bluuebunny/crossref_metadata_2025 huggingface dataset:
Deglycosylation of the 45/47-Kilodalton Antigen Complex ofMycobacterium tuberculosisDecreases Its Capacity To Elicit In Vivo or In Vitro Cellular Immune Responses
Authors: Romain Félix , Horn Cynthia , Pescher Pascale , Namane Abdelkader , Riviere Michel , Puzo Germain , Barzu Octavian , Marchal Gilles
Published: December 2019

A protection against a challenge withMycobacterium tuberculosisis induced by previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guérin (BCG). The 45/47-kDa antigen complex (Apa) present in culture filtrates of BCG ofM. tuberculosishas been identified and isolated based on its ability to interact mainly with T lymphocytes and/or antibodies induced by immunization with living bacteria. The protein is glycosylated. A large batch of Apa was purified fromM. tuberculosisculture filtrate to determine the extent of glycosylation and its role on the expression of the immune responses. Mass spectrometry revealed a spectrum of glycosylated molecules, with the majority of species bearing six, seven, or eight mannose residues (22, 24, and 17%, respectively), while others three, four, or five mannoses (5, 9, and 14%, respectively). Molecules with one, two, or nine mannoses were rare (1.5, 3, and 3%, respectively), as were unglycosylated species (in the range of 1%). To eliminate the mannose residues linked to the protein, the glycosylated Apa molecules were chemically or enzymatically treated. The deglycosylated antigen was 10-fold less active than native molecules in eliciting delayed-type hypersensitivity reactions in guinea pigs immunized with BCG. It was 30-fold less active than native molecules when assayed in vitro for its capacity to stimulate T lymphocytes primed in vivo. The presence of the mannose residues on the Apa protein was essential for the antigenicity of the molecules in T-cell-dependent immune responses in vitro and in vivo.
CD4+T-Cell- and Gamma Interferon-Dependent Protection against Murine Malaria by Immunization with Linear Synthetic Peptides from aPlasmodium yoelii17-Kilodalton Hepatocyte Erythrocyte Protein
Authors: Charoenvit Yupin , Majam Victoria Fallarme , Corradin Giampietro , Sacci John B. , Wang Ruobing , Doolan Denise L. , Jones Trevor R. , Abot Esteban , Patarroyo Manuel E. , Guzman Fanny , Hoffman Stephen L.
Published: December 2019

Most work on protective immunity against the pre-erythrocytic stages of malaria has focused on induction of antibodies that prevent sporozoite invasion of hepatocytes, and CD8+T-cell responses that eliminate infected hepatocytes. We recently reported that immunization of A/J mice with an 18-amino-acid synthetic linear peptide fromPlasmodium yoeliisporozoite surface protein 2 (SSP2) in TiterMax adjuvant induces sterile protection that is dependent on CD4+T cells and gamma interferon (IFN-γ). We now report that immunization of inbred A/J mice and outbred CD1 mice with each of two linear synthetic peptides from the 17-kDaP. yoeliihepatocyte erythrocyte protein (HEP17) in the same adjuvant also induces protection against sporozoite challenge that is dependent on CD4+T cells and IFN-γ. The SSP2 peptide and the two HEP17 peptides are recognized by B cells as well as T cells, and the protection induced by these peptides appears to be directed against the infected hepatocytes. In contrast to the peptide-induced protection, immunization of eight different strains of mice with radiation-attenuated sporozoites induces protection that is absolutely dependent on CD8+T cells. Data represented here demonstrate that CD4+T-cell-dependent protection can be induced by immunization with linear synthetic peptides. These studies therefore provide the foundation for an approach to pre-erythrocytic-stage malaria vaccine development, based on the induction of protective CD4+T-cell responses, which will complement efforts to induce protective antibody and CD8+T-cell responses.
Human Onchocerciasis and Tetanus Vaccination: Impact on the Postvaccination Antitetanus Antibody Response
Authors: Cooper Philip J. , Espinel Ivan , Wieseman Moira , Paredes Wilson , Espinel Mauricio , Guderian Ronald H. , Nutman Thomas B.
Published: December 2019

To investigate whether helminth infections may affect the efficacy of vaccines by impairing the immune response to nonparasite vaccine antigens, we compared the antibody responses to tetanus toxoid (TT) after tetanus vaccination in 193 subjects withOnchocerca volvulusinfection with 85 comparable noninfected controls. After vaccination, the proportions of subjects in each group attaining protective levels of antitetanus antibodies were similar (96.9% infected versus 97.6% noninfected). Postvaccination increases in antitetanus immunoglobulin G (IgG) and the predominant IgG isotype, IgG1, were equivalent in both groups, as were increases in specific IgG4 and IgE; however, significantly greater increases in specific IgG2 (P< 0.05) and IgG3 (P< 0.001) were observed in the noninfected group. Stratification of theO. volvulus-infected group into two groups representing light and heavy infections revealed a significantly impaired antitetanus IgG response in those with heavy infections compared to those with light infections (P< 0.01) or no infection (P< 0.05). The impact of concurrent intestinal helminth infections on the antitetanus response was also examined; an increased IgG4/IgE ratio was seen in those infected withStrongyloides stercoralis(P< 0.05) and when all helminth infections were combined as a single group (P< 0.05). These findings indicate that concurrent infection withO. volvulusdoes not prevent the development of a protective antitetanus response, although heavierO. volvulusinfections are able to alter the magnitude of this response, and concurrent helminth infections (O. volvulusand intestinal helminths) may alter TT-specific antibody isotype responses.
Bordetella bronchiseptica
-Mediated Cytotoxicity to Macrophages Is Dependent on
bvg
-Regulated Factors, Including Pertactin
Authors: Forde Catrina B. , Shi Xiaoju , Li Jingli , Roberts Mark
Published: December 2019

The effect of
Bordetella bronchiseptica
infection on the viability of murine macrophage-like cells and on primary porcine alveolar macrophages was investigated. The bacterium was shown to be cytotoxic for both cell types, particularly where tight cell-to-cell contacts were established. In addition,
bvg
mutants were poorly cytotoxic for the eukaryotic cells, while a
prn
mutant was significantly less toxic than wild-type bacteria.
B. bronchiseptica
-mediated cytotoxicity was inhibited in the presence of cytochalasin D or cycloheximide, an inhibitor of microfilament-dependent phagocytosis or de novo eukaryotic protein synthesis, respectively. The mechanism of eukaryotic cell death was examined, and cell death was found to occur primarily through a necrotic pathway, although a small proportion of the population underwent apoptosis.
Determination of Antibody Responses of Elderly Adults to All 23 Capsular Polysaccharides after Pneumococcal Vaccination
Authors: Rubins Jeffrey B. , Alter Michael , Loch Joyce , Janoff Edward N.
Published: December 2019

The 23-valent pneumococcal polysaccharide vaccine was formulated to prevent invasive infection in the elderly and other high-risk populations from the most prevalent
Streptococcus pneumoniae
serotypes. However, the immunogenicity of all 23 vaccine polysaccharides has not been fully characterized in elderly adults. We previously reported that whereas the majority of elderly subjects had vigorous immune responses to selected pneumococcal vaccine polysaccharides, a subset of elderly individuals responded to fewer than two of seven vaccine serotypes after immunization. To determine whether these elderly low responders have a general inability to respond to pneumococcal vaccine and to determine whether elderly low responders might be identified by their responses to a few polysaccharides, we measured antibody responses of elderly adults to all 23 vaccine polysaccharides after pneumococcal immunization. As a group, elderly subjects showed a significant rise after immunization in geometric mean antibody levels to all 23 vaccine serotypes. However, when individual rather than group immune responses were assessed, the 23-valent vaccine did not appear to be uniformly immunogenic in these elderly subjects. Eleven elderly subjects (20%) had twofold increases in specific antibody after vaccination to only 5 or fewer of the 23 vaccine polysaccharides, and they did not respond to the most prevalent serotypes causing invasive disease. Antibody responses to serotype 9N were found to reliably distinguish low vaccine responders from other elderly subjects. However, no particular group of vaccine polysaccharides could be used as a marker for adequate immune responses if only postvaccination sera were analyzed.
De Novo Induction of Atherosclerosis by
Chlamydia pneumoniae
in a Rabbit Model
Authors: Fong Ignatius W. , Chiu Brian , Viira Esther , Jang Dan , Mahony James B.
Published: December 2019

Chlamydia pneumoniae
, a bacterial respiratory tract pathogen, has been associated with atherosclerosis in humans.
C. pneumoniae
infection of the respiratory tracts of rabbits fed a noncholesterol diet induced changes of atherosclerosis of the aorta in 6 (26.1%) of 23 animals after a single inoculum at 3 months. Multiple inocula given three times within 6 weeks resulted in grade III atherosclerosis in 8 (34.8%) of 23 rabbits, with an additional 5 (21.7%) showing increased myxoid changes in the intima-media junction and exhibiting 8 (34.8%) focal periaortitis. Control animals inoculated with carrier broth (
n
= 24), HEp-2 cells (
n
= 12), or another respiratory pathogen,
Mycoplasma pneumoniae
(
n
= 32), produced no changes of atherosclerosis after 3 months. The histological changes were dissimilar (fewer foam cells) from those of rabbits fed a 0.5% cholesterol diet but were highly similar to or indistinguishable from changes in rabbits fed a 0.15% cholesterol diet (similar to that of humans). Proinflammatory cytokines and tissue growth factors were more consistently detected in cholesterol-induced aortic lesions than those induced by
C. pneumoniae
. These data are compatible with de novo induction of atherogenesis by
C. pneumoniae
in rabbits and suggest that
C. pneumoniae
may be important in the pathogenesis of atherosclerosis in humans.
Binding of Pili from Uropathogenic
Escherichia coli
to Membranes Secreted by Human Colonocytes and Enterocytes
Authors: Goetz G. S. , Mahmood A. , Hultgren S. J. , Engle M. J. , Dodson K. , Alpers D. H.
Published: December 2019

PapG adhesins mediate the binding of uropathogenic
Escherichia coli
. Although receptors for these adhesins have not been demonstrated in intestinal epithelia, the colonic microflora includes strains of uropathogenic
E. coli
. We now report that surfactant-like particles secreted by the human intestine contain receptors for PapG adhesins and may provide an intestinal habitat for uropathogenic bacteria.
Role of the Amino-Terminal Region of
Porphyromonas gingivalis
Fimbriae in Adherence to Epithelial Cells
Authors: Sojar Hakimuddin T. , Han Yiping , Hamada Nobushiro , Sharma Ashu , Genco Robert J.
Published: December 2019

Porphyromonas gingivalis
fimbriae elicit many responses in eukaryotic cells, including mitogenicity, cytokine production, epithelial cell invasion, and cellular immune response. Specific domains of the major fimbrial protein (FimA) have been shown to be important in triggering some of these functions. The goal of the present study was to identify the domain(s) of
P. gingivalis
FimA responsible for specific interaction with human mucosal epithelial cells. Fimbriated
P. gingivalis
strains have been shown to bind to buccal epithelial cells, whereas nonfimbriated strains bind at low levels or not at all. This and other studies provide evidence that FimA mediates the adherence of
P. gingivalis
to oral epithelial cells. To determine the specific region(s) of
P. gingivalis
FimA involved in epithelial cell binding, specific antipeptide antibodies were used to inhibit the binding of iodinated purified fimbriae as well as the binding of
P. gingivalis
cells to epithelial cells. Antibodies directed against peptides 49 to 68 (VVMANTAGAMELVGKTLAEVK) and 69 to 90 (ALTTELTAENQEAAGLIMTAEP) were found to highly inhibit both the binding of fimbriae and the binding of
P. gingivalis
cells to epithelial cells. The antibody against FimA peptides 69 to 90 also reacted with
P. gingivalis
fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain is exposed on the surface of fimbriae. Our results suggest that the amino-terminal domain corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain of
P. gingivalis
fimbriae.
Superantigen Immune Stimulation Evokes Epithelial Monocyte Chemoattractant Protein 1 and RANTES Production
Authors: Jedrzkiewicz Sean , Kataeva Galina , Hogaboam Cory M. , Kunkel Steven L. , Strieter Robert M. , McKay Derek M.
Published: December 2019

Bacterial superantigens (SAgs) have been implicated in inflammatory disease, and SAg-treated mice have increased jejunal T cells. Here we show that T84 cells (a human epithelial cell line) display increased MCP-1 and RANTES mRNA expression and protein production in response to conditioned medium fromStaphylococcus aureusenterotoxin B (SEB; a model SAg)-activated immune cells. Also, MCP-1 and RANTES mRNAs were increased in jejunal enterocytes isolated from SEB-treated mice. We suggest that T-cell recruitment to the gut following SAg immune activation could be partially due to epithelium-derived chemokines.
Intracellular Delivery of a Cytolytic T-Lymphocyte Epitope Peptide by Pertussis Toxin to Major Histocompatibility Complex Class I without Involvement of the Cytosolic Class I Antigen Processing Pathway
Authors: Carbonetti Nicholas H. , Irish Teresa J. , Chen Carrie H. , O’Connell Colin B. , Hadley Gregg A. , McNamara Ulrike , Tuskan Robert G. , Lewis George K.
Published: December 2019

A CD8+cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion byBordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin andN-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.
Early Resistance of Interleukin-10 Knockout Mice to Acute Systemic Candidiasis
Authors: Vazquez-Torres Andres , Jones-Carson Jessica , Wagner R. Doug , Warner Thomas , Balish Edward
Published: December 2019

In contrast to immunocompetent controls, interleukin-10 (IL-10) knockout (KO) mice eliminated an experimental intravenous inoculation withCandida albicansfrom their kidneys. Improved clearance ofC. albicansfrom the kidneys of IL-10 KO mice was evident at 24 h after intravenous challenge with the fungus. Conversely, mice with a deletion of the IL-4 cytokine gene were more susceptible to systemic candidiasis than were immunocompetent controls. The hyperresistance of IL-10 KO mice to acute systemic candidiasis did not seem to correlate with nitric oxide-mediated immunity, but rather, it appeared to be associated with more efficient effector function of innate cells, possibly neutrophils. In support of the latter hypothesis, we observed that neutrophils from IL-10 KO mice were more efficient at killingC. albicansblastoconidia and hyphae than were neutrophils from immunocompetent control mice. Neither IL-10 KO nor IL-4 KO mice that were monoassociated withC. albicansfor 4 weeks showed any histologic evidence of systemic candidiasis of endogenous origin. In contrast to systemic candidiasis, we observed no significant (P< 0.05) differences in susceptibility among IL-10 KO, IL-4 KO, and wild-type (immunocompetent) mice to orogastric candidiasis. Our results suggest that IL-10 exerts a negative effect on the early, innate response to acute systemic candidiasis; however, in comparison to immunocompetent control (wild-type) mice, neither IL-10 nor IL-4 deficiency enhanced susceptibility to orogastric candidiasis.
Regulatory T Cells in the Antibody Response toHaemophilus influenzaeType b Polysaccharide
Authors: Breukels Mijke A. , Rijkers Ger T. , Voorhorst-Ogink Marleen M. , Zegers Ben J. M.
Published: December 2019

An in vitro culture system for the induction of an antipolysaccharide response was used to study the cellular interactions which determine the magnitude and nature of this B-lymphocyte response. Healthy adult volunteers were vaccinated with theHaemophilus influenzaetype b polysaccharide (PRP)-tetanus toxoid (TT) conjugate vaccine. Optimal in vitro anti-PRP and anti-TT antibody responses were obtained when B cells were cultured with equal amounts of T cells. The in vitro response is antigen dependent and antigen specific. Culturing with PRP mixed with TT in the presence of T cells induces the highest number of anti-PRP antibody-secreting cells (ASC) (128.4 ×/÷ 15.9 [geometric mean ×/÷ standard deviation] immunoglobulin M [IgM] anti-PRP ASC/106cells; 9.3 ×/÷ 7.6 IgG anti-PRP ASC/106cells). Culturing without T cells induced no anti-PRP ASC; culturing with only PRP, in the presence of T cells, yielded low numbers of anti-PRP ASC (3.7 ×/÷ 5.2 IgM anti-PRP ASC/106cells and 1.2 ×/÷ 2.2 IgG anti-PRP ASC/106cells). Transwell studies showed that the requirements for the antibody response against the polysaccharide are different from those of an antiprotein response. Cytokines formed as a consequence of contact between protein-specific B and T cells were on their own not sufficient to activate TT-specific B cells (8.4 ×/÷ 1.4 anti-TT ASC/106cells); direct contact between T and B cells appeared to be an absolute requirement. However, physical contact between B and T cells in one compartment of the Transwell system resulted in the release of soluble factors able to stimulate B cells in the other compartment to secrete antipolysaccharide antibodies (164 ×/÷ 1.6 anti-PRP ASC/106cells).
Cholera Toxin B Subunit Activates Arachidonic Acid Metabolism
Authors: Peterson Johnny W. , Finkelstein Richard A. , Cantu Juan , Gessell Deborah L. , Chopra Ashok K.
Published: December 2019

Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E
2
), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin’s activation of phospholipase A
2
, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT’s known enzymatic activity (i.e., ADP-ribosylation of G
Sα
), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc
−
murine Theta (Th)1.2-positive lymphoma cells deficient in G
Sα
. The experimental results showed that native CT stimulated the release of [
3
H[AA from S49 cyc
−
cells at a level similar to that for S49 WT cells, indicating that G
Sα
is not essential for this process. Further, levels of cAMP in the CT-treated cyc
−
cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [
3
H]AA metabolites from both S49 WT and cyc
−
cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [
3
H]AA from S49 cyc
−
cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.
In Vivo Blockage of Nitric Oxide with Aminoguanidine Inhibits Immunosuppression Induced by an Attenuated Strain ofSalmonella typhimurium, PotentiatesSalmonellaInfection, and Inhibits Macrophage and Polymorphonuclear Leukocyte Influx into the Spleen
Authors: MacFarlane Amanda Shearer , Schwacha Martin G. , Eisenstein Toby K.
Published: December 2019

Our laboratory has previously shown that after immunization with a strain ofSalmonella typhimurium, SL3235, made avirulent by a blockage in the pathway of aromatic synthesis, murine splenocytes were profoundly suppressed in their capacity to mount an in vitro antibody plaque-forming cell (PFC) response to sheep erythrocytes. Evidence indicated that suppression was mediated by nitric oxide (NO), since the in vitro addition ofNG-monomethyl-l-arginine blocked suppression. The present studies examined the effect of blocking NO production onSalmonella-induced immunosuppression by in vivo administration of aminoguanidine hemisulfate (AG). AG was administered to C3HeB/FeJ mice in their drinking water (2.5% solution) for 7 days prior to intraperitoneal inoculation with SL3235. AG treatment inhibited the increase in nitrate and nitrite levels in plasma and nitrite levels in the spleen seen in immunized mice. Importantly, AG treatment completely blocked suppression of the splenic PFC response and markedly attenuated the suppression of the response to concanavalin A in immunized mice, providing further evidence thatSalmonella-induced immunosuppression is mediated by NO. AG treatment also alleviated the majority of the splenomegaly associated with SL3235 inoculation, which correlated with a blockage of influx of neutrophils and macrophages into spleens, as assessed by flow cytometry. AG treatment unexpectedly resulted in 90% mortality in mice injected with the highly attenuated vaccine strain ofSalmonella, SL3235. Increased mortality in AG-treated mice correlated with inability to clear organisms from the spleen by day 15 postinoculation and with persistent bacteremia, compared with control mice. Collectively, these in vivo results underscore the dual biological consequences of NO production followingSalmonellainfection, with NO being necessary for host defense, but also having the potentially adverse effect of immunosuppression. A unifying hypothesis to explain how these seemingly paradoxical effects could both result from NO production is presented.
Regulation of ExoS Production and Secretion by
Pseudomonas aeruginosa
in Response to Tissue Culture Conditions
Authors: Vallis Amy J. , Yahr Timothy L. , Barbieri Joseph T. , Frank Dara W.
Published: December 2019

This study was initiated to characterize the regulation and secretion of ExoS by
Pseudomonas aeruginosa
during contact with eukaryotic cells. The production of ExoS was monitored by a sensitive ADP-ribosyltransferase activity assay, and specific activities were calculated for supernatant and cell-associated fractions. Time course analysis indicated that ExoS was produced after a lag period, suggesting that induction of the regulon is necessary for the expression of detectable amounts of enzyme activity. Under tissue culture growth conditions, ExoS was induced when
P. aeruginosa
was in contact with Chinese hamster ovary (CHO) cells or after growth in tissue culture medium with serum. The serum induction of ExoS appeared to result in generalized type III secretion, while induction by contact with CHO cells appeared to result in polarized type III secretion. Mutants in the type III secretory system that express a null phenotype for ExoS production in bacteriological medium produced but did not secrete the enzyme when
P. aeruginosa
was grown under inducing conditions in tissue culture medium. These results suggest that both induction and secretion of ExoS may differ when the bacteria are exposed to different growth environments. The putative type III translocation proteins and secretion apparatus of
P. aeruginosa
were required for translocation of bacterial factors that mediate changes in CHO cell morphology during infection.
Fas-FasL Interaction Involved in Pathogenesis of Ocular Toxoplasmosis in Mice
Authors: Hu Mark S. , Schwartzman Joseph D. , Yeaman Grant R. , Collins Jane , Seguin Rosanne , Khan Imtiaz A. , Kasper Lloyd H.
Published: December 2019

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation ofToxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8+and CD4+T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000T. gondiitachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8+than CD4+T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lprand B6-gldmice than in wild-type B6 mice (P< 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.
Functional Analysis of the Carboxy-Terminal Domain of
Bacillus anthracis
Protective Antigen
Authors: Brossier Fabien , Sirard Jean-Claude , Guidi-Rontani Chantal , Duflot Edith , Mock Michele
Published: December 2019

Protective antigen (PA) is the common receptor-binding component of the two anthrax toxins. We investigated the involvement of the PA carboxy-terminal domain in the interaction of the protein with cells. A deletion resulting in removal of the entire carboxy-terminal domain of PA (PA608) or part of an exposed loop of 19 amino acids (703 to 722) present within this domain was introduced into the
pag
gene. PA608 did not induce the lethal-factor (LF)-mediated cytotoxic effect on macrophages because it did not bind to the receptor. In contrast, PA711- and PA705-harboring lethal toxins (9- and 16-amino-acid deletions in the loop, starting after positions 711 and 705, respectively) were 10 times less cytotoxic than wild-type PA. After cleavage by trypsin, the mutant PA proteins formed heptamers and bound LF. The capacity of PA711 and PA705 to interact with cells was 1/10 that of wild-type PA. In conclusion, truncation of the carboxy-terminal domain or deletions in the exposed loop resulted in PA that was less cytotoxic or nontoxic because the mutated proteins did not efficiently bind to the receptor.
Role of DnaK in In Vitro and In Vivo Expression of Virulence Factors ofVibrio cholerae
Authors: Chakrabarti Sabyasachi , Sengupta Nilanjan , Chowdhury Rukhsana
Published: December 2019

ThednaKgene ofVibrio choleraewas cloned, sequenced, and used to construct adnaKinsertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes ofV. choleraeis ToxR, a transmembrane DNA binding protein. TheV. cholerae dnaKmutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis andtoxRtranscriptional fusions, we demonstrated a reduction in expression of thetoxRgene in thednaKmutant strain together with a concomitant increase in expression of ahtpG-like heat shock gene that is located immediately upstream and is divergently transcribed fromtoxR. This may be due to increased heat shock induction in thednaKmutant. In vivo, however, although expression from heat shock promoters in thednaKmutant was similar to that observed in vitro, expression of bothtoxRandhtpGwas comparable to that by the parental strain. In both strains, in vivo expression oftoxRwas significantly higher than that observed in vitro, but no reciprocal decrease inhtpGexpression was observed. These results suggest that the modulation oftoxRexpression in vivo may be different from that observed in vitro.
The
Pseudomonas aeruginosa
Secretory Product Pyocyanin Inactivates α
1
Protease Inhibitor: Implications for the Pathogenesis of Cystic Fibrosis Lung Disease
Authors: Britigan Bradley E. , Railsback Michelle A. , Cox Charles D.
Published: December 2019

α
1
Protease inhibitor (α
1
PI) modulates serine protease activity in the lung. Reactive oxygen species inactivate α
1
PI, and this process has been implicated in the pathogenesis of a variety of forms of lung injury. An imbalance of protease-antiprotease activity is also detected in the airways of patients with cystic fibrosis-associated lung disease who are infected with
Pseudomonas aeruginosa. P. aeruginosa
secretes pyocyanin, which, through its ability to redox cycle, induces cells to generate reactive oxygen species. We tested the hypothesis that redox cycling of pyocyanin could lead to inactivation of α
1
PI. When α
1
PI was exposed to NADH and pyocyanin, a combination that results in superoxide production, α
1
PI lost its ability to form an inhibitory complex with both porcine pancreatic elastase (PPE) and trypsin. Similarly, addition of pyocyanin to cultures of human airway epithelial cells to which α
1
PI was also added resulted in a loss of the ability of α
1
PI to form a complex with PPE or trypsin. Neither superoxide dismutase, catalase, nor dimethylthiourea nor depletion of the media of O
2
to prevent formation of reactive oxygen species blocked pyocyanin-mediated inactivation of α
1
PI. These data raise the possibility that a direct interaction between reduced pyocyanin and α
1
PI is involved in the process. Consistent with this possibility, pretreatment of α
1
PI with the reducing agent β-mercaptoethanol also inhibited binding of trypsin to α
1
PI. These data suggest that pyocyanin could contribute to lung injury in the
P. aeruginosa
-infected airway of cystic fibrosis patients by decreasing the ability of α
1
PI to control the local activity of serine proteases.
Treponema denticola
Outer Membrane Enhances the Phagocytosis of Collagen-Coated Beads by Gingival Fibroblasts
Authors: Battikhi Tulin , Lee Wilson , McCulloch Christopher A. G. , Ellen Richard P.
Published: December 2019

Human gingival fibroblasts (HGFs) degrade collagen fibrils in physiological processes by phagocytosis. Since
Treponema denticola
outer membrane (OM) extract perturbs actin filaments, important structures in phagocytosis, we determined whether the OM affects collagen phagocytosis in vitro by HGFs. Phagocytosis was measured by flow cytometric assessment of internalized collagen-coated fluorescent latex beads. Confluent HGFs pretreated with
T. denticola
ATCC 35405 OM exhibited an increase in the percentage of collagen phagocytic cells (phagocytosis index [PI]) and in the number of beads per phagocytosing cell (phagocytic capacity [PC]) compared with untreated controls. The enhancement was swift (within 15 min) and was still evident after 1 day. PI and PC of HGFs for bovine serum albumin (BSA)-coated beads were also increased, indicating a global increase in phagocytic processes. These results contrasted those for control OM from
Veillonella atypica
ATCC 17744, which decreased phagocytosis. The
T. denticola
OM-induced increase in bead uptake was eliminated by heating the OM and by depolymerization of actin filaments by cytochalasin D treatment of HGFs. Fluid-phase accumulation of lucifer yellow was enhanced in a saturable, concentration-dependent, transient manner by the
T. denticola
OM. Our findings were not due to HGF detachment or cytotoxicity in response to the
T. denticola
OM treatment since the HGFs exhibited minimal detachment from the substratum; they did not take up propidium iodide; and there was no change in their size, granularity, or content of sub-G
1
DNA. We conclude that a heat-sensitive component(s) in
T. denticola
OM extract stimulates collagen phagocytosis and other endocytic processes such as nonspecific phagocytosis and pinocytosis by HGFs.
Association of a Myosin Immunoanalogue with Cell Envelopes of
Aspergillus fumigatus
Conidia and Its Participation in Swelling and Germination
Authors: Esnault Karine , el Moudni Brahim , Bouchara Jean-Philippe , Chabasse Dominique , Tronchin Guy
Published: December 2019

A myosin immunoanalogue was identified in conidia of
Aspergillus fumigatus
by Western blotting, indirect immunofluorescence assay, and gold immunoelectron microscopy with two different antimyosin antibodies. The distribution pattern of this protein was followed during the early stages of germination. A single 180-kDa polypeptide, detected predominantly in a cell envelope extract, was found to cross-react with monoclonal and polyclonal antibodies raised against vertebrate muscle myosin. Immunoelectron microscopy permitted precise localization of this polypeptide, indicating that myosin analogue was mainly distributed along the plasma membrane of resting and swollen conidia. In germinating conidia, indirect immunofluorescence microscopy revealed myosin analogue at the periphery of germ tubes, whereas actin appeared as dispersed punctate structures in the cytoplasm that were more concentrated at the site of germ tube emergence. A myosin ATPase inhibitor, butanedione monoxime, greatly reduced swelling and blocked germination. In contrast, when conidia were treated with cytochalasin B, an inhibitor of actin polymerization, swelling was not affected and germination was only partially reduced. Butanedione monoxime-treated conidia showed accumulation of cytoplasmic vesicles and did not achieve cell wall reorganization, unlike swollen conidia. Collectively, these results suggest an essential role for this myosin analogue in the deposition of cell wall components during germination of
A. fumigatus
conidia and therefore in host tissue colonization.
Noncompetitive Expansion of Cytotoxic T Lymphocytes Specific for Different Antigens during Bacterial Infection
Authors: Vijh Sujata , Pilip Ingrid M. , Pamer Eric G.
Published: December 2019

Listeria monocytogenesis an intracellular bacterium that elicits complex cytotoxic T-lymphocyte (CTL) responses in infected mice. The responses of CTL populations that differ in antigen specificity range in magnitude from large, dominant responses to small, subdominant responses. To test the hypothesis that dominant T-cell responses inhibit subdominant responses, we eliminated the two dominant epitopes ofL. monocytogenesby anchor residue mutagenesis and measured the T-cell responses to the remaining subdominant epitopes. Surprisingly, the loss of dominant T-cell responses did not enhance subdominant responses. While mice immunized with bacteria lacking dominant epitopes developedL. monocytogenes-specific immunity, their ability to respond to dominant epitopes upon rechallenge with wild-type bacteria was markedly diminished. Recall responses in mice immunized with wild-type or epitope-deficientL. monocytogenesshowed that antigen presentation during recall infection is sufficient for activating memory cells yet insufficient for optimal priming of naive T lymphocytes. Our findings suggest that T-cell priming to different epitopes duringL. monocytogenesinfection is not competitive. Rather, T-cell populations specific for different antigens but the same pathogen expand independently.
Hyperproduction of Alpha-Hemolysin in a
sigB
Mutant Is Associated with Elevated SarA Expression in
Staphylococcus aureus
Authors: Cheung Ambrose L. , Chien Yueh-tyng , Bayer Arnold S.
Published: December 2019

To evaluate the role of SigB in modulating the expression of virulence determinants in
Staphylococcus aureus
, we constructed a
sigB
mutant of RN6390, a prototypic
S. aureus
strain. The mutation in the
sigB
gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate ςB-dependent promoters (e.g., the
sarC
promoter) of
S. aureus
. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of the
sigB
mutation on the enhanced expression of the alpha-hemolysin gene (
hla
) was primarily transcriptional. Upon complementation with a plasmid containing the
sigB
gene,
hla
expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of the
sigB
mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the
agr
locus remained unaltered in the
sigB
mutant. Because of a lack of perturbation in
agr
, we hypothesize that inactivation of
sigB
leads to increased expression of SarA which, in turn, modulates target genes via an
agr
-independent but SarA-dependent pathway.
Role of Iron inNramp1-Mediated Inhibition of Mycobacterial Growth
Authors: Zwilling Bruce S. , Kuhn Donald E. , Wikoff Lisa , Brown David , Lafuse William
Published: December 2019

Innate resistance to mycobacterial growth is mediated by a gene,Nramp1. We have previously reported thatNramp1mRNA from macrophages ofMycobacterium bovisBCG-resistant (Bcgr) mice is more stable thanNramp1mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely relatedNramp2gene is a metal ion transporter, we evaluated the effect of iron on the growth ofMycobacterium aviumwithin macrophages as well as on the stability ofNramp1mRNA. The addition of iron to macrophages fromBcgsmice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages fromBcgrmice to control the growth ofM. avium. When we treated recombinant gamma interferon (IFN-γ)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-γ-activated macrophages fromBcgsmice but that it did not affect the capacity of macrophages fromBcgrmice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages fromBcgrmice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages fromBcgrmice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth.
Molecular and Evolutionary Analysis of
Borrelia burgdorferi
297 Circular Plasmid-Encoded Lipoproteins with OspE- and OspF-Like Leader Peptides
Authors: Akins Darrin R. , Caimano Melissa J. , Yang Xiaofeng , Cerna Felix , Norgard Michael V. , Radolf Justin D.
Published: December 2019

We previously described two OspE and three OspF homologs in
Borrelia burgdorferi
297 (D. R. Akins, S. F. Porcella, T. G. Popova, D. Shevchenko, S. I. Baker, M. Li, M. V. Norgard, and J. D. Radolf, Mol. Microbiol. 18:507–520, 1995; D. R. Akins, K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf, J. Clin. Investig. 101:2240–2250, 1998). In this study, we characterized four additional lipoproteins with OspE/F-like leader peptides (Elps) and demonstrated that all are encoded on plasmids homologous to cp32 and cp18 from the B31 and N40 strains, respectively. Statistical analysis of sequence similarities using the binary comparison algorithm revealed that the nine lipoproteins from strain 297, as well as the OspE, OspF, and Erp proteins from the N40 and B31 strains, fall into three distinct families. Based upon the observation that these lipoproteins all contain highly conserved leader peptides, we now propose that the ancestors of each of the three families arose from gene fusion events which joined a common N terminus to unrelated proteins. Additionally, further sequence analysis of the strain 297 circular plasmids revealed that rearrangements appear to have played an important role in generating sequence diversity among the members of these three families and that recombinational events in the downstream flanking regions appear to have occurred independently of those within the lipoprotein-encoding genes. The association of hypervariable regions with genes which are differentially expressed and/or subject to immunological pressures suggests that the Lyme disease spirochete has exploited recombinatorial processes to foster its parasitic strategy and enhance its immunoevasiveness.
Interleukin-12 Is Capable of Generating an Antigen-Specific Th1-Type Response in the Presence of an Ongoing Infection-Driven Th2-Type Response
Authors: Schopf Lisa R. , Bliss Judy L. , Lavigne Liz M. , Chung Charles L. , Wolf Stanley F. , Sypek Joseph P.
Published: December 2019

Previously we demonstrated that recombinant murine interleukin-12 (rmIL-12) administration can promote a primary Th1 response while suppressing the Th2 response in mice primed with 2,4,6-trinitrophenyl–keyhole limpet hemocyanin (TNP-KLH). The present studies examined the capacity of rmIL-12 to drive a Th1 response to TNP-KLH in the presence of an ongoing Th2-mediated disease. To establish a distinct Th2 response, we used a murine model of leishmaniasis. Susceptible BALB/c mice produce a strong Th2 response when infected withLeishmania majorand develop progressive visceral disease. On day 26 postinfection, when leishmaniasis was well established, groups of mice were immunized with TNP-KLH in the presence or absence of exogenous rmIL-12. Even in the presence of overt infection, TNP-KLH-plus-rmIL-12-immunized mice were still capable of generating KLH-specific gamma interferon (IFN-γ) as well as corresponding TNP-specific immunoglobulin G2a (IgG2a) titers. In addition, the KLH-specific IL-4 was suppressed in infected mice immunized with rmIL-12. However, parasite-specific IL-4 and IgG1 production with a lack of parasite-specific IFN-γ secretion were maintained in all infected groups of mice including those immunized with rmIL-12. These data show that despite the ongoing infection-driven Th2 response, rmIL-12 was capable of generating an antigen-specific Th1 response to an independent immunogen. Moreover, rmIL-12 administered with TNP-KLH late in infection did not alter the parasite-specific cytokine or antibody responses.
Antibody Response to
Cryptococcus neoformans
Proteins in Rodents and Humans
Authors: Chen Lin-Chi , Goldman David L. , Doering Tamara L. , Pirofski Liise-anne , Casadevall Arturo
Published: December 2019

The prevalence and specificity of serum antibodies to
Cryptococcus neoformans
proteins was studied in mice and rats with experimental infection, in individuals with or without a history of potential laboratory exposure to
C. neoformans
, human immunodeficiency virus (HIV)-positive individuals who developed cryptococcosis, in matched samples from HIV-positive individuals who did not develop cryptococcosis, and in HIV-negative individuals. Rodents had little or no serum antibody reactive with
C. neoformans
proteins prior to infection. The intensity and specificity of the rodent antibody response were dependent on the species, the mouse strain, and the viability of the inoculum. All humans had serum antibodies reactive with
C. neoformans
proteins regardless of the potential exposure, the HIV infection status, or the subsequent development of cryptococcosis. Our results indicate (i) a high prevalence of antibodies reactive with
C. neoformans
proteins in the sera of rodents after cryptococcal infection and in humans with or without HIV infection; (ii) qualitative and quantitative differences in the antibody profiles of HIV-positive individuals; and (iii) similarities and differences between humans, mice, and rats with respect to the specificity of the antibodies reactive with
C. neoformans
proteins. The results are consistent with the view that
C. neoformans
infections are common in human populations, and the results have implications for the development of vaccination strategies against cryptococcosis.
Bovine γδ T-Cell Responses to the Intracellular Protozoan ParasiteTheileria parva
Authors: Daubenberger Claudia A. , Taracha Evans L. N. , Gaidulis Laima , Davis William C. , McKeever Declan J.
Published: December 2019

T cells bearing the γδ antigen receptor (γδ T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that γδ T cells are involved in immune responses againstTheileria parva. γδ T cells isolated from peripheral blood mononuclear cells (PBMC) ofT. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologousT. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of γδ T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the γδ T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the γδ T-cell lines and clones. These γδ T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of γδ T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine γδ T cells are activated by and lyseT. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion.