Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.     

Modulation of Murine Lyme Borreliosis by Interruption of the B7/CD28 T-Cell Costimulatory Pathway

Recent studies have implicated cytokines associated with Th2 cells in the genetic resistance to murine Lyme borreliosis. Because the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th-cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to the Th2 cytokine profile and development of arthritis in BALB/c mice infected with Borrelia burgdorferi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin 4 (IL-4) and upregulation of gamma interferon (IFN-γ) responses by B. burgdorferi-specific T cells and by reduction of B. burgdorferi-specific immunoglobulin G. Despite the shift toward a Th1 cytokine pattern, which others have associated with disease susceptibility, the severity of arthritis was unchanged. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies or CTLA-4Ig enhanced IFN-γ production over that seen with CD86 blockade alone, yet augmentation of this Th1-associated cytokine did not enhance disease. These results demonstrate that IL-4 production by T cells in B. burgdorferi-infected BALB/c mice is dependent upon CD86/CD28 interaction and that this cytokine does not contribute significantly to host resistance to the development of arthritis. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-γ-producing T cells in B. burgdorferi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. These studies may provide insight into the role of the B7/CD28 pathway in other infectious and autoimmune diseases in which deviation of Th cell immune responses occurs and antigen is persistently present.Lyme disease is a multisystem illness due to infection with the tick-transmitted spirochete Borrelia burgdorferi. Experimental infection of laboratory mice with B. burgdorferi results in acute arthritis and carditis that reproducibly peak at 2 to 4 weeks of infection and then resolve within 3 months despite spirochete persistence (4). Studies using SCID mice, which lack functional T and B cells, have demonstrated that disease is due to the innate immunity of the host and can occur in the absence of specific immune responses (6, 28). The persistent and progressive nature of disease manifestations in SCID mice underscores the importance of T and B cells in initiating disease regression (5, 6, 28). Recent studies support the additional role of specific immunity in modulating disease severity via direct effects on spirochete burden through B. burgdorferi-specific antibodies (5) and indirectly through Th cell-associated cytokines that influence the activation of innate immune cells (14, 23). In particular, the dominance of Th1-type responses, which support macrophage activation, in patients with chronic Lyme arthritis has implicated this T-cell phenotype in the development and perpetuation of severe inflammatory disease (32, 37). Th1-type responses have also been observed during B. burgdorferi infection of C3H mice, a disease-susceptible strain, whereas Th2 responses, which promote B-cell functions, can be detected in BALB/c mice, a comparatively disease-resistant strain (14, 23). Despite the greater inflammatory response in C3H mice, their pathogen burden as assessed by quantitative PCR of spirochete DNA remains higher than that of disease-resistant mouse strains (36), suggesting that the recruitment of innate immune cells is appropriate yet ineffective at controlling infection (29).In addition to signals provided by T-cell antigen receptor engagement, the interaction of costimulatory molecules present on antigen-presenting cells (APCs) with their ligands on T cells is believed to be necessary for the initial priming of naive T cells. In particular, the B7/CD28 costimulatory pathway has been implicated in the differentiation of naive Th0 cells into Th1 and Th2 subsets (33). The mechanisms by which these molecules assist in the priming of the T-cell immune response are complex and poorly understood. Two members of the B7 family have been characterized, CD80 and CD86 (also known as B7-1 and B7-2, respectively), and differ not only in their binding properties to CD28 on T cells but also in the timing of their appearance on conventional APCs during the initiation of an immune response (11). CD86 appears earlier on the surface of mitogen-activated APCs and has a lower affinity for CD28 than does CD80. Once activated, T cells express CTLA-4, a second receptor to which both CD80 and CD86 bind with greater affinity than they bind CD28 (21). Interaction of CD80/CD86 with CTLA-4 can downregulate the T-cell immune response (35). Blockade of CD86 during the initiation of a T-cell response results in an immune response oriented toward a Th1 phenotype, whereas a similar blockade of CD80 does not consistently favor a Th2 phenotype (20). Experiments using mutant mice deficient in CD80 and/or CD86 reveal the important role of these molecules in sustaining a Th-cell phenotype and, in the case of CD86 expression, in the development of a Th2 response (20). Costimulation through the B7/CD28 pathway contributes to the expansion of autoimmune disease processes seen in experimental autoimmune encephalitis (17, 27), a predominantly Th1-associated disease, and autoimmune diabetes (19). Studies using a soluble recombinant form of CTLA-4 designated CTLA-4Ig have supported many of the observations made with anti-B7 antibodies (13, 19, 26).We have recently reported that the Th2 response of B. burgdorferi-infected BALB/c mice is preceded by a Th1 response and that the presence of interleukin 4 (IL-4) is associated with accelerated resolution of arthritis (12). A hind-foot inoculation route was used in that study so that T-cell responses could be examined in lymph nodes adjacent to joints afflicted with arthritis. We demonstrated that this route of inoculation induces moderately severe arthritis in BALB/c mice at day 14 of infection that undergoes more rapid regression than the arthritis seen in similarly infected C3H mice, in which IL-4 responses are not detectable. Previous studies have shown that treatment of mice with anti-IL-4 monoclonal antibody (MAb) exacerbates arthritis in BALB/c mice assessed at intervals corresponding to the plateau and resolution phases of disease, providing evidence that IL-4 modulates the severity of established arthritis (14, 23). The influence of Th2 cell effector functions on the development of arthritis remains unknown. In the current study, we have examined the effects of interruption of Th2 cell differentiation by B7/CD28 blockade with anti-CD80 and/or anti-CD86 MAb or CTLA-4Ig on the cytokine profiles and development of arthritis in BALB/c mice infected with B. burgdorferi.     

Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation

The attenuated S. typhimurium SL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-A^d/I-E^d antigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80⁺ and CD11b⁺ spleen cells.Salmonella infections still pose a serious health hazard worldwide, affecting both humans and animals. Salmonella typhi, the agent of human typhoid fever, is not pathogenic for common laboratory animals. Therefore, natural resistance and acquired immunity to Salmonella are studied mainly in the mouse model by using host-adapted salmonellae which cause systemic infections believed to mimic the human disease.In mice, early bacterial growth in the reticuloendothelial system (RES) is controlled by the innate resistance Nramp (Ity) gene, which is expressed in macrophages (22). In lethal infections, salmonellae rapidly reach large numbers in the tissues and death occurs presumably by endotoxin poisoning when bacterial counts reach levels of ca. 10⁸ CFU per organ (30). In sublethal infections, survival requires a host response that suppresses the exponential growth of the organisms in the RES towards the end of the first week, resulting in a plateau phase (17, 25). The establishment of the plateau phase does not require functional T cells. In fact, nude (T-cell-deficient) mice and mice depleted of T cells by administration of anti-CD4 and anti-CD8 antibodies can still suppress Salmonella growth in infected tissues (17). A bone marrow-dependent influx of radiation-sensitive cells is required for the plateau phase and for the formation of granulomas rich in mononuclear cells (17, 32). Most of the salmonellae in the spleens and livers of the infected animals are localized within the phagocytes present in the focal lesions (38). Tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and nitric oxide (NO) derivatives appear to be required for the suppression of salmonella growth in the RES (27, 28, 32, 36, 37, 48). TNF-α is needed for the recruitment of mononuclear cells in the tissues and for granuloma formation (32); IFN-γ can activate macrophages to kill salmonellae in vitro (20).The establishment of the plateau phase coincides with the development of hypersusceptibility to the toxic and lethal effects of bacterial lipopolysaccharide (LPS) (29, 33). We have previously shown that mice immunized with a live attenuated aromatic-dependent Salmonella vaccine strain show transient hypersusceptibility to LPS, which can be prevented by treatment with anti-TNF-α antibodies (29). The role of other cytokines in this phenomenon is not known.Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine produced by macrophages, B cells, polymorphonuclear leukocytes, and dendritic cells in response to a variety of stimuli including products of bacterial origin (5, 10). IL-12 mediates resistance to intracellular organisms including Listeria, Toxoplasma, Candida, Leishmania, Mycobacterium tuberculosis, and Brucella abortus (8, 13, 18, 23, 39, 46, 50). IL-12 is generally believed to mediate host resistance by inducing IFN-γ production by NK and T cells as well as by contributing to the establishment of protective Th1 antigen-specific responses (5, 6, 9, 10, 12, 13, 24, 34, 39, 43, 47).Evidence for IL-12 induction in salmonellosis has been provided. IL-12 and IL-12-specific mRNA have been detected in vivo and in vitro in response to Salmonella. Elicited peritoneal mouse macrophages stimulated with Salmonella dublin express elevated levels of IL-12 p40-specific mRNA (4, 7). Oral infection with virulent or live attenuated S. dublin induces early (6 and 20 h postinfection) production of IL-12-specific mRNA in Peyer’s patches and mesenteric lymph nodes (3); biologically active IL-12 in lymph node homogenates has been documented 36 h after S. dublin infection (21). We and others previously reported that in vivo IL-12 neutralization reduces the ability of the host to suppress the growth of virulent salmonellae in the tissues and impairs IFN-γ production (21, 31). A recent report indicates that a mutation in the IL-12 receptors renders humans more susceptible to salmonellosis (11). Nevertheless, the mechanisms by which IL-12 mediates host resistance to Salmonella are still unclear.In the present study, we attempted to clarify the mechanisms by which IL-12 contributes to host resistance in mice infected with Salmonella. We investigated the role of IL-12 in survival, granuloma formation, and macrophage activation in mice infected with an attenuated Salmonella strain that normally causes very mild infections in BALB/c mice. We also investigated the involvement of IL-12 in the toxic and lethal effects of high bacterial loads in the tissues as well as in the expression of hypersusceptibility to LPS normally seen in mice infected with salmonellae. We also wished to clarify the involvement of IFN-γ in IL-12-mediated resistance to salmonellosis.     

In Vivo Regulation of Replicative Legionella pneumophila Lung Infection by Endogenous Interleukin-12

The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth of Legionella pneumophila was assessed by using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of A/J mice with virulent bacteria (10⁶ L. pneumophila cells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreated L. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, which regulate L. pneumophila growth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicative L. pneumophila lung infection were subsequently assessed. Results of these experiments demonstrated that TNF-α activity was significantly lower, while protein levels of IFN-γ and IL-10 in the lung were similar, in L. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicative L. pneumophila lung infection and suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is mediated, at least in part, by TNF-α.

Legionella pneumophila, the causative agent of Legionnaires’ disease, is an intracellular pathogen of mononuclear phagocytic cells (MPCs) (37, 43, 45). Pulmonary infection usually develops following inhalation of L. pneumophila-contaminated water aerosols or microaspiration of contaminated water sources (9). Following inhalation, the bacteria invade and replicate in host MPCs, primarily in alveolar MPCs (34, 36, 37, 43, 45). Intracellular growth of L. pneumophila results in eventual lysis of infected MPCs, the release of bacterial progeny, and reinfection of additional pulmonary cells (34, 36). Severe lung damage, mediated by tissue-destructive substances likely derived from both damaged host cells and the bacteria, ensues (20, 21).Previous studies have demonstrated that resistance to primary replicative L. pneumophila lung infection is dependent on the induction of cellular immunity and is mediated in part by cytokines including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) (8, 12, 14, 15, 23, 27, 28, 35, 57). Growth of L. pneumophila within permissive MPCs requires iron. IFN-γ limits MPC iron, thereby converting the MPC intracellular environment from one that is permissive to one that is nonpermissive for L. pneumophila replication (14, 15). IFN-γ in combination with other cytokines including TNF-α facilitates elimination of L. pneumophila from infected MPCs, likely through the induction of effector molecules including nitric oxide (12). In contrast, other cytokines including interleukin 10 (IL-10) facilitate growth of L. pneumophila in permissive MPCs, due in part to IL-10-mediated inhibition of TNF-α secretion and IFN-γ-mediated MPC activation (46).IL-12 is a recently described cytokine with pleiotropic effects on T cells and natural killer (NK) cells which include (i) regulation of expression of cytokines including IFN-γ, TNF-α, and IL-10 by T cells and/or NK cells, (ii) induction of T-cell and/or NK cell proliferation and/or differentiation, and (iii) enhancement of NK cell and T-cell cytotoxic activity (4, 5, 19, 32, 33, 39, 44, 47, 48, 50, 56). While systemic administration of exogenous IL-12 has been demonstrated to increase host resistance to several intracellular pathogens, including Leishmania major, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, and Plasmodium chabaudi, in mice (26, 29, 33, 40, 51, 52, 55), the role of endogenous IL-12 in innate immunity to intracellular pathogens including L. pneumophila has not been thoroughly investigated. We have recently developed a model of replicative L. pneumophila lung infection in A/J mice inoculated intratracheally with virulent bacteria and have used this model system to identify immune responses which mediate host resistance to legionellosis (10–12). Using this murine model of Legionnaires’ disease, we assessed the biologic relevance and immunomodulatory role of endogenous IL-12 in innate immunity to replicative L. pneumophila lung infection.     

Distinct Characteristics of Resistance to Borrelia burgdorferi-Induced Arthritis in C57BL/6N Mice

Studies of mice infected with Borrelia burgdorferi have indicated that the severity of arthritis is influenced by the genetic composition of the host: the C3H mouse develops severe arthritis while BALB/c and C57BL/6 mice develop mild arthritis. In this study, the effects of increasing infectious dose on the severity of arthritis were determined in these three mouse strains. C3H/He mice developed severe arthritis at all infectious doses, with 100% infection requiring 200 spirochetes. In BALB/cAnN mice, arthritis severity was dependent on infectious dose; symptoms were mild with infection by 200 B. burgdorferi and progressively more severe with increasing infectious dose. Infection of BALB/cAnN mice with 2 × 10⁴ B. burgdorferi resulted in arthritis with severity identical to that in C3H/He mice. Spirochete levels in rear ankle joints of C3H/HeJ and C3H/HeN mice were relatively high, as detected by PCR, and did not increase with infectious dose. Spirochete levels in joints from BALB/cAnN mice increased with increasing infectious dose to levels found in severely arthritic C3H/He mice. Thus, resistance to severe arthritis in BALB/cAnN mice was conditional: it could be overcome by high infectious dose and the arthritis became severe when high levels of B. burgdorferi were present in joints. A unique response to increasing infectious dose was seen in C57BL/6N mice, which displayed mild to moderate arthritis at all doses of B. burgdorferi tested, up to 2 × 10⁵. At all infectious doses, the levels of spirochetes in ankle joints of C57BL/6N mice were high, equivalent to those found in the severely arthritic C3H/He mice. The arthritis observed in infected (C57BL/6N × C3H/HeN)F₁ mice was of severity intermediate between those of the two parental strains. The finding that resistance to severe arthritis in C57BL/6N mice could not be overcome by high infectious doses and was independent of spirochete levels in joints suggested that it was mediated by a distinct mechanism from that operating in BALB/cAnN mice.Lyme disease is caused by infection with the tick-transmitted spirochete Borrelia burgdorferi and is characterized by multisystem involvement (14, 16, 24). Many tissues may display disease involvement, and there is variability in the degree to which patients are affected. This variability could be due to host, microbial, or environmental factors. In fact, infection in Europe by related members of the B. burgdorferi sensu lato group is more frequently associated with chronic skin abnormalities and central nervous system involvement, while infection by B. burgdorferi sensu stricto in the United States is more commonly associated with arthritis (4, 38). Studies using the murine model of Lyme disease, developed by Barthold and colleagues, indicate host factors also influence disease outcome. Arthritis seen in this model is representative of human disease and is characterized by tendonitis, synovial hyperproliferation, and infiltration of neutrophils and other leukocytes (7). Interestingly, a spectrum of arthritis severity has been observed among inbred strains of mice in response to infection by B. burgdorferi. Infected C3H mice develop severe arthritis, whereas infected BALB/c and C57BL/6 mice develop only mild to moderate arthritis (8). Thus, inbred strains of mice provide opportunities to study host influences on disease severity.The results of several studies using the mouse model suggest the presence of inflammatory and/or anti-inflammatory cytokines can influence disease development and resolution. For example, manipulations of interleukin 12, interleukin 4, and gamma interferon levels by treating infected mice with neutralizing antibodies can influence disease severity and alter its resolution (2, 17, 21). The acquired defenses, particularly antibody production, are clearly involved in disease resolution (9, 30) but do not appear to be required for arthritis and carditis development. Not only does disease develop in scid mice, which lack mature T and B lymphocytes, but the relative differences in severity of arthritis in C3H/He and BALB/c mice is maintained in the presence of the scid mutation (12). Finally, studies with congenic mice expressing distinct major histocompatibility complex haplotypes on resistant or susceptible backgrounds suggest that the major histocompatibility complex itself had little influence on disease severity, but rather, that genes located at distinct chromosomal locations were important determinants of disease (41). These studies suggest that genes independent of acquired defenses play a large role in determining severity of disease in infected mice.In order to identify host genes that influence disease severity, the phenotypes of severe and mild arthritis must be well characterized. We previously compared B. burgdorferi levels in many tissues of C3H/HeJ and BALB/cJ mice, at several times following infection (42). Quantitative PCR demonstrated that the highest levels of spirochetes were found in the hearts and ankle joints at most time points. C3H/HeJ mice harbored 5- to 10-fold more B. burgdorferi in ankles and hearts than did BALB/cJ mice. This suggested that the severity of arthritis in C3H/HeJ mice was directly related to the high levels of spirochetes in tissues and that the relative resistance in BALB/cJ mice was associated with more restricted growth of the spirochetes.In this study we report that there are at least two different mechanisms for resistance to severe arthritis in mice. Resistance in BALB/cAnN mice could be overcome by increasing the infectious dose of B. burgdorferi and was associated with low levels of spirochetes in tissues. In contrast, resistance to severe arthritis in C57BL/6N mice was not overcome by increasing infectious dose and did not require the levels of spirochetes in joints to be low. F₁ mice from BALB/cAnN × C3H/HeJN mating developed severe arthritis upon infection, suggesting that resistance in BALB/cAnN mice could be masked by alleles from C3H/HeN mice (42). In contrast, infection of F₁ mice from a C57BL/6N × C3H/HeN cross resulted in arthritis of intermediate severity, suggesting more equal contribution by C57BL/6N and C3H/HeN genes.     

d-Lactate Production and [14C]Succinic Acid Uptake by Adherent and Nonadherent Escherichia coli

Escherichia coli isolates of different adherence phenotypes produced different amounts of d-lactate. Alterations of culture conditions did not influence the amount of d-lactate produced. The observed pH decreases in tissue culture medium corresponded with increases in d-lactate concentration. Very little [¹⁴C]succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E. coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed. The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E. coli.One of the virulence associated properties of enteropathogenic Escherichia coli (5, 13, 14) is the ability to adhere to small intestinal mucosa (3, 11, 12, 21, 24, 26, 27). Although this adherence is an important event in the induction of diarrhea, the mechanism by which adherent E. coli mediates pathogenicity remains uncertain (1, 2, 7, 18, 26, 27).Several studies have shown that the in vitro adherence of E. coli to HEp-2 or HeLa cells in tissue culture can be used as a marker of enteroadherence (4, 6, 8, 9, 15, 16, 19, 22, 23, 28, 29). We used the HeLa assay (20) to detect this virulence characteristic in E. coli isolates from infants with acute diarrhea and, during the 3-h assay, observed E. coli-induced changes in the pH of the tissue culture medium (17). The pH changes induced by organisms with different adherence phenotypes differed. Since the characteristic end products of E. coli fermentation include lactic acid, succinic acid, and acetic acid, the pH changes could be explained by differences in the production of organic acids. Other plausible explanations are differences in the removal of organic acids from the medium and interactions between bacteria and HeLa cells during adherence.This paper describes two sets of experiments, one based on the production of lactic acid and the other on the removal of succinic acid from the medium. The objectives were to determine (i) whether there is a metabolic difference between localized, diffuse, and nonadherent isolates in the amount of lactate produced or succinate removed from the incubation medium, (ii) whether E. coli changes from aerobic to anaerobic metabolism during incubation periods of up to 5 h under different culture conditions, (iii) whether an increase in lactate production or succinate removal coincides with the drop in pH previously observed, and (iv) whether the pH changes can be attributed to differences in bacterial growth rates between isolates with different in vitro adherence patterns and nonadherent strains.     

Mannose Induces the Release of Cytopathic Factors from Acanthamoeba castellanii

Acanthamoeba keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoeba keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, using Acanthamoeba castellanii trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be ≥100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding of Acanthamoeba to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.Acanthamoeba spp. are protozoal parasites capable of infecting the skin, brain, and eye (10, 15, 17, 31, 32, 37). Corneal inflammation produced by Acanthamoeba was first recognized in 1973 and has since been intimately associated with contact lens wear (15, 31). Often the disease displays a ring-like neutrophilic stromal infiltrate with an overlying epithelial ulcer. The epithelium often undergoes a recurrent cycle of healing and breakdown during the progression of the disease. Topical or systemic treatment with antibiotics, antifungals, corticosteroids, and antivirals is often ineffectual (2). Typical treatment consists of around-the-clock hourly topical treatments with propamidine isothionate, polyhexamethylene biguanide, neomycin, or chlorhexidine, alone or in combination. This therapeutic regimen may continue for weeks. Many patients receive therapeutic corneal transplants, which can be reinfected by quiescent parasites residing in the periphery of the cornea.Parasite binding to the corneal epithelium is believed to be an important first step in the infectious cascade of Acanthamoeba keratitis. We have shown that adherence of Acanthamoeba to corneal buttons in vitro varies among mammalian species and correlates with susceptibility to experimental Acanthamoeba keratitis (14, 19, 35). Parasitic infections, such as Acanthamoeba keratitis, often occur in a sequential manner and are initiated by the pathogen’s adherence to host cells. Bacteria, fungi, and amoebae have been shown to bind to epithelial cells via lectin-glycoprotein interactions (5, 6, 11, 18, 20–22, 26, 27, 40). The cell surface of Pseudomonas aeruginosa is decorated with lectins which bind surface glycoproteins of the epithelium to be invaded (30, 39). Entamoeba histolytica also utilizes glycoproteins as receptor ligands for adherence to the gastrointestinal epithelium (6, 16, 25–29). Binding of Acanthamoeba polyphaga and A. castellanii to corneal epithelial cells in culture is inhibited by mannose (18, 40). Subsequent studies have indicated that the binding of A. castellanii to corneal epithelial cells is mediated by a 136-kDa mannose-binding protein on the trophozoite cell membrane (40).The pathophysiology of Acanthamoeba keratitis is poorly understood. Several studies have demonstrated that Acanthamoeba trophozoites can induce either cytolysis or apoptosis of target cells in vitro (1, 7, 24, 33, 34). Pathogenic Acanthamoeba trophozoites produce a variety of proteases which are believed to facilitate parasite penetration into the corneal stroma (9). Once in the stroma, Acanthamoeba trophozoites secrete collagenolytic enzymes which contribute to the dissolution of the stromal matrix (13).This study was undertaken to examine the cytopathic mechanisms employed by Acanthamoeba during the initial phase of ocular infection. We tested the hypothesis that blocking parasite binding to corneal epithelial cells with mannose would prevent parasite-mediated cytolysis and invasion of the corneal stroma. The results, however, indicate that although mannose blocks parasite binding, it also facilitates the release of cytolytic factors which kill corneal epithelial cells.     

Two new insulator proteins,Pita and ZIPIC,target CP190 to chromatin

Insulators are multiprotein–DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.Insulators in the Drosophila and vertebrate genomes have been identified based on their ability to disrupt the communication between an enhancer and a promoter when inserted between them (Raab and Kamakaka 2010; Ghirlando et al. 2012; Herold et al. 2012; Matzat and Lei 2013; Chetverina et al. 2014; Kyrchanova and Georgiev 2014). The growing amount of data show that insulator proteins fulfil an architectural function in mediating inter- and intrachromosomal interactions and in contacting regulatory elements such as promoters or enhancers (Maksimenko and Georgiev 2014).The best studied Drosophila insulator proteins, dCTCF (homolog of vertebrate insulator protein CTCF) and Su(Hw) are DNA-binding zinc-finger proteins (Herold et al. 2012; Matzat and Lei 2013; Kyrchanova and Georgiev 2014). Binding sites for dCTCF have been identified in the insulators that separate functional regulatory domains of the bithorax complex and in many promoter regions (Moon et al. 2005; Holohan et al. 2007; Mohan et al. 2007; Nègre et al. 2010, 2011; Ni et al. 2012). The Su(Hw) protein more frequently associates with intergenic sites (Adryan et al. 2007; Bushey et al. 2009; Nègre et al. 2010, 2011; Soshnev et al. 2012, 2013). As shown in a transgenic assay, dCTCF and Su(Hw) binding sites can support specific distant interactions (Kyrchanova et al. 2008a,b), which suggests a key role for these proteins in organizing chromatin architecture.The Su(Hw), dCTCF, and BEAF-32 proteins interact with Centrosomal Protein 190 kD, named CP190 (Pai et al. 2004; Gerasimova et al. 2007; Mohan et al. 2007; Bartkuhn et al. 2009; Oliver et al. 2010; Liang et al. 2014). CP190 (1096 amino acids) contains an N-terminal BTB/POZ domain, an aspartic-acid-rich D-region, four C2H2 zinc-finger motifs, and a C-terminal E-rich domain (Oliver et al. 2010; Ahanger et al. 2013). The BTB domain of CP190 forms stable homodimers that may be involved in protein–protein interactions (Oliver et al. 2010; Bonchuk et al. 2011). In addition to these motifs, CP190 also contains a centrosomal targeting domain (M) responsible for its localization to centrosomes during mitosis (Butcher et al. 2004). It has been shown that CP190 is recruited to chromatin via its interaction with the Su(Hw) and dCTCF proteins (Pai et al. 2004; Mohan et al. 2007). Inactivation of CP190 affects the activity of the dCTCF-dependent insulator Fab-8 from the bithorax complex (Gerasimova et al. 2007; Mohan et al. 2007; Moshkovich et al. 2011) and the gypsy insulator, which contains 12 binding sites for the Su(Hw) protein (Pai et al. 2004). Binding of Su(Hw) and CP190 at gypsy-like sites is mutually dependent, indicating a stabilizing role of CP190 in these cases (Schwartz et al. 2012).Recent genome-wide ChIP-chip studies provide evidence for an extensive overlap of the CP190 distribution pattern with dCTCF, BEAF-32, and Su(Hw) insulator proteins and the promoters of active genes (Bartkuhn et al. 2009; Bushey et al. 2009; Nègre et al. 2010, 2011; Schwartz et al. 2012; Soshnev et al. 2012). Very recently, it has been demonstrated that CP190 bridges DNA-bound insulator factors with promoters (Liang et al. 2014). These data support the model that CP190 has a global role in the function of insulator proteins. However, there are a number of sites in the Drosophila genome where CP190 does not colocalize with any known insulator DNA binding protein (IBP), suggesting that there may be some other proteins that recruit CP190 to chromatin (Schwartz et al. 2012).To identify new factors that associate with CP190, we purified the FLAG-tagged CP190 protein from S2 cells and identified two zinc-finger proteins, CG7928 and Pita, which were shown to interact with CP190 in vivo and in vitro. Genome-wide identification of binding sites for Pita and CG7928 in S2 cells revealed their extensive colocalization with CP190, providing evidence for direct interactions between these proteins, which was supported by binding and in vivo functional assays. Based on these results we termed CG7928 the “zinc-finger protein interacting with CP190” (ZIPIC).     

Isotype Switching Increases Efficacy of Antibody Protection against Cryptococcus neoformans Infection in Mice

The isotype and epitope specificities of antibodies both contribute to the efficacy of antibodies that mediate immunity to Cryptococcus neoformans, but the relationship between these properties is only partially understood. In this study, we analyzed the efficacy of protection of two sets of immunoglobulin G (IgG) isotype switch variants from two IgG3 monoclonal antibodies (MAbs) which are either not protective or disease enhancing, depending on the mouse model used. The two IgG3 MAbs 3E5 and 4H3 have different epitope specificities. Protection experiments were done with A/JCr mice infected intravenously with C. neoformans and administered with 3E5 IgG3 and its IgG1, IgG2a, and IgG2b switch variants. These experiments revealed that IgG1, IgG2b, and IgG2a were each more effective than IgG3. For 4H3 IgG3 and its IgG1 and IgG2b switch variants, the relative efficacy was IgG2b > IgG1 >> IgG3. The combination of 3E5 IgG3 and 4H3 IgG3 was more deleterious than either IgG3 alone. All IgG isotypes were opsonic for mouse bronchoalveolar cells, with the relative efficacy being IgG2b > IgG2a > IgG1 > IgG3. These results (i) confirm that a nonprotective IgG3 MAb can be converted to a protective MAb by isotype switching, (ii) indicate that the efficacy of protection of an IgG1 MAb can be increased by isotype switching to another subclass, (iii) show that protective and nonprotective IgG MAbs are opsonic, and (iv) provide additional evidence for the concept that the efficacy of the antibody response to C. neoformans is dependent on the type of MAb elicited.Cryptococcus neoformans is a fungus which is a frequent cause of life-threatening meningoencephalitis in patients with impaired immunity (22, 25). Cryptococcosis has been reported to occur in 6 to 8% of patients with AIDS (7). In immunocompromised individuals, C. neoformans infections are often incurable with conventional antifungal agents, and these patients frequently require lifelong therapy (45). The difficulties involved in the management of cryptococcosis in immunocompromised individuals have led to a reexamination of the potential of antibody-mediated immunity for prevention and therapy of cryptococcal infections. A polysaccharide-tetanus toxoid (TT) conjugate vaccine which is highly immunogenic and can elicit protective antibodies in mice has been made (3, 8, 9). In addition, several monoclonal antibodies (MAbs) have been shown to modify the course of infection in mice, and these may be useful in therapy of human infection (12, 14, 28, 42, 43).Cell-mediated immunity is generally acknowledged to provide important host defense against C. neoformans infection (4, 20, 26, 31, 42). In contrast, the role of antibody-mediated immunity in host resistance is less certain (2), but there is considerable evidence that administration of some MAbs can modify the course of infection in mice (8, 12, 14, 16, 28, 33). C. neoformans is unusual among fungal pathogens in that it has a polysaccharide capsule composed primarily of glucuronoxylomannan (GXM) (6), which is important for virulence (5). The capsular polysaccharide has been shown to produce a variety of deleterious effects including inhibition of phagocytosis (21), interference with antigen presentation (39), shedding of adhesion molecules (11), inhibition of leukocyte migration (10), and alterations in cytokine production by host effector cells (24, 40, 41). Antibodies to the C. neoformans capsular polysaccharide may contribute to host defense through multiple effects including enhanced opsonization (13, 18, 23, 30, 44), clearance of polysaccharide antigen (15), promotion of granuloma formation (14), and release of oxygen- and nitrogen-derived oxidants (27, 38).In previous studies, we demonstrated that immunoglobulin G3 (IgG3) MAbs are not protective in various mouse models of cryptococcal infection (32, 42). When one of these nonprotective IgG3 MAbs was switched to IgG1, the IgG1 significantly prolonged animal survival (32, 42). In the present study, we analyzed two families of IgG switch variants generated in vitro from two nonprotective IgG3 MAbs with different epitope specificities. We found that MAbs with different isotypes have different protective efficacies and that switching of nonprotective IgG3 MAbs to IgG1, IgG2b, and IgG2a significantly increased antibody protective efficacy. These studies demonstrate a complex relationship among efficacy of antibody protection, epitope specificity, and isotype.     

Streptococcal Histone-Like Protein: Primary Structure of hlpA and Protein Binding to Lipoteichoic Acid and Epithelial Cells

In addition to its role in the nucleoid, the histone-like protein (HlpA) of Streptococcus pyogenes is believed to act as a fortuitous virulence factor in delayed sequelae by binding to heparan sulfate-proteoglycans in the extracellular matrix of target organs and acting as a nidus for in situ immune complex formation. To further characterize this protein, the hlpA genes were cloned from S. pyogenes, S. gordonii, S. mutans, and S. sobrinus, using PCR amplification, and sequenced. The encoded HlpA protein of S. pyogenes has 91 amino acids, a predicted molecular mass of 9,647 Da, an isoelectric point of 9.81, and 90% to 95% sequence identity with HlpA of several oral streptococci. The consensus sequence of streptococcal HlpA has 69% identity with the consensus sequence of the histone-like HB protein of Bacillus species. Oral viridans group streptococci, growing in chemically defined medium at pH 6.8, released HlpA into the milieu during stationary phase as a result of limited cell lysis. HlpA was not released by these bacteria when grown at pH 6.0 or below. S. pyogenes did not release HlpA during growth in vitro; however, analyses of sera from 155 pharyngitis patients revealed a strong correlation (P < 0.0017) between the production of antibodies to HlpA and antibodies to streptolysin O, indicating that the histone-like protein is released by group A streptococci growing in vivo. Extracellular HlpA formed soluble complexes with lipoteichoic acid in vitro and bound readily to heparan sulfate on HEp-2 cell surfaces. These results support a potential role for HlpA in the pathogenesis of streptococcus-induced tissue inflammation.

Prokaryotes contain several small, basic, heat-stable proteins in association with the nucleoid. These proteins bind to single- and double-stranded DNA without obvious sequence specificity and are termed histone-like proteins; however, they do not have sequence homology with eukaryotic histones (for reviews, see references 13, 19, 33, and 37). The best-studied histone-like proteins are HU of Escherichia coli (4, 15, 29, 35, 38) and HB of Bacillus species (10, 23, 24, 31, 44). HU is a heterodimer of HU1 and HU2 proteins, which contain 90 amino acid residues each and have 70% sequence identity. HB is a protein highly homologous to HU but existing as a homodimer of a 92-amino-acid subunit (10, 23, 24, 31). Although the biological functions of histone-like proteins are not fully understood, they are known to wrap DNA and restrain negative supercoiling (4, 35). The resulting alterations in DNA structure and topology affect several cellular processes, including initiation of DNA replication (11, 51), DNA partitioning and cell division (12, 50), binding of repressors (3, 17, 30, 34), and transposition of bacteriophage Mu (43).In addition to the physiological functions of bacterial histone-like proteins, HlpA (previously called GAG-BP and HBP) of Streptococcus species may contribute fortuitously to the virulence of these bacteria when the protein is released into the tissues during infection. Purified HlpA binds selectively in vitro to heparan sulfate in proteoglycans of heart and kidney basement membranes (1, 5, 6, 49). The accumulation of intravenously administered HlpA on renal basement membranes of mice and rabbits and the ensuing in situ immune complex formation (7, 20) indicate that it might be an important virulence factor in acute poststreptococcal glomerulonephritis and the glomerulonephritis that is often associated with streptococcal endocarditis in humans (21, 47). Tissue-bound HlpA may serve as a nidus for in situ immune complex formation leading to the inflammation and immunopathology that typify these diseases. The HlpAs of Streptococcus pyogenes, S. mutans, S. gordonii, and S. mitis are immunologically cross-reactive and exhibit identical binding activities for basement membranes in animal tissues (5, 6, 49).This study was undertaken to clone and sequence hlpA from group A and viridans group streptococci, to compare the primary structure of HlpAs, and to evaluate the ability of these bacteria to release HlpA protein into the culture medium during growth. The hlpA genes of four Streptococcus species encode proteins of 91 amino acids that have at least 90% sequence identities. Members of the viridans group streptococci released more HlpA during stationary phase of growth than did the group A streptococci, and extracellular HlpA was complexed with soluble lipoteichoic acid (LTA). These antigen complexes bind to the surfaces of human epithelial cells in vitro and can lead to immune complex formation in situ.     

Effect of Prior Experimental Human Enteropathogenic Escherichia coli Infection on Illness following Homologous and Heterologous Rechallenge

Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.

Enteropathogenic Escherichia coli (EPEC) strains are one of several categories of pathogenic E. coli strains that cause diarrhea. EPEC infections are prevalent on six continents (5, 22–24, 28, 43). In many parts of the world, EPEC strains are the most common bacterial cause of diarrhea in infants (7, 21, 43). Disease due to EPEC can be severe, refractory to oral rehydration, protracted, and lethal (3, 14, 21, 45, 48).The pathogenesis of EPEC infection involves three distinct stages, initial adherence, signal transduction, and intimate attachment (12). Initial adherence is associated with the production of a type IV fimbria, the bundle-forming pilus (BFP) (20), that is encoded on the large EPEC adherence factor (EAF) plasmid (50). EPEC uses a type III secretion apparatus to export several proteins, including EspA, EspB, and EspD, that are required for tyrosine kinase-mediated signal transduction within the host cell (17, 25, 30, 31). This signaling leads to phosphorylation and activation of a 90-kDa protein that is a putative receptor for the bacterial outer membrane protein intimin (44). Intimin, the product of the eae gene, is required for intimate attachment of bacteria to the host cell membrane and for full virulence in volunteers (13, 26, 27). The interaction between EPEC and host cells results in the loss of microvilli and the formation of adhesion pedestals containing numerous cytoskeletal proteins (16, 33, 34, 39, 46). This interaction between bacteria and host cells is known as the attaching and effacing effect (40).One of the most striking clinical features of EPEC infections is the remarkable propensity of these strains to cause disease in very young infants. Rare reports of disease in older children and adults usually reflect common-source outbreaks that probably involve large inocula (47, 53). In contrast, in nosocomial outbreaks among neonates, EPEC spreads rapidly by person-to-person contact, apparently involving low inocula (54). The incidence of community-acquired EPEC infection is highest in the first 6 months after birth (4, 7, 21). EPEC infection is also more severe in younger children (8). Infants are more likely to develop diarrhea during the first episode of colonization with EPEC than they are during subsequent encounters (8). Whether the low incidence of EPEC diarrhea in older children and adults is due to acquired immunity or decreased inherent susceptibility is not known.The immune response to EPEC infection remains poorly characterized. It has previously been demonstrated that volunteers convalescing from experimental EPEC infection develop antibodies to the O antigen component of lipopolysaccharide (LPS) of the infecting strain, to intimin, and to type I-like fimbriae (13, 15, 29, 38). Antibodies to common EPEC O antigens are found more often in children of greater than 1 year in age than they are in younger children (42). Breast-feeding is protective against EPEC infection (2, 19, 43, 52). Breast milk contains antibodies against EPEC O antigens and outer membrane proteins and inhibits EPEC adherence to tissue culture cells (6, 9, 49).In an earlier study, it was reported that volunteers infected with EPEC developed antibodies to a 94-kDa outer membrane protein (38). Subsequently, it was determined that this antigen was intimin (26). Interestingly, the lone volunteer in that earlier study who did not have diarrhea after challenge with a wild-type EPEC strain had prechallenge serum antibodies to intimin. This led to the hypothesis that antibodies to intimin are protective against EPEC infection. To test this hypothesis and to test the more general hypothesis that EPEC infection induces protective immunity, two volunteer studies were performed. The first was a heterologous-challenge study performed in 1986, in which volunteers were infected with an O55:H6 EPEC strain and challenged, along with a naive cohort, with an O127:H6 EPEC strain. The second was a homologous-challenge study performed in 1991, in which veterans of a study comparing the virulence of a wild-type EPEC O127:H6 strain with that of an isogenic eae mutant (13) were rechallenged, along with a naive cohort, with the homologous wild-type strain. The availability of new purified antigens allowed us to analyze data from these studies in the context of humoral immune responses.