serological determinants of HLA-DR, MB, and MT and were OKT3+, OKT4+, OKT8-. They did not induce cytolysis of autologous monocytes, B lymphoblasts, or PHA blasts nor did they express natural killer-like activity toward K562 cells. Several autoactivated clones (irradiated with 500 R) expressed helper activity, shown by an enhancing effect on AMLR proliferation. Furthermore, many irradiated clones were capable of inducing proliferation of autologous T cells in the absence of accessory cells. These observations suggest that autoactivated clones generated from soft agar colonies may interact with autologous T lymphocytes.     

Effects of amino acids on colony phenotype of Neisseria gonorrhoeae.

The effects of amino acid supplementation of commercially available semidefined GC media (Oxoid GC medium and Difco GC base) on the stability of the virulent colony phenotype of Neisseria gonorrhoeae were studied. The -SH-containing amino acids, together with glycine, were found to cause segregation of avirulent colony types followed by growth inhibition after addition of 40 mg/100 ml. Proline and arginine were found to change the colony phenotype profile significantly without reducing the viable count. Arginine supplementation in both media caused a progressive loss of virulent colony types, whereas proline had the opposite effect.     

Cytokine production of CD8+ immune T cells but not of CD4+ T cells from Toxoplasma gondii-infected mice is polarized to a type 1 response following stimulation with tachyzoite-infected macrophages.

To examine whether cytokine production of CD4(+)immune T cells and CD8(+)immune T cells in Toxoplasma gondii-infected mice differ in their responses to infected cells and to soluble antigens of the parasite, we compared the production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-10 by the immune T cell populations following in vitro stimulation with tachyzoite-infected macrophages and tachyzoite lysate antigens (TLA). Both CD4(+)and CD8(+)immune T cells produced large amounts of IFN-gamma in response to either infected macrophages or TLA, but the CD4(+)T cells produced greater amounts of the cytokine than did the CD8(+)T cells with both stimulations. Both T cell populations also produced IL-2 after stimulation with infected macrophages, whereas only CD4(+)T cells did when stimulated with TLA. CD4(+)immune T cells also produced large amounts of IL-4 and IL-10 after stimulation with infected macrophages, but CD8(+)T cells did not. These results indicate that CD4(+)immune T cells produce IFN-gamma, IL-2, IL-4, and IL-10 in response to infected macrophages, whereas CD8(+)immune T cells produce predominantly IFN-gamma and IL-2. Since IL-4 and IL-10 could suppress IFN-gamma-mediated protective mechanisms against the parasite, the production of these cytokines by CD4(+)immune T cells in response to infected cells could negatively affect their protective activity in vivo.     

Mediation of immunity to Eimeria vermiformis in mice by L3T4+ T cells.

Immunity to infection with Eimeria vermiformis was transferred in NIH mice by both the nylon wool-adherent (B-cell-enriched) and nonadherent (T-cell-enriched) fractions of lymphocytes (spleen and mesenteric lymph node) taken from infected donors. Transfer was more variable with the adherent fraction, and when contaminating T cells were removed by treatment with anti-Thy1 monoclonal antibody (MAb) and complement, this fraction lost all protective activity. The protective effect of T-cell-enriched populations of mesenteric lymphocytes was abrogated by treatment with anti-L3T4 MAb and complement in vitro before transfer or by opsonization with this MAb in vitro before intravenous inoculation into recipients. Similar treatments of cells with anti-Lyt2 MAb did not have this effect, confirming that Thy1+ L3T4+ cells mediate the adoptive transfer of immunity to E. vermiformis. Thy1+ L3T4+ cells were also shown to limit the replication of E. vermiformis in primary infections: mice depleted of this subset (by thymectomy followed by intravenous injection of anti-L3T4 MAb) passed greater numbers of oocysts over a longer period of time than did mice similarly depleted of Lyt2+ cells.     

Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes

Infection with Neisseria gonorrhoeae can trigger an intense inflammatory response, yet there is little specific immune response or development of immune memory. In addition, gonorrhea typically correlates with a transient reduction in T lymphocyte counts in blood, and these populations recover when gonococcal infection is resolved. Such observations suggest that the gonococci have a suppressive effect on the host immune response. We report here that N. gonorrhoeae Opa proteins were able to bind CEACAM1 expressed by primary CD4+ T lymphocytes and suppress their activation and proliferation. CEACAM1 bound by gonococcal Opa52 associated with the tyrosine phosphatases SHP-1 and SHP-2, which implicates the receptor's ITIM (immunoreceptor tyrosine-based inhibitory motif) in this effect.