bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of a prominent Th2 cytokine response in human tuberculosis
Authors: Lin Y , Zhang M , Hofman F M , Gong J , Barnes P F
Published: January 2020

Depressed Th1 responses are a prominent feature of human tuberculosis, but an enhanced Th2 response has not been detected in peripheral blood T cells stimulated in vitro with Mycobacterium tuberculosis. In disease due to Mycobacterium leprae, Th2 cells predominate in tissue lesions of patients with extensive disease but are absent from peripheral blood. To determine if Th2 cells are present in tissue lesions of tuberculosis patients, we evaluated patterns of cytokine expression in lymph nodes from tuberculosis patients with or without human immunodeficiency virus infection and in controls without tuberculosis. Gamma interferon and interleukin-10 (IL-10) mRNA expression in tuberculosis patients with or without human immunodeficiency virus infection was high, whereas IL-4 expression in the same patients was low. Immunolabeling studies showed that macrophage production of IL-12 was increased in lymph nodes from tuberculosis patients, that gamma interferon was produced by T cells, and that IL-10 was produced by macrophages rather than Th2 cells. These results indicate that Th2 responses are not enhanced either systemically or at the site of disease in human tuberculosis.
Mycobacterium tuberculosis invades and replicates within type II alveolar cells
Authors: Bermudez L E , Goodman J
Published: January 2020

Although Mycobacterium tuberculosis is assumed to infect primarily alveolar macrophages after being aspirated into the lung in aerosol form, it is plausible to hypothesize that M. tuberculosis can come in contact with alveolar epithelial cells upon arrival into the alveolar space. Therefore, as a first step toward investigation of the interaction between M. tuberculosis and alveolar epithelial cells, we examined the ability of M. tuberculosis to bind to and invade alveolar epithelial cells in vitro. The H37Rv and H37Ra strains of M. tuberculosis were cultured to mid-log phase and used in both adherence and invasion assays. The A549 human type II alveolar cell line was cultured to confluence in RPMI 1640 supplemented with 5% fetal bovine serum, L-glutamine, and nonessential amino acids. H37Rv was more efficient in entering A549 cells than H37Ra, Mycobacterium avium, and Escherichia coli Hb101, and nonpiliated strain (4.7% +/- 1.0% of the initial inoculum in 2 h compared with 3.1% +/- 0.8%, 2.1% +/- 0.9%, and 0.03% +/- 0.0%, respectively). The invasion was more efficient at 37 degrees C than 30 degrees C (4.7% +/- 1.0% compared with 2.3% +/- 0.8%). H37Rv and H37Ra were both capable of multiplying intracellularly at a similar ration over 4 days. Binding was inhibited up to 55.7% by anti-CD51 antibody (antivitronectin receptor), up to 55% with anti-CD29 antibody (beta(1) integrin), and 79% with both antibodies used together. Update of M. tuberculosis H37Rv was microtubule and microfilament dependent. It was inhibited by 6l.4% in the presence of 10 micron colchicine and by 72.3% in the presence of 3 micron cytochalasin D, suggesting two separate pathways for uptake. Our results show that M. tuberculosis is capable of invading type II alveolar epithelial cells and raise the possibility that invasion of alveolar epithelial cells is associated with the pathogenesis of lung infection.
Binding between outer membrane proteins of nontypeable Haemophilus influenzae and human nasopharyngeal mucin
Authors: Reddy M S , Bernstein J M , Murphy T F , Faden H S
Published: January 2020

Bacterial colonization of the epithelial cells precedes infection. Mucins of the epithelial cell secretions modulate bacterial colonization. This study was designed to understand the mechanism of mucin-bacterial interactions and in particular binding between nontypeable Haemophilus influenzae and nasopharyngeal mucin(s). In an overlay assay, binding appears to be mediated by outer membrane proteins P2 and P5 of bacteria and sialic acid-containing oligosaccharides of mucin.
Hyaluronate capsule and surface M protein in resistance to opsonization of group A streptococci
Authors: Dale J B , Washburn R G , Marques M B , Wessels M R
Published: January 2020

The major virulence determinant of group A streptococci is the ability to resist opsonization and phagocytic ingestion. The present studies were performed to compare the mechanisms of resistance to opsonization of type 18 and type 24 streptococci and to determine the relative roles of M protein-fibrinogen interaction and the hyaluronate capsule in preventing phagocytic ingestion and killing. By use of parent strains and acapsular transposon mutants in the presence and absence of fibrinogen, we show that type 18 and type 24 streptococci rely on somewhat different mechanisms for resistance to opsonization. Type 24 streptococci bound fibrinogen avidly to their surfaces, and encapsulated organisms were completely resistant to opsonization only in the presence of fibrinogen. In contrast, type 18 streptococci bound 10-fold less fibrinogen than type 24 streptococci and were fully resistant to phagocytosis only when they expressed capsule. The general structural characteristics of the amino-terminal halves of type 18 and type 24 M proteins differed in that type 18 M protein contained only one complete B repeat, whereas type 24 M protein contained five complete B repeats, a structural difference which could potentially be related to the differences in fibrinogen binding between the two serotypes. Immunofluorescence assays of complement deposition were used in combination with 125I-C3 binding assays to show that encapsulated type 24 streptococci were fully resistant to opsonization by C3 only in the presence of plasma. Encapsulated and unencapsulated type 18 streptococci were equally opsonized by C3 in either plasma or serum, yet only encapsulated organisms resisted phagocytic killing in blood. The results of this study indicate that opsonization by C3 does not necessarily lead to phagocytic ingestion and that the hyaluronate capsule and M proteins are variably important in resistance to different group A streptococci to opsonization and phagocytic killing.
Interleukin-1 alpha production during Rickettsia rickettsii infection of cultured endothelial cells: potential role in autocrine cell stimulation
Authors: Sporn L A , Marder V J
Published: January 2020

Rickettsia rickettsii infection results in numerous responses by cultured endothelial cells, among them a rapid, transient increase in steady-state levels of tissue factor mRNA (L.A. Sporn, P.J. Haidaris, R.-J. Shi, Y. Nemerson, D.J. Silverman, and V.J. Marder, Blood 83:1527-1534, 1994). In this study, production of interleukin-1 (IL-1) was measured during infection and its potential role in autocrine cell stimulation was investigated. A fivefold increase in levels of IL-1 alpha antigen was measured in cell lysate samples by enzyme-linked immunosorbent assay at 18 h of infection. The majority of IL-1 alpha remained cell associated, as no significant increase was detected in culture medium. No IL-1 beta antigen was detected in cell lysates or culture medium from either control or infected cultures. A dramatic increase in the levels of IL-1 alpha mRNA occurred following infection, as measured by reverse transcriptase PCR, which revealed the appearance of the expected 421-kb product with RNA extracted from cells infected for 4 h and no detectable product from control cell samples. The presence of functional, cell-associated IL-1 alpha activity in infected cells was confirmed, following disruption, by the ability of the infected cells to induce tissue factor expression in target endothelial cells. Such induction was eliminated by pretreatment of the disrupted cell samples with neutralizing antibodies against IL-1 alpha but not against IL-1 beta. To investigate whether endogenously produced IL-1 participates in the stimulation of tissue factor expression, neutralizing antibodies against IL-1 or the IL-1 receptor antagonist were added to culture medium during infection. Both anti-IL-1 alpha and the IL-1 receptor antagonist resulted in approximately 40% inhibition of tissue factor expression, thus implicating IL-1 alpha in autocrine cell stimulation.
Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells
Authors: Gaydos C A , Summersgill J T , Sahney N N , Ramirez J A , Quinn T C
Published: January 2020

Chlamydia pneumoniae has recently been associated with atherosclerotic lesions in coronary arteries. To investigate the biological basis for the dissemination and proliferation of this organism in such lesions, the in vitro growth of C. pneumoniae was studied in two macrophage cell lines, peripheral blood monocyte-derived macrophages, human bronchoalveolar lavage macrophages, several endothelial cell lines, and aortic smooth muscle cells. Five strains of C. pneumoniae were capable of three passages in human U937 macrophages and in murine RAW 246.7 macrophages. Titers were suppressed in both macrophage types with each passage, as compared with growth titers in HEp-2 cells. Both human bronchoalveolar lavage macrophages and peripheral blood monocyte-derived macrophages were able to inhibit C. pneumoniae after 96 h of growth. Eleven C. pneumoniae strains were capable of replicating in normal human aortic artery-derived endothelial cells, umbilical vein-derived endothelial cells, and pulmonary artery endothelial cells. Infection in human aortic artery smooth muscle cells was also established for 13 strains of C. pneumoniae. The in vitro ability of C. pneumoniae to maintain infections in macrophages, endothelial cells, and aortic smooth muscle cells may provide support for the hypothesis that C. pneumoniae can infect such cells and, when infection is followed by an immune response, may contribute to atheroma formation in vivo. More studies are needed to investigate the complex relationship between lytic infection and persistence and the potential for C. pneumoniae to influence the generation of atheromatous lesions.
Gonococcal opacity protein promotes bacterial entry-associated rearrangements of the epithelial cell actin cytoskeleton
Authors: Grassmé H U , Ireland R M , van Putten J P
Published: January 2020

Neisseria gonorrhoeae enters cultured human mucosal cells following binding of a distinct gonococcal opacity (Opa) outer membrane protein to cell surface proteoglycan receptors. We examined the route of internalization that is activated by Opa-expressing gonococci (strain VP1). Microscopy of infected Chang epithelial cells showed that gonococcal uptake was insensitive to monodansylcadaverine (150 microM), which interferes with clathrin-mediated endocytosis. Similarly, indirect immunofluorescence staining for clathrin in infected cells showed distribution of cellular clathrin unaltered from the distribution in noninfected cells. The microtubule inhibitors colchicine (50 microM) and nocodazole (20 microM) but not the microtubule-stabilizing agent taxol (10 microM) caused a moderate (30 to 50%) reduction in gonococcal entry without affecting bacterial adherence. The most dramatic effects were obtained with the microfilament-disrupting agent cytochalasin D (3 microM), which totally blocked bacterial entry into the cells. Double immunofluorescence staining of gonococci and actin filaments in infected cells demonstrated bacterium-associated accumulations of F-actin as an early signal of bacterial entry. The recruitment of F-actin was transient and disappeared once the bacteria were inside the cells. Cytochalasin D disrupted the actin cytoskeleton architecture but did not prevent the recruitment of F-actin by the bacteria. Adherent, noninvasive gonococcal Opa variants lacked the ability to mobilize F-actin. Recombinant Escherichia coli expressing the gonococcal invasion-promoting Opa of gonococcal strain MS11 (Opa50) adhered to the epithelial cells in an Opa-dependent fashion but was not internalized and did not recruit detectable amounts of F-actin. Coinfection with the E. coli recombinant strain and gonococci resulted in specific entry of the diplococci, despite the presence of large numbers of adherent E. coli cells. Together, our results indicate that Opa-mediated gonococcal entry into Chang cells resembles phagocytosis rather than macropinocytosis reported for Salmonella spp. and sequentially involves gonococcal adherence to the cell surface, Opa-dependent and cytochalasin-insensitive recruitment of F-actin, and cytochalasin D-sensitive bacterial internalization.
Upregulation of tumor necrosis factor alpha and interleukin-1 beta in Q fever endocarditis
Authors: Capo C , Zugun F , Stein A , Tardei G , Lepidi H , Raoult D , Mege J L
Published: January 2020

The occurrence of Q fever endocarditis likely involves some alterations in the responses of monocytes, the in vivo targets of Coxiella burnetii. To test this hypothesis, the production of the inflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 was assessed in monocytes from patients with Q fever endocarditis. Spontaneous transcription and secretion of tumor necrosis factor and interleukin-1 were significantly higher in patient monocytes than in healthy controls. The interleukin-6 transcripts were also upregulated in patient cells. Moreover, in patients with recent endocarditis exhibiting high titers of immunoglobulin G directed to C. burnetii in phase I, monocytes released significantly higher levels of tumor necrosis factor and interleukin-1 than in patients with stabilized endocarditis. Immunoglobulin G titers and the overproduction of tumor necrosis factor and interleukin-1 were significantly correlated. Hence, the overproduction of inflammatory cytokines might be a marker of disease activity.
Mitotic block and delayed lethality in HeLa epithelial cells exposed to Escherichia coli BM2-1 producing cytotoxic necrotizing factor type 1
Authors: De Rycke J , Mazars P , Nougayrede J P , Tasca C , Boury M , Herault F , Valette A , Oswald E
Published: January 2020

The cytopathic effect (CPE) of Escherichia coli producing cytotoxic necrotizing factor type 1 (CNF1) was investigated by using a human epithelial cell (HeLa) model of infection with CNF1-producing E. coli BM2-1. This strain was shown to bind loosely, but massively, to HeLa cells. A 4-h interaction between bacteria and eukaryotic cells triggered the delayed appearance of a progressive dose-dependent CPE characterized by (i) intense swelling of cells accompanied by the formation of a dense network of actin stress fibers, (ii) inhibition of cell division due to a complete block in the G2 phase of the cell cycle, and (iii) nucleus swelling and chromatin fragmentation. These alterations resulted in cell death starting about 5 days after interaction. The absence of multinucleation clearly distinguished the CPE from the effect produced by cell-free culture supernatants of infected cells nor prevented by a CNF1-neutralizing antiserum. Pathogenicity was completely abolished after Tn5::phoA insertion mutagenesis in the cnf-1 structural gene but not restored by trans complementation with a recombinant plasmid containing intact cnf-1 and its promoter. These results suggest that a gene downstream of cnf-1, essential to the induction of the CPE, was affected by the mutation. On the other hand, transformation of the wild-type strain BM2-1 with the same recombinant plasmid leads to a significant increase in both CNF1 activity and CPE, demonstrating the direct contribution of CNF1 to the CPE. In conclusion, the pathogenicity of E. coli BM2-1 for HeLa cells results from a complex interaction involving cnf-1 and associated genes and possibly requiring a preliminary step of binding of bacterial organisms to target cells.
Interaction of verotoxin 2e with pig intestine
Authors: Waddell T E , Lingwood C A , Gyles C L
Published: January 2020

In pigs with edema disease, verotoxin 2e (VT2e) is produced in the intestine and transported to tissues, but neither the mechanism by which toxin passes through the intestine nor its failure to induce an enterotoxic reaction is understood. Binding of VT2e to pig intestine was examined by enzyme-linked immunosorbent assay involving microvillus membranes (MVM) and crude mucus; thin-layer chromatographic overlay immunoassay with total lipids extracted from MVM; and indirect immunofluorescence of toxin bound to thin sections of jejunum, ileum, and colon. VT2e bound significantly to MVM from pig jejunum and ileum but not to crude mucus. Verotoxin 2e-binding glycolipids, globotetraosylceramide and globotriaosylceramide, were detected by thin-layer chromatographic overlay immunoassay in extracts of MVM from jejunum and ileum. Indirect immunofluorescence showed that VT2e bound to vessels within the submucosa and muscularis mucosa of the jejunum, ileum, and colon and to enterocytes at the lower portion but not at the tips of villi in the jejunum and ileum. Receptors for VT2e are therefore present in the intestine of the pig, but their role in absorption of VT2e is unclear since intraintestinal inoculation of pigs with large quantities of VT2e does not result in edema disease. Previously reported lack of enterotoxicity of verotoxins in pig intestine may be explained by the absence of toxin receptors in the villus absorptive enterocytes.
ADP-ribosylation of an approximately 70-kilodalton protein of Klebsiella pneumoniae
Authors: Geipel U , Just I , Aktories K
Published: January 2020

An approximately 70-kDa protein in the culture supernatant of a human pathogenic strain of Klebsiella pneumoniae was labeled in the presence of [32P-adenylate]NAD. Labeling was significantly increased by the addition of dithiothreitol ( > 1 mM) but prevented by treatment of the culture supernatant for 3 min at 56 degrees C. The addition of unlabeled NAD, but not of ADP-ribose, blocked labeling of the approximately 70-kDa protein. The radioactive label was released by formic acid but not by HgCl2 (1 mM) or neutral hydroxylamine (0.5 M). The addition of homogenates of human platelets, human neutrophils, rat brain, rat lung, or rat spleen tissues to the culture supernatant did not induce labeling of eukaryotic proteins. The data indicate that the K. pneumoniae strain produces ADP-ribosyltransferase which modifies an endogenous protein.
Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock
Authors: Cluss R G , Goel A S , Rehm H L , Schoenecker J G , Boothby J T
Published: January 2020

The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry. The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C. Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature. Spheroplasts of B. burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction. Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5. A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature. Some of these putative degradation products were recognized by monoclonal antibodies directed against the B. burgdorferi DnaK protein. These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer. These findings provide a new interpretation of previous work suggesting that 10 to 15 B. burgdorferi polypeptides, including DnaK have a common epitope.
Human monocyte CD14 is upregulated by lipopolysaccharide
Authors: Landmann R , Knopf H P , Link S , Sansano S , Schumann R , Zimmerli W
Published: January 2020

Membrane CD14 is involved in lipopolysaccharide (LPS)-induced monocyte activation; it binds LPS, and antibodies against CD14 block the effects of low-dose LPS. It is unknown how LPS regulates its own receptor CD14 in vitro. Therefore, we investigated the effects of LPS on CD14 mRNA and membrane and soluble CD14 (mCD14 and sCD14, respectively) in human monocytes and macrophages. No changes were observed during the first 3 h of LPS stimulation. After 6 to 15 h, LPS weakly reduced CD14 mRNA and mCD14 and transiently enhanced sCD14 release. A 2-day incubation with LPS caused increases in the levels of CD14 mRNA (2-fold), mCD14 (2-fold), sCD14 (1.5-fold), and LPS-fluorescein isothiocyanate binding (1.5-fold); a 5-h incubation with LPS was sufficient to induce the late effects on mCD14 and sCD14. The maximal effect on mCD14 and sCD14 was reached with > or = 1 ng of LPS per ml; the proportional distribution of the two sCD14 isoforms was not modified by LPS. Besides rough and smooth LPS, lipid A, heat-killed Escherichia coli, lipoteichoic acid, and Staphylococcus aureus cell wall extract (10 micrograms/ml) caused similar increases of mCD14. The LPS effect was blocked by polymyxin B but not by anti-tumor necrosis factor alpha, anti-interleukin-6, anti-gamma interferon, and anti-LPS-binding protein. LPS-induced tumor necrosis factor alpha production was abolished after a second 4-h challenge. In contrast, the LPS-induced increases CD14 mRNA, mCD14, and sCD14 were stronger and appeared earlier after a second LPS challenge. In conclusion, CD14 is transcriptionally upregulated by LPS and other bacterial cell wall constituents.
Developmental changes in the expression of Leishmania chagasi gp63 and heat shock protein in a human macrophage cell line
Authors: Streit J A , Donelson J E , Agey M W , Wilson M E
Published: January 2020

The ability of the protozoan Leishmania chagasi to infect a vertebrate host depends on its ability to survive intracellularly in a mammalian macrophage. Novel patterns of gene expression are probably important for conversion from the extracellular promastigote to the obligate intracellular amastigote parasite form. We found that the human macrophage-like cell line U937 provided an in vitro model of phagocytosis of L. chagasi promastigotes and intracellular conversion to amastigotes, allowing examination of parasite protein and RNA expression. The Leishmania surface protease gp63 assumed three isoforms during stage conversion, and a 64-kDa form of gp63 not present in promastigotes became the most prominent form in amastigotes. gp63 RNAs derived from the three different classes of msp genes (mspS, mspL, and mspC) were also differentially expressed. Infectious promastigotes contained mRNAs from mspS and mspC genes, whereas converting parasites expressed only mspL and mspC mRNAs. Sequence analysis of clones from an amastigote cDNA library confirmed the presence of gp63 mRNAs only from mspL and mspC class genes in tissue-derived amastigotes. Finally, 24 h after phagocytosis, there was a transient increase in the level of hsp70 and hsp90 proteins that subsequently decreased to baseline; this increase was not due to heat shock alone. We conclude that a unique pattern of selected L. chagasi proteins and RNAs is induced following phagocytosis by macrophages.
Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model
Authors: Menzies B E , Kernodle D S
Published: January 2020

A nonhemolytic, nonlethal variant of Staphylococcus aureus alpha-toxin constructed via oligonucleotide-directed mutagenesis and containing a single amino acid substitution (H-35 to L) was used to immunize a rabbit. The resulting antiserum was cross-reactive with wild-type alpha-toxin and neutralized its hemolytic activity in vitro. Passive immunization of mice with rabbit antiserum conferred protection against lethal challenge with wild-type alpha-toxin and against acute lethal challenge with a high-alpha-toxin -producing S. aureus strain. H35L alpha-toxin may be useful as a protective immunogen in S. aureus vaccine studies.
Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells
Authors: Sambri V , Aldini R , Massaria F , Montagnani M , Casanova S , Cevenini R
Published: January 2020

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae. Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed. The liver uptakes ranged between 65.9% for B. burgdorferi IRS and 40.5% for B. turicatae. Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium. The in vitro uptake of B. burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B. burgdorferi antibody. The reculturing of B. burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative. The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors.
Serum antibody responses of cattle following experimental infection with Escherichia coli O157:H7
Authors: Johnson R P , Cray W C , Johnson S T
Published: January 2020

Oral inoculation of calves and steers with 10(10) CFU of Escherichia coli O157:H7 induced prompt and sustained increases in serum antibodies to O157 lipopolysaccharide. Neutralizing antibodies to verotoxin 1 also increased rapidly in most steers but more gradually in calves. None of the animals developed neutralizing antibodies to verotoxin 2. These serological responses were not correlated with elimination of infection in calves or steers or protection of calves against reinfection with the same strain.
In vitro simulation of immunosuppression caused by Trypanosoma brucei: active involvement of gamma interferon and tumor necrosis factor in the pathway of suppression
Authors: Darji A , Beschin A , Sileghem M , Heremans H , Brys L , De Baetselier P
Published: January 2020

Experimental infections of mice with the African trypanosome Trypanosoma brucei lead to a profound state of T-cell unresponsiveness in the lymph node cell (LNC) compartment. This suppression is mediated by macrophage-like cells which inhibit interleukin 2 (IL-2) secretion and down-regulate IL-2 receptor expression (M. Sileghem, A. Darji, R. Hamers, M. Van de Winkel, and P. De Baetselier, Eur. J. Immunol. 19:829-835, 1989). Similar suppressive cells can be generated in vitro by pulsing 2C11-12 macrophage hybridoma cells with opsonized T. brucei parasites (2C11-12P cells). Cocultures of 2C11-12P cells and LNCs secrete higher levels of gamma interferon (IFN-gamma), and the hyperproduction of IFN-gamma was found to be confined to CD8+ lymphoid cells. Elimination of CD8+ cells from cocultures of 2C11-12P cells and LNCs restores the T-cell proliferative response. Furthermore, addition of neutralizing anti-IFN-gamma antibodies to the cocultures reduces the level of suppression and concomitantly restores the level of IL-2 receptor expression. Hence, IFN-gamma plays a cardinal role in this in vitro model for T. brucei-elicited immunosuppression. Cocultures of LNCs and 2C11-12P cells in a two-chamber culture system further demonstrated that cell-cell contact is required for hyperproduction of IFN-gamma and, moreover, that IFN-gamma cooperates with a 2C11-12P-derived diffusible factor to exert its suppressive activity. Finally, tumor necrosis factor alpha (TNF-alpha produced by 2C11-12P cells was found to be implicated in the hyperproduction of IFN-gamma, since addition of neutralizing anti-TNF-alpha antibodies to cocultures reduced the level of suppression and concomitantly abrogated the hyperproduction of IFN-gamma. Collectively, our findings indicate that T. brucei-elicited suppressive 2C11-12 macrophage cells differentially influence T-cell subpopulations: (i) CD8+ cells are signaled via cell-cell contact to produce IFN-gamma, and TNF-alpha is implicated in this process, and (ii) locally produced IFN-gamma and macrophage-released factors act in concert to inhibit CD4+ and CD8+ T-cell proliferative responses.
Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes
Authors: Aliberti J C , Cardoso M A , Martins G A , Gazzinelli R T , Vieira L Q , Silva J S
Published: January 2020

Host resistance to infection by Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the first week of infection in mice, NK cell-derived gamma interferon (IFN-gamma) is involved in controlling intracellular parasite replication, mainly through the induction of NO biosynthesis by activated macrophages. Interleukin-12 (IL-12) has been shown to be a powerful cytokine in inducing IFN-gamma synthesis by NK cells, as well as in mediating resistance to different intracellular protozoa. We have therefore studied the ability of T. cruzi to elicit IL-12 synthesis by macrophages and the role of this cytokine in controlling parasite replication during acute infection in mice. Our results show that macrophages cultured in the presence of live trypomastigote forms (but not epimastigotes) release IL-12 that can induce IFN-gamma production by normal spleen cells. IL-12 was detected in as little as 12 h after the addition of the trypomastigotes, and the level of IL-12 peaked at 48 h after the initial macrophage-parasite incubation. The addition of anti-IL-12 monoclonal antibody to macrophage-trypomastigote supernatants dose-dependently inhibited IFN-gamma production by naive splenocytes. Finally, the in vivo role of IL-12 in resistance to infection by T. cruzi was analyzed. Mice treated with anti-IL-12 monoclonal antibody had significantly increased parasitemia and mortality in comparison with those of control infected mice treated with control antibody. Together, these results suggest that macrophage-derived IL-12 plays a major role in controlling the parasitemia in T. cruzi-infected mice and that the animal's resistance during the acute phase of infection may, at least in part, be a consequence of postinfection levels of IL-12.
Expression of the CopB outer membrane protein by Moraxella catarrhalis is regulated by iron and affects iron acquisition from transferrin and lactoferrin
Authors: Aebi C , Stone B , Beucher M , Cope L D , Maciver I , Thomas S E , McCracken G H , Sparling P F , Hansen E J
Published: January 2020

The amino acid sequence of the cell-surface-exposed, 81-kDa CopB outer membrane protein of Moraxella catarrhalis was found to be similar to those of TonB-dependent outer membrane proteins of other gram-negative bacteria. Expression of CopB was affected by the availability of iron in the growth medium, and the extent of overexpression of CopB in response to iron limitation varied widely among the M. catarrhalis strains tested. Wild-type M. catarrhalis strains were found to be able to utilize ferric citrate, transferrin, lactoferrin, and heme as sources of iron for growth in vitro. However, an isogenic copB mutant was severely impaired in its ability to utilize transferrin and lactoferrin as sole sources of iron for growth, whereas this same mutant grew similarly to the wild-type parent strain when supplied with ferric citrate as the iron source. The copB mutant was not significantly different from its wild-type parent strain in its ability to bind transferrin and lactoferrin. In addition, the wild-type parent strain and the copB mutant exhibited equivalent rates of uptake of 55Fe from ferric citrate. However, the copB mutant was markedly less able than the wild-type strain to take up 55Fe from transferrin and lactoferrin. These results indicate that lack of expression of the CopB protein exerts a direct or indirect effect on the ability of M. catarrhalis to utilize iron bound to certain carrier proteins.
Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species
Authors: Shang E S , Summers T A , Haake D A
Published: January 2020

We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens.
Binding of hemoglobin to the envelope of Porphyromonas gingivalis and isolation of the hemoglobin-binding protein
Authors: Fujimura S , Shibata Y , Hirai K , Nakamura T
Published: January 2020

The binding activity of the Porphyromonas gingivalis envelope and hemoglobin was examined over a wide range of pH values from 4.5 to 9.0. The binding activity in low-pH buffers was much higher than that at high pH; the optimum pHs for the binding were found to be 4.5 and 5.0. Since the hemoglobin bound to the envelope was found to dissociate in the pH 8.5 and 9.0 buffers, the binding is reversible. We hypothesized that hemoglobin-binding protein (HbBP), responsible for the binding to hemoglobin, exists in the envelope and confirmed its presence by dot blot determination with peroxidase-conjugated hemoglobin. Then we attempted to isolate HbBP from the solubilized (by a detergent) materials of the envelope by affinity chromatography. The molecular mass of HbBP was 19 kDa, and the isoelectric point was 4.3.
Listeriolysin O activates mitogen-activated protein kinase in eucaryotic cells
Authors: Tang P , Rosenshine I , Cossart P , Finlay B B
Published: January 2020

Infection with Listeria monocytogenes induces the activation of mitogen-activated protein (MAP) kinase in several tissue culture cell lines (P.Tang, I. Rosenshine, and B. B. Finlay, Mol. Biol. Cell 5:455-464, 1994). After various mutants were examined, the bacterial factor responsible for MAP kinase activation was identified as listeriolysin O (LLO). Growth supernatant containing LLO or purified LLO alone can induce MAP kinase tyrosine phosphorylation in HeLa cells. Single-amino-acid mutations in LLO that do not affect its membrane binding capacity but reduce its cytolytic activity also reduced its ability to induce MAP kinase activity in HeLa cells. Streptolysin O, another sulfhydryl-activated hemolysin, and the detergent saponin are also able to activate MAP kinase in target cells. Thus, the increased MAP kinase activity observed in L. monocytogenes-infected cells is most likely a result of the permeabilization of the host cell membrane by LLO and may not be linked with invasion.
New evidence for an inflammatory component in diarrhea caused by selected new, live attenuated cholera vaccines and by El Tor and Q139 Vibrio cholerae
Authors: Silva T M , Schleupner M A , Tacket C O , Steiner T S , Kaper J B , Edelman R , Guerrant R
Published: January 2020

Using a lactoferrin latex agglutination assay, we have compared the inflammatory responses to a cholera vaccine candidate, CVD 110, in which all known toxin genes have been deleted or mutated yet still produced significant diarrhea, with a less reactive vaccine strain and wild-type El Tor and 0139 Vibrio cholerae strains. Data suggest that diarrhea due to attenuated and wild-type El Tor V. cholerae, and to a lesser extent 0139 V. cholerae, involves an inflammatory response. Further study is required to further elucidate the mechanism of the process(es) involved.
Purification and biochemical characterization of a 65-kilodalton mannoprotein (MP65), a main target of anti-Candida cell-mediated immune responses in humans
Authors: Gomez M J , Torosantucci A , Arancia S , Maras B , Parisi L , Cassone A
Published: January 2020

A 65 kDa-constituent (MP65) of a whole-cell mannoprotein (MP) fraction of Candida albicans was purified by immunoaffinity chromatography with monoclonal antibodies directed against periodate-insensitive, protease-sensitive MP epitopes, putatively polypeptide in nature. These antibodies were obtained by immunization of mice with concanavalin A bead-coupled, low-glycosylated MP from hyphal cells of C. albicans grown in the presence of a subinhibitory dose of tunicamycin. The immunoaffinity-purified MP65 molecule had a pI of 4.1 and a protein/polysaccharide ratio of 1.8:1. It was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, or N-glycoffanases but still reactive with concanavalin A. The polysaccharide moiety of MP65 was composed exclusively of mannose and glucose at a ratio of 12.7 to 1. The protein moiety showed numerous potential O-glycosidic linkage sites as suggested by the high proportion of serine and threonine (together accounting for more than 20% of the total amino acid composition) and susceptibility to diluted alkali. This treatment and digestion with alpha-mannosidase caused a reduction in the MP65 molecular mass to around 54 kDa. The N-terminal sequence of MP65 protein moiety was rich in alanine and valine (7 of 13 amino acids) and did not show any significant homology with deposited sequences in data banks. Purified MP65, at doses of a few nanograms, induced extensive T-cell proliferation of human peripheral blood mononuclear cells. This proliferation was specifically inhibited, in a dose-response fashion, by the antigen-binding fragment of the monoclonal antibody used for immunoaffinity purification. Overall, these results highlight biochemical and molecular details of MP65, a main target of human T-cell response to C.albicans.
Infection of rabbit Peyer's patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on follicle-associated epithelium
Authors: Sansonetti P J , Arondel J , Cantey J R , Prévost M C , Huerre M
Published: January 2020

In order to invade the colonic mucosa, the bacterial pathogen Shigella flexneri must find a site of entry. Experiments with the rabbit ligated intestinal loop model described here confirm that M cells of the follicle-associated epithelium (FAE) that covers lymphoid structures of the Peyer's patches represent a major site of entry for invasive microorganisms. In addition, in an isogenic Shigella background, expression of an adhesive phenotype, or of an invasive phenotype, is required for bacteria to efficiently colonize the FAE. A nonadhesive, noninvasive mutant barely interacted with FAE. Adhesive and invasive strains induced dramatic but different alterations on FAE. Invasive strain M90T caused major inflammation-mediated tissue destruction after 8 h of infection. Adhesive strain BS15 caused limited inflammation, but major architectural changes, characterized by an increase in the size of M cells that became stretched over large pockets containing an increased number of mononuclear cells, were observed. M cells progressively occupied large surface areas of the FAE at the expense of enterocytes. This contributed to enterocytes losing contact with the lumen. These experiments demonstrate that various remodeling patterns may occur in Peyer's patches in response to bacterial pathogens, depending on the virulence phenotype expressed by the pathogenic strain.
Species-, serogroup-, and serovar-specific epitopes are juxtaposed in variable sequence region 4 of the major outer membrane proteins of some Chlamydia trachomatis serovars
Authors: Batteiger B E , Lin P M , Jones R B , Van Der Pol B J
Published: January 2020

Synthetic peptides and murine monoclonal antibodies were used to map cross-reactive chlamydial epitopes. A species-specific epitope in the central region of variable sequence region 4 abuts the amino-terminal end of a B-serogroup-specific or F/G-serogroup-specific epitope, which in turn abuts known serovar-specific epitopes. The carboxyl-terminal portion of variable sequence region 4 (residues 297 to 314) comprises a region of end-to-end B-cell epitopes in some serovars of the B and F/G serogroups.
Chronic experimental Chagas' disease: functional syngeneic T-B-cell cooperation in vitro in the absence of an exogenous stimulus
Authors: Freire-de-Lima C , Peçanha L M , Dos Reis G A
Published: January 2020

We have investigated CD4+ T-cell autoreactivity to normal syngeneic B cells in vitro in chronic experimental Chagas' disease. Resting B cells induced an intense proliferative response and lymphokine secretion by splenic CD4+ T cells from Trypanosoma cruzi-infected (8 months or more of infection) donors, compared to much lower responses by uninfected controls. On the other hand, lipopolysaccharide-activated B cells induced syngeneic CD4+ T-cell activation in both control and infected groups. The observed syngeneic T-B-cell cooperation was bidirectional. In the absence of any exogenous stimulus, CD4+ T cells from T. cruzi-infected animals induced much higher production of all tested immunoglobulin (Ig) isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) by syngeneic B cells, compared to T cells from uninfected donors. When lipopolysaccharide-treated B cells were used, CD4+ T cells from either control or infected donors enhanced IgG1 and IgG3 production, but only CD4+ T cells of infected origin induced IgG2a production in this system without addition of exogenous gamma interferon. Enhanced T-cell proliferation and Ig production were also observed with highly purified CD4+ T cells and in serum-free medium. Both proliferation and Ig production could be blocked with anti-major histocompatibility complex class II monoclonal antibodies. Enhanced reactivity and help for Ig production were seen only in response to syngeneic BALB B cells and not in response to allogeneic B10 B cells. These results indicate that chronic infection with T. cruzi results in increased CD4+ T-cell reactivity towards syngeneic B cells, which leads to spontaneous Ig production. These autoreactive T cells might play a role in polyclonal autoantibody production in chronic Chagas' disease.
Induction of an extracellular esterase from Candida albicans and some of its properties
Authors: Tsuboi R , Komatsuzaki H , Ogawa H
Published: January 2020

An extracellular esterase from Candida albicans A-714 was found to be induced in a medium containing 0.7% yeast nitrogen base and 2.5% Tween 80 (polyoxyethylenesorbitan compounds). Enzyme activity, which exists predominantly in the extracellular space, was measured by a colorimetric method using alpha-naphthyl palmitate as a substrate. The induction level of the esterase activity was found to be well correlated with fungal growth and was dependent on the Tween 80 concentration. Such esterase activity was observed only in medium containing Tween 80 or other Tweens as the sole carbon source and therefore was not observed in either peptone-glucose medium or peptone-glucose medium supplemented with Tween 80. The induced esterase was heat labile and had maximum activity at pH 5.5. Enzyme activity was stimulated by the addition of sodium taurocholate, an activator of lipase. Thin-layer chromatography revealed that this enzyme does not hydrolyze triolein and L-alpha-lecithin, suggesting that it is a monoester hydrolase (not a lipase in the strict sense of the word). Esterase activity was examined in 85 clinical isolates of Candida species; C. albicans, C. tropicalis, and C. parapsilosis tended to have higher enzyme activities than C. kefyr, C. krusei, C. glabrata, and C. guilliermondii. Although the physiological properties of this esterase are not clear at present, it was found to be crucial for fungal growth under specific conditions.
Cytokine production by human epithelial and endothelial cells following exposure to oral viridans streptococci involves lectin interactions between bacteria and cell surface receptors
Authors: Vernier A , Diab M , Soell M , Haan-Archipoff G , Beretz A , Wachsmann D , Klein J P
Published: January 2020

In order to examine the possible implication of human epithelial and endothelial cells in the pathogenesis of various diseases associated with oral viridans streptococci, we tested the immunomodulatory effects of 11 representative strains of oral viridans streptococci on human epithelial KB cells and endothelial cells. We then examined the possible role of two major adhesins from oral viridans streptococci, protein I/II and rhamnose-glucose polymers (RGPs), in this process. In this study we demonstrate that oral viridans streptococci are potent stimulators of interleukin-8 (IL-8) production from KB cells and of IL-6 and IL-8 production from endothelial cells. The ability of protein I/II and RGPs to contribute to these effects was then examined. Using biotinylated protein I/IIf and RGPs from Streptococcus mutans OMZ 175, we showed that these adhesins bind to KB and endothelial cells through specific interactions and that the binding of these molecules initiates the release of IL-8 from KB cells and of IL-6 and IL-8 from endothelial cells. These results suggest that protein I/IIf and RGPs play an important role in the interactions between bacteria and KB and endothelial cells in that similar cytokine profiles are obtained when cells are stimulated with bacteria or surface components. We also provide evidence that protein I/IIf binds to and stimulates KB and endothelial cells through lectin interactions and that N-acetyl neuraminic acid (NANA) and fucose present on cell surface glycoproteins may form the recognition site since binding and cytokine release can be inhibited by dispase and periodate treatment of cells and by NANA and fucose. These results demonstrate that oral viridans streptococci, probably by engaging two cell surface adhesins, exert immunomodulatory effects on human KB and endothelial cells.