bluuebunny/crossref_metadata_2025 huggingface dataset:
Borrelia burgdorferiStimulates the Production of Interleukin-10 in Peripheral Blood Mononuclear Cells from Uninfected Humans and Rhesus Monkeys
Authors: Giambartolomei Guillermo H. , Dennis Vida A. , Philipp Mario T.
Published: December 2019

Heat-killedBorrelia burgdorferispirochetes stimulate in vitro production of interleukin-10 (IL-10) at both mRNA and protein levels in peripheral blood mononuclear cells (PBMC) of uninfected rhesus monkeys. A concomitant down-modulation of IL-2 gene transcription was observed. Neither IL-4 nor gamma interferon gene expression was ostensibly affected byB. burgdorferispirochetes. These phenomena were observed regardless of whether the stimulating spirochetes belonged to theB. burgdorferisensu stricto,Borrelia afzelii, orBorrelia gariniigenospecies, the three main species that cause Lyme disease.B. burgdorferialso induced production of IL-10 in uninfected human PBMC, indicating that this effect might play a role in human Lyme disease. Purified lipidated outer surface protein A (OspA), but not its unlipidated form, induced the production of high levels of IL-10 in uninfected human PBMC. Thus, the lipid moiety is essential in the induction of IL-10 in these PBMC.B. burgdorferiM297, a mutant strain that lacks the plasmid that encodes OspA and OspB, also induced IL-10 gene transcription in PBMC, indicating that this phenomenon is not causally linked exclusively to OspA and its lipid moiety. These results demonstrate thatB. burgdorferican stimulate the production of an antiinflammatory, immunosuppressive cytokine in naive cells and suggest that IL-10 may play a role both in avoidance by the spirochete of deleterious immune responses and in limiting the inflammation that the spirochete is able to induce.
Augmentation of Human Macrophage Candidacidal Capacity by Recombinant Human Myeloperoxidase and GranulocyteMacrophage Colony-Stimulating Factor
Authors: Maródi László , Tournay Christophe , Káposzta Rita , Johnston Richard B. , Moguilevsky Nicole
Published: December 2019

Phagocyte myeloperoxidase (MPO) is believed to be particularly important in defense against candida infection. We reported earlier that monocytes, rich in MPO, killedCandida albicansat a significantly higher rate and extent than did monocyte-derived macrophages, known to lack MPO, and thatC. albicansis less resistant to MPO-dependent oxidants than less pathogenicCandidaspecies. We hypothesized, therefore, that the capacity of macrophages to killC. albicansmight be improved in the presence of MPO. In this study, we evaluated the ability of recombinant human MPO (rhMPO) to augment the killing ofC. albicansby resident macrophages and macrophages activated by recombinant human granulocyte-macrophage colony-stimulating factor. Addition of rhMPO (concentration range, 0.8 to 6.4 U/ml) to suspensions of resident and activated macrophages and opsonizedC. albicansresulted in concentration-dependent and significant increases in candida killing. This enhancement was particularly pronounced with activated macrophages, whetherC. albicanswas opsonized or unopsonized and ingested through the macrophage mannose receptor. rhMPO did not affect the killing ofC. albicansby monocytes, nor did it affect phagocytosis of opsonized or unopsonizedC. albicans. These results indicate that exogenous rhMPO can augment the candidacidal capacity of both resident and activated macrophages, with a more profound effect on activated cells. We suggest that rhMPO may be effective in the treatment of invasive candidiasis.
Expanded CD14+CD16+Monocyte Subpopulation in Patients with Acute and Chronic Infections Undergoing Hemodialysis
Authors: Nockher Wolfgang Andreas , Scherberich Jürgen E.
Published: December 2019

Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections. The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcγ receptor type III (CD16). We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients. In healthy controls CD14+CD16+monocytes account for 8% ± 4% of CD14+monocytes, with an absolute number of 29 ± 14 cells/μl. In stable HD patients the CD14+CD16+subpopulation was significantly elevated (14% ± 3%, or 66 ± 28 cells/μl), while the number of CD14++monocytes (monocytes strongly positive for CD14) remained constant. In HD patients suffering from chronic infections a further rise in CD14+CD16+monocytes was observed (128 ± 71 cells/μl;P< 0.01) such that this subpopulation constituted 24% of all blood monocytes. In contrast, numbers of CD14++cells did not change compared to those for stable HD patients, indicating that the CD14+CD16+monocyte subpopulation was selectively expanded. During acute infections the CD14+CD16+cell subpopulation always expanded. A whole-blood assay revealed that CD14+CD16+monocytes exhibited a higher phagocytosis rate forEscherichia colibacteria than CD14++monocytes, underlining their role during host defense. In addition, CD14+CD16+monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC). Thus, CD14+CD16+cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients.
Pasteurella haemolyticaA1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways
Authors: Hsuan S. L. , Kannan M. S. , Jeyaseelan S. , Prakash Y. S. , Sieck G. C. , Maheswaran S. K.
Published: December 2019

Leukotoxin and endotoxin derived fromPasteurella haemolyticaserotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca2+is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+in a concentration-dependent fashion by influx of extracellular Ca2+through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.
Expression of Two Members of the pMGA Gene Family of
Mycoplasma gallisepticum
Oscillates and Is Influenced by pMGA-Specific Antibodies
Authors: Markham Philip F. , Glew Michelle D. , Browning Glenn F. , Whithear Kevin G. , Walker Ian D.
Published: December 2019

Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of
Mycoplasma gallisepticum
, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the expression of pMGA and instead expressed an antigenically unrelated new polypeptide (p82). Upon the removal of antibody, the progeny of these cells regained pMGA expression and produced antigenically sectored colonies. The basis of this switch between pMGA
+
and pMGA
−
states was shown to be transcriptional. The p82 polypeptide, the expression of which resulted from growth of cells in antibodies, was another member of the pMGA gene family and was located just downstream from the pMGA gene normally expressed by the
M. gallisepticum
cells used. Collectively the results of this work suggest that this organism has evolved an unusual means of altering the antigenic composition of its surface in response to antibodies or to other environmental cues.
Isotypes and Opsonophagocytosis of Pneumococcus Type 6B Antibodies Elicited in Infants and Adults by an Experimental Pneumococcus Type 6B-Tetanus Toxoid Vaccine
Authors: Vidarsson Gestur , Sigurdardottir Sigurveig T. , Gudnason Thorolfur , Kjartansson Sveinn , Kristinsson Karl G. , Ingolfsdottir Gunnhildur , Jonsson Steinn , Valdimarsson Helgi , Schiffman Gerald , Schneerson Rachel , Jonsdottir Ingileif
Published: December 2019

Streptococcus pneumoniaeis a major respiratory pathogen of infants, children, and the elderly. Polysaccharide vaccines have been useful in adult populations but do not elicit protective immunity in infants and young children. To enhance their immunogenicity, vaccines of pneumococcal polysaccharides conjugated to proteins are being developed. In this study antibody levels and opsonic activities were compared in sera of infants and adults injected with pneumococcal polysaccharide type 6B (Pn6B) conjugated to tetanus toxoid (TT) (Pn6B-TT). Healthy infants were injected with Pn6B-TT; group A was injected at 3, 4, and 6 months of age, and group B was injected at 7 and 9 months of age. A booster injection was given at 18 months. Adults were injected once. Antibodies were measured by enzyme-linked immunosorbent assay and radioimmunoassay, and their functional activities were measured by opsonophagocytosis of radiolabelled pneumococci. In adults, increases in immunoglobulin M (IgM), IgG, IgA, IgG1, and IgG2 to Pn6B were observed. Infants reached adult levels of IgG1 anti-Pn6B after the primary injections. After the booster injection the infant groups had total IgG- and IgM-Pn6B antibody levels similar to those of adults. After the booster injection, IgG1 was the dominant infant anti-Pn6B isotype and at a level higher than in vaccinated adults, but IgA and IgG2 antibodies remained at very low levels. Opsonic activity increased significantly after Pn6B-TT injections; the highest infant sera showed opsonic activity comparable to that of vaccinated adults. Overall, opsonic activity correlated best with total and IgG anti-Pn6B antibodies (r= 0.741,r= 0.653, respectively;n= 35) and was highest in sera with high levels of all Pn6B antibody isotypes. The results indicate the protective potential of a pneumococcal 6B polysaccharide protein conjugate vaccine for young infants.
Rectal and Intranasal Immunizations with Recombinant Urease Induce Distinct Local and Serum Immune Responses in Mice and Protect against
Helicobacter pylori
Infection
Authors: Kleanthous Harry , Myers Gwendolyn A. , Georgakopoulos Kathleen M. , Tibbitts Timothy J. , Ingrassia Jennifer W. , Gray Heather L. , Ding Ru , Zhang Zhen-Zi , Lei Wende , Nichols Richard , Lee Cynthia K. , Ermak Thomas H. , Monath Thomas P.
Published: December 2019

To determine the optimal inductive sites for immunization against
Helicobacter pylori
infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route. When mice were immunized with rUre (25 μg p.o. or rectally or 10 μg i.n.) plus heat-labile toxin from
Escherichia coli
as the mucosal adjuvant, all routes afforded protection against challenge with
H. pylori
, as indicated by a significant reduction in gastric urease activity (
P
< 0.0005) compared to that of sham-immunized controls. Quantitative
H. pylori
culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (
P
< 0.05). Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o. immunization was greater than after i.n. administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with
H. pylori
. However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40.89 ng/ml), which was detectable prior to challenge with
H. pylori
. Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4
+
T cells showed levels of recruitment to be dependent upon challenge with
H. pylori
and equivalent for all routes. These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.
Haemophilus ducreyiInfection Causes Basal Keratinocyte Cytotoxicity and Elicits a Unique Cytokine Induction Pattern in an In Vitro Human Skin Model
Authors: Hobbs Marcia M. , Paul Terry R. , Wyrick Priscilla B. , Kawula Thomas H.
Published: December 2019

Haemophilus ducreyiis the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Predominantly a cutaneous pathogen,H. ducreyiis present in chancroid ulcers that are characterized by extensive neutrophil accumulation in intraepidermal lesions accompanied by a mononuclear infiltrate in the dermis. We used an in vitro human skin model composed of foreskin fibroblasts and keratinocytes to examine host skin cell interactions withH. ducreyi35000. Bacteria replicated and persisted in artificial skin for at least 14 days. We observedH. ducreyiinside suprabasal keratinocytes using transmission electron microscopy. Although no bacteria were seen in the basal keratinocyte region, these cells were disrupted in infected cocultures.H. ducreyiinfection stimulated increased secretion of interleukin-6 (IL-6) and IL-8 by skin cells. Conversely, tumor necrosis factor alpha and IL-1α levels were not elevated. IL-8 produced in response toH. ducreyiinfection may be involved in recruiting polymorphonuclear leukocytes and other inflammatory cells, thereby contributing to the tissue necrosis and ulcer formation characteristic of chancroid.
Evaluation of New Vaccines in the Mouse and Guinea Pig Model of Tuberculosis
Authors: Baldwin Susan L. , D’Souza Celine , Roberts Alan D. , Kelly Brian P. , Frank Anthony A. , Lui Margaret A. , Ulmer Jeffrey B. , Huygen Kris , McMurray David M. , Orme Ian M.
Published: December 2019

The results of this study provide the first evidence that two completely separate vaccine approaches, one based on a subunit vaccine consisting of a mild adjuvant admixed with purified culture filtrate proteins and enhanced by the cytokine interleukin-2 and the second based on immunization with DNA encoding the Ag85A protein secreted byMycobacterium tuberculosis, could both prevent the onset of caseating disease, which is the hallmark of the guinea pig aerogenic infection model. In both cases, however, the survival of vaccinated guinea pigs was shorter than that conferred byMycobacterium bovisBCG, with observed mortality of these animals probably due to consolidation of lung tissues by lymphocytic granulomas. An additional characteristic of these approaches was that neither induced skin test reactivity to commercial tuberculin. These data thus provide optimism that development of nonliving vaccines which can generate long-lived immunity approaching that conferred by the BCG vaccine is a feasible goal.
LcrG is Required for Efficient Translocation of
Yersinia
Yop Effector Proteins into Eukaryotic Cells
Authors: Sarker Mahfuzur R. , Sory Marie-Paule , Boyd Aoife P. , Iriarte Maite , Cornelis Guy R.
Published: December 2019

Extracellular
Yersinia
disables the immune system of its host by injecting effector Yop proteins into host cells. We show that a
Yersinia enterocolitica
nonpolar
lcrG
mutant is severely impaired in the translocation of YopE, YopH, YopM, YpkA/YopO, and YopP into eukaryotic cells. LcrG is thus required for efficient internalization of all the known Yop effectors.
Entamoeba histolyticaInduces Host Cell Death in Amebic Liver Abscess by a Non-Fas-Dependent, Non-Tumor Necrosis Factor Alpha-Dependent Pathway of Apoptosis
Authors: Seydel Karl B. , Stanley Samuel L.
Published: December 2019

Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and fewEntamoeba histolyticatrophozoites.E. histolyticaproduces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report thatE. histolyticainduces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found thatE. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown thatE. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest thatE. histolyticainduces cell death without using two common pathways for apoptosis.
Extracellular Release of Antigenic Proteins by
Helicobacter pylori
Authors: Cao Ping , McClain Mark S. , Forsyth Mark H. , Cover Timothy L.
Published: December 2019

Screening a
Helicobacter pylori
genomic library with antisera raised against
H. pylori
broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA).
H. pylori
antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation.
Acute and Chronic Phases ofToxoplasma gondiiInfection in Mice Modulate the Host Immune Responses
Authors: Nguyen T. D. , Bigaignon G. , Van Broeck J. , Vercammen M. , Nguyen T. N. , Delmee M. , Turneer M. , Wolf S. F. , Coutelier J. P.
Published: December 2019

Murine antibody responses to soluble proteins are generally restricted to the immunoglobulin G1 (IgG1) isotype. When mice were infected withToxoplasma gondiiBeverley and concomitantly immunized with a soluble unrelated protein antigen, a modification in the isotypic distribution of antibodies directed against this nonparasite antigen was observed, with a preferential production of IgG2a. Interestingly, when mice were immunized with a soluble protein antigen during the chronic phase (day 40) of infection withT. gondiiBeverley, a similar modification in the isotypic distribution of antiprotein antibodies was observed.
Phase 1 Evaluation ofVibrio choleraeO1, Serotype Inaba, Polysaccharide-Cholera Toxin Conjugates in Adult Volunteers
Authors: Gupta Rajesh K. , Taylor David N. , Bryla Dolores A. , Robbins John B. , Szu Shousun C.
Published: December 2019

Conjugate vaccines were prepared by binding hydrazine-treated lipopolysaccharide (DeALPS) fromVibrio choleraeO1, serotype Inaba, to cholera toxin (CT) variants CT-1 and CT-2. Volunteers (n= 75) were injected with either 25 μg of DeALPS, alone or as a conjugate, or the licensed cellular vaccine containing 4 × 109organisms each of serotypes Inaba and Ogawa per ml. No serious adverse reactions were observed. DeALPS alone did not elicit serum LPS or vibriocidal antibodies in mice and only low levels of immunoglobulin M (IgM) anti-LPS in the volunteers. Recipients of the cellular vaccine had the highest IgM anti-LPS levels, but the difference was not statistically significant from that elicited by the conjugates. The conjugates elicited the highest levels of IgG anti-LPS (DeALPS-CT-2 > DeALPS-CT-1 > cellular vaccine). Both conjugates and the cellular vaccine elicited vibriocidal antibodies: after 8 months, recipients of cellular vaccine had the highest geometric mean titer (1,249), followed by DeALPS-CT-2 (588) and DeALPS-CT-1 (330). The correlation coefficient between IgG anti-LPS and 2-mercaptoethanol (2-ME)-resistant vibriocidal antibodies was 0.81 (P= 0.0004). Convalescent sera from cholera patients had a mean vibriocidal titer of 2,525 that was removed by treatment with 2-ME. The vibriocidal activities of sera from all vaccine groups and from the patients were absorbed (>75%) by LPS but not by either CT-1 or CT-2. Conjugate-induced IgG vibriocidal antibodies persisted longer than those elicited by the whole-cell vaccine. Both conjugates, but not the cellular vaccine, elicited IgG anti-CT.
Purification of the
inlB
Gene Product of
Listeria monocytogenes
and Demonstration of Its Biological Activity
Authors: Müller Simone , Hain Torsten , Pashalidis Philippos , Lingnau Andreas , Domann Eugen , Chakraborty Trinad , Wehland Jürgen
Published: December 2019

Entry of
Listeria monocytogenes
into nonphagocytic cells requires the
inlAB
gene products. InlA and InlB are bacterial cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating agents including mutanolysin, thiol reagents, sodium chloride, and detergents like Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate did not release substantial amounts of InlB from the bacterial cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or other protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purification. A panel of monoclonal antibodies against listerial Tris-Cl extracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enabled purification of the InlB protein to homogeneity with relatively high yields. When added externally, purified InlB associated with the surface of noninvasive bacteria such as
Listeria innocua
or an
L. monocytogenes inlB2
mutant, where it promoted entry of these strains into Vero cells >300- and 17-fold, respectively. This effect was even more dramatic for HeLa cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified native, invasion-competent InlB will allow analysis of the molecular basis of InlB-mediated entry into tissue culture cell lines in greater detail.
Cloning and Characterization of an Outer Membrane Protein of
Vibrio vulnificus
Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence
Authors: Litwin Christine M. , Byrne Burke L.
Published: December 2019

Vibrio vulnificus
is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For
V. vulnificus
, the ability to acquire iron from the host has been shown to correlate with virulence.
V. vulnificus
is able to use host iron sources such as hemoglobin and heme. We previously constructed a
fur
mutant of
V. vulnificus
which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the
V. vulnificus fur
mutant had 67% homology with the first 15 amino acids of the mature protein of the
Vibrio cholerae
heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of
V. vulnificus
. DNA sequencing of
hupA
demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of
V. cholerae
HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the
V. vulnificus hupA
gene. Analysis of the promoter region of
V. vulnificus hupA
showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the
V. vulnificus hupA
gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The
hupA
deletion mutant of
V. vulnificus
will be helpful in future studies of the role of heme iron in
V. vulnificus
pathogenesis.
Chronic Active Hepatitis Induced byHelicobacter hepaticusin the A/JCr Mouse Is Associated with a Th1 Cell-Mediated Immune Response
Authors: Whary M. T. , Morgan T. J. , Dangler C. A. , Gaudes K. J. , Taylor N. S. , Fox J. G.
Published: December 2019

Helicobacter hepaticusinfection in A/JCr mice results in chronic active hepatitis characterized by perivascular, periportal, and parenchymal infiltrates of mononuclear and polymorphonuclear cells. This study examined the development of hepatitis and the immune response of A/JCr mice toH. hepaticusinfection. The humoral and cell-mediated T helper immune response was profiled by measuring the postinfection (p.i.) antibody response in serum, feces, and bile and by the production of cytokines and proliferative responses by splenic mononuclear cells toH. hepaticusantigens. Secretory immunoglobulin A (IgA) and systemic IgG2a antibody developed by 4 weeks p.i. and persisted through 12 months. Splenocytes from infected mice proliferated and produced more gamma interferon (IFN-γ) than interleukin-4 (IL-4) or IL-5 when cultured withH. hepaticusouter membrane proteins. The predominantly IgG2a antibody response in serum and the in vitro production of IFN-γ in excess of IL-4 or IL-5 are consistent with a Th1 immune response reported in humans and mice infected withHelicobacter pyloriandHelicobacter felis, respectively. Mice infected withH. hepaticusdeveloped progressively severe perivascular, periportal, and hepatic parenchymal lesions consisting of lymphohistiocytic and plasmacytic cellular infiltrates. In addition, transmural typhlitis was observed at 12 months p.i. The characterization of a cell-mediated Th1 immune response toH. hepaticusinfection in the A/JCr mouse should prove valuable as a model for experimental regimens which manipulate the host response toHelicobacter.
Pet, an Autotransporter Enterotoxin from Enteroaggregative
Escherichia coli
Authors: Eslava Carlos , Navarro-García Fernando , Czeczulin John R. , Henderson Ian R. , Cravioto Alejandro , Nataro James P.
Published: December 2019

Enteroaggregative
Escherichia coli
(EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic
E. coli
and to EspC of enteropathogenic
E. coli
, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects.
Induction of Protective Immune Responses by Immunization with Linear Multiepitope Peptides Based on Conserved Sequences fromPlasmodium falciparumAntigens
Authors: Bharadwaj Ashima , Sharma Pawan , Joshi Sunil K. , Singh Balwan , Chauhan V. S.
Published: December 2019

A cysteine-containing peptide motif, EWSPCSVTCG, is found highly conserved in the circumsporozoite protein (CSP) and the thrombospondin-related anonymous protein (TRAP) of all thePlasmodiumspecies analyzed so far and has been shown to be crucially involved in the sporozoite invasion of hepatocytes. We have recently shown that peptide sequences containing this motif, and also the antibodies raised against the motif, inhibit the merozoite invasion of erythrocytes. However, during natural infection, and upon immunization with recombinant CSP, this motif represents a cryptic epitope. Here we present the results of immunization studies with two linear multiepitopic constructs, a 60-residue (P60) and a 32-residue (P32) peptide, containing the conserved motif sequence. Both the peptides per se generated high levels of specific antibodies in BALB/c mice. P32 was found to be genetically restricted toH-2dandH-2bhaplotypes of mice, whereas P60 was found to be immunogenic in five different strains of mice. The antibody response was predominantly targeted to the otherwise cryptic, conserved motif sequence in P60. Anti-P60 antibodies specifically stained the asexual blood stages ofPlasmodium falciparumandPlasmodium yoeliiin an immunofluorescence assay, recognized a 60- to 65-kDa parasite protein in an immunoblot assay, and blockedP. falciparummerozoite invasion of erythrocytes in a dose-dependent manner. Immunization with P60 also induced significant levels of the cytokines interleukin-2 (IL-2), IL-4, and gamma interferon in BALB/c mice. Moreover, >60% of mice immunized with P60 survived a heterologous challenge infection with a lethal strain ofP. yoelii. These results indicate that appropriate medium-sized synthetic peptides might prove useful in generating specific immune responses to an otherwise cryptic but critical and putatively protective epitope in an antigen and could form part of a multicomponent malaria vaccine.
In Vitro Cellular Toxicity Predicts
Pseudomonas aeruginosa
Virulence in Lung Infections
Authors: Sawa Teiji , Ohara Maria , Kurahashi Kiyoyasu , Twining Sally S. , Frank Dara W. , Doroques David B. , Long Taicy , Gropper Michael A. , Wiener-Kronish Jeanine P.
Published: December 2019

The role of quorum sensing by
Pseudomonas aeruginosa
in producing cytotoxicity has not been fully investigated. Strains of
P. aeruginosa
have been characterized as having an invasive or a cytotoxic phenotype (S. M. J. Fleiszig et al., Infect. Immun. 65:579–586, 1997). We noted that the application of a large inoculum of the invasive strain 6294 caused cytotoxicity of cultured epithelial cells. To investigate this dose-related cytotoxicity, we compared the behavior of 6294 to that of another invasive strain, PAO1, and determined whether the cytotoxicity could be related to quorum sensing. Both invasive strains, 6294 and PAO1, appear to have quorum-sensing systems that were operative when large doses of bacteria were applied to cultured lung epithelial cells or instilled into the lungs of animals. Nonetheless, only 6294 was cytotoxic. Cytotoxicity induced by 6294 correlated with increased elastase production. These experiments suggest that there are multiple mechanisms for the induction of cytotoxicity, pathology, and mortality in vivo. However, in vivo cytotoxicity and mortality, but not pathology, could be predicted by quantitative in vitro cellular damage experiments utilizing a range of bacteria-to-cell ratios. It appears that quorum sensing may inversely correlate with virulence in that strains that produced PAI [
N
-(3-oxododecanoyl) homoserine lactone] also appeared to attract more polymorphonuclear leukocytes in vivo and were possibly eliminated more quickly. In addition, exoproduct production in bacteriological medium in vitro may differ significantly from exoproduct expression from infections in vivo or during cocultivation of bacteria with tissue culture cells.
Bradyzoite Development in
Toxoplasma gondii
and the hsp70 Stress Response
Authors: Weiss Louis M. , Ma Yan Fen , Takvorian Peter M. , Tanowitz Herbert B. , Wittner Murray
Published: December 2019

Toxoplasma gondii
is a well-described ubiquitous Apicomplexan protozoan parasite that is an important opportunistic pathogen. The factors affecting the transition of tachyzoites to the latent bradyzoite stage remain to be defined. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite, and many of the other stressors associated with heat shock protein (hsp) induction. There is evidence for other organisms that hsps are developmentally regulated. Therefore, we examined whether hsp induction is an early event in bradyzoite differentiation. Extracellular and intracellular
T. gondii
cells, after exposure to pH 8.1 or 7.1, were analyzed for the expression of inducible hsp70 by using monoclonal antibody C92F3A-5 (specific to hsp70). Western blotting demonstrated that a 72-kDa protein reactive with C92F3A-5 (hsp70), which we believe is part of the hsp70 family, is induced during bradyzoite development. By immunofluorescence and immunoelectron microscopy, we were able to demonstrate that hsp70 staining colocalized to
T. gondii
expressing bradyzoite-specific antigens and the presence of hsp70 in bradyzoites isolated from mouse brain. Quercetin, a bioflavonoid which inhibits the synthesis of hsp90, hsp70, and hsp27, suppresses the induction of bradyzoite development in vitro. Reverse transcription-PCR with conserved hsp70 primers demonstrated an increase in hsp70 in
T. gondii
on exposure to conditions which induce bradyzoite formation. A
T. gondii
hsp70 was subsequently cloned and sequenced by using this amplified fragment. We believe our evidence suggests that hsps are important in the process of bradyzoite differentiation.
Identification of Three Highly Attenuated
Salmonella typhimurium
Mutants That Are More Immunogenic and Protective in Mice than a Prototypical
aroA
Mutant
Authors: Valentine Peter J. , Devore Brian P. , Heffron Fred
Published: December 2019

A panel of
Salmonella typhimurium
14028s mutants, which were previously shown to be highly attenuated in the BALB/c mouse model of infection, were analyzed for their potential as live
Salmonella
oral-vaccine candidates. A prototypical
aroA
mutant was chosen as a basis of comparison. From the panel of mutants initially chosen for this study, three mutants with comparable levels of attenuation elicited higher
Salmonella
-specific serum immunoglobulin G (IgG) and/or mucosal secretory-IgA antibody titers than the
aroA
vaccine strain. The three mutants, CL288, CL401, and CL554, also elicited a better protective immune response than the
aroA
control strain, after a single oral dose of 1 × 10
9
to 2 × 10
9
bacteria.
Immunogenicity of FourPlasmodium falciparumPreerythrocytic Antigens inAotus lemurinusMonkeys
Authors: Perlaza Blanca Liliana , Arévalo-Herrera Myriam , Brahimi Karima , Quintero Gustavo , Palomino Julio Cesar , Gras-Masse Hélène , Tartar André , Druilhe Pierre , Herrera Sócrates
Published: December 2019

Aotus lemurinusmonkeys were immunized with pools of either lipid-tailed peptides injected in PBS or peptides in Montanide ISA-51, all derived from fourPlasmodium falciparumpre-erythrocytic antigens, namely, LSA1, LSA3, SALSA, and STARP. These formulations were well tolerated. Their immunogenicity was demonstrated by the induction of both B- and T-cell responses to most of the peptides studied (of the 12, 10 induced antibody production, 9 induced T-cell proliferative responses, and all 12 induced gamma interferon secretion). Immune responses proved to be long lasting, since some were still detectable 210 days after immunization. Of particular importance is the fact that B- and T-cell responses elicited in this way by synthetic peptides were specific for native parasite proteins onP. falciparumsporozoites and liver stage parasites.
In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein Is Associated with Enhanced Progression and Hematogenous Dissemination in Experimental
Staphylococcus aureus
Infective Endocarditis
Authors: Dhawan Vinod K. , Bayer Arnold S. , Yeaman Michael R.
Published: December 2019

We examined the influence of thrombin-induced platelet microbicidal protein 1 (tPMP-1) on the progression and hematogenous dissemination of experimental endocarditis caused by isogenic
Staphylococcus aureus
strains differing in tPMP susceptibility (tPMP
s
) or resistance (tPMP
r
) in vitro. Following simultaneous challenge of animals with both strains, significantly higher tPMP
r
bacterial densities were present in vegetations (
P
< 0.0001), kidneys (
P
< 0.0001), and spleens (
P
< 0.0001) compared with those for the tPMP
s
strain. These data indicate that tPMP-1 limits the intravegetation proliferation and hematogenous dissemination of a tPMP
s
strain in experimental endocarditis, while the tPMP
r
phenotype confers a selective advantage associated with the enhanced progression of this infection.
Protection against Ascending Infection of the Genital Tract byChlamydia trachomatisIs Associated with Recruitment of Major Histocompatibility Complex Class II Antigen-Presenting Cells into Uterine Tissue
Authors: Stagg A. J. , Tuffrey M. , Woods C. , Wunderink E. , Knight S. C.
Published: December 2019

A mouse model of ascending infection following intravaginal inoculation with a strain ofChlamydia trachomatisisolated from humans has been used to identify immune mechanisms associated with protection against genital infection. BALB/c and C3H mice differed in their susceptibilities to infection and inflammatory disease. In both mouse strains, ascension of the organism and recruitment of bone marrow-derived mononuclear leukocytes were evident in uterine tissue 1 week postinfection. By 3 weeks the organism had been cleared and inflammation had been resolved in the BALB/c mice, but both persisted in the C3H animals. In athymic nude BALB/c mice both the organism and inflammation persisted, indicating the influence of the hosts’ immune response on the outcome of infection. Both BALB/c and C3H mice had a Th1 response in draining lymph nodes, with predominant production of gamma interferon and tumor necrosis factor alpha, low levels of interleukin-10, and no detectable levels of interleukin-4. However, the composition of the early uterine infiltrate differed in these two mouse strains. Cell surface labeling and analysis of light scatter properties by flow cytometry identified a population of large, CD45+major histocompatibility complex class II mononuclear cells, which were a prominent feature of the infiltrates in BALB/c mice but were present in significantly lower numbers in C3H mice. These cells expressed the costimulatory molecules CD86 and CD40 and stimulated allogeneic T cells, suggesting that these mononuclear cells are a population of antigen-presenting cells and that they may play a role in clearing antigen and protecting against inflammatory disease in BALB/c mice. An additional level of immunological control may thus exist in genital chlamydial infection.
Iron Starvation ofBordetella aviumStimulates Expression of Five Outer Membrane Proteins and Regulates a Gene Involved in Acquiring Iron from Serum
Authors: Connell Terry D. , Dickenson Amy , Martone Andrew J. , Militello Kevin T. , Filiatraut Melanie J. , Hayman Mark L. , Pitula Joseph
Published: December 2019

Iron starvation ofBordetella aviuminduced expression of five outer membrane proteins with apparent molecular masses of 95, 92, 91.5, 84, and 51 kDa. Iron-responsive outer membrane proteins (FeRPs) of similar sizes were detected in six of six strains ofB. avium, suggesting that the five FeRPs are common constituents of the outer membrane of most, if not all, strains ofB. avium. Iron-regulated genes ofB. aviumwere targeted for mutagenesis with the transposon TnphoA. Two mutants with iron-responsive alkaline phosphatase activities were isolated from the transposon library. The transposon insertion did not alter the iron-regulated expression of the five FeRPs in mutant Pho-6. The mutant Pho-20 exhibited a loss in expression of the 95-kDa FeRP and the 84-kDa FeRP. Both Pho-6 and Pho-20 were able to use free iron as a nutrient source. However, Pho-20 was severely compromised in its ability to use iron present in turkey serum. The data indicated that the mutation in Pho-20 affected expression of one or more components of an uptake machinery that is involved in acquisition of iron from organic ferricomplexes.
The Role of Protegrins and Other Elastase-Activated Polypeptides in the Bactericidal Properties of Porcine Inflammatory Fluids
Authors: Shi Jishu , Ganz Tomas
Published: December 2019

The mammalian host response to infection includes the production and secretion of antimicrobial peptides from phagocytes and epithelial cells. Protegrins, a group of broadly microbicidal peptides isolated originally from porcine neutrophil lysates, were found to be stored as inactive proforms in porcine neutrophil granules but could be activated extracellularly by neutrophil elastase. We assessed the biological role of protegrins and other elastase-activated polypeptides in the microbicidal activity of neutrophil secretions and inflammatory fluids. When stimulated with phorbol myristate acetate (PMA), neutrophils generated stable microbicidal activity against bothEscherichia coliandListeria monocytogenesunder normal-salt conditions and in the presence of 0 to 10% serum. The generation of these antimicrobial substances was dependent on neutrophil elastase, since it was inhibited by 1 mMN-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone when it was present during activation, but not when this inhibitor was added afterwards. However, elastase-dependent activation of proprotegrins to protegrins in PMA-stimulated neutrophils was not inhibited by the presence of 1 to 2% serum. Porcine neutrophils also released antibacterial activity during phagocytosis of latex beads, and this too was dependent in large part on elastase-activated polypeptides, including protegrins. Moreover, protegrins were found at bactericidal concentrations in cell-free abscess fluid from naturally infected pigs. Taken together, these studies show that protegrins and other elastase-activated polypeptides are important stable antibacterial factors in porcine neutrophil secretions. The potential host defense role of elastase as an activating enzyme for the precursors of microbicidal peptides must be taken into account when therapeutic inhibitors of neutrophil elastase are evaluated for clinical use as anti-inflammatory agents.
An Epitope Delivery System for Use with Recombinant Mycobacteria
Authors: Hetzel Charlotte , Janssen Riny , Ely Sarah J. , Kristensen Nanna M. , Bunting Karen , Cooper Jonathan B. , Lamb Jonathan R. , Young Douglas B. , Thole Jelle E. R.
Published: December 2019

We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains,Mycobacterium bovisbacille Calmette-Guérin (BCG) andMycobacterium vaccae. The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens. We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed inM. vaccae. The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice. This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles.
Antibody Responses to Capsular Polysaccharide Backbone and O-Acetate Side Groups of
Streptococcus pneumoniae
Type 9V in Humans and Rhesus Macaques
Authors: McNeely Tessie B. , Staub Joan M. , Rusk Cynthia M. , Blum Michael J. , Donnelly John J.
Published: December 2019

Streptococcus pneumoniae
is responsible for high rates of pneumococcal bacteremia, meningitis, pneumonia, and acute otitis media worldwide. Protection from disease is conferred by antibodies specific for the polysaccharide (Ps) capsule of the bacteria. Of the four types of group 9 pneumococci, types 9N and 9V cause the most disease, and both types are included in the polyvalent pneumococcal vaccine. The type 9V capsule consists of repeating pentasaccharide units linearly arranged, with an average of 1 to 2 mol of O-acetate side chains per mol of repeat units, added in a complex pattern in which not all repeat units are alike. α-GlcA residues may be O-acetylated in the 2 (17%) or 3 (25%) position and β-ManNAc residues may be O-acetylated in the 4 (6%) or 6 (55%) position. Under certain conditions, the O-acetate side chains are subject to oxidation, which results in subsequent de-O-acetylation of a significant number of the repeat units. This de-O-acetylation could adversely affect the efficacy of a vaccine containing the 9V Ps. A study was undertaken to compare the relative contributions of O-acetate and Ps backbone epitopes in the immune response to
S. pneumoniae
9V type-specific Ps. In both an infant rhesus monkey model and humans, antibodies against the non-O-acetylated 9V backbone as well as against O-acetylated 9V Ps were detected. Functional (opsonophagocytic) activity was observed in antisera in which the predominant species of antibody recognized de-O-acetylated 9V Ps. We concluded that the O-acetate side groups, while recognized, are not essential to the ability of the 9V Ps to induce functional antibody responses.