bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a
Mycobacterium bovis
BCG Auxotrophic Mutant That Protects Guinea Pigs against
M. bovis
and Hematogenous Spread of
Mycobacterium tuberculosis
without Sensitization to Tuberculin
Authors: Chambers Mark A. , Williams Ann , Gavier-Widén Dolores , Whelan Adam , Hall Graham , Marsh Philip D. , Bloom Barry R. , Jacobs William R. , Hewinson R. Glyn
Published: July 2002

Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from
Mycobacterium bovis
and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with
M. tuberculosis
relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with
M. bovis
and protection of the spleens of guinea pigs infected with
M. tuberculosis
in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.
The Arginine Finger Domain of ExoT Contributes to Actin Cytoskeleton Disruption and Inhibition of Internalization ofPseudomonas aeruginosaby Epithelial Cells and Macrophages
Authors: Garrity-Ryan L. , Kazmierczak B. , Kowal R. , Comolli J. , Hauser A. , Engel J. N.
Published: July 2002

Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence. Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive. Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system. In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells. ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor. ExoT contains a signature arginine finger domain found in GTPase-activating proteins. Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton. Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation. In a mouse model of acute pneumonia, PA103ΔUΔT reached the lungs as efficiently as PA103ΔU but showed reduced colonization of the liver. This finding suggests that the ability to resist internalization may be important for virulence in vivo.
Identification ofCowdria ruminantiumAntigens That Stimulate Proliferation of Lymphocytes from Cattle Immunized by Infection and Treatment or with Inactivated Organisms
Authors: Van Kleef Mirinda , Gunter Nico J. , Macmillan Henriette , Allsopp Basil A. , Shkap Varda , Brown Wendy C.
Published: July 2002

Cowdria ruminantiumis an obligate intracellular pathogen that causes heartwater in ruminants. Several findings suggest that T cells play an important role in protection against the disease. In order to identify which proteins are involved in T-cell immunity,C. ruminantiumproteins were fractionated by continuous-flow electrophoresis and tested for their ability to stimulate lymphocyte proliferation in vitro.C. ruminantium-infected endothelial cell lysates were fractionated at between 11 and 38 kDa and 50 and 168 kDa on 15 and 7% acrylamide gels, respectively. In an attempt to stimulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with fractionated proteins. In a parallel study, four cattle were immunized with inactivatedC. ruminantiumto determine whether their lymphocytes also responded to fractionated proteins. Proliferation assays after immunization by infection and treatment detected noC. ruminantium-specific proliferation in vitro after one vaccination. Proliferation was observed, however, between 1 and 4 weeks after challenge. This was followed by a period of no detectable response, after which the response reappeared. PBMC from animals immunized with inactivated organisms proliferated specifically in response to antigen soon after the first immunization. OnlyC. ruminantiumproteins with low molecular masses of 11, 12, 14 to 17, and 19 to 23 kDa induced proliferative responses by lymphocytes from all six animals. These protein fractions may have potential as vaccine antigens.
Matrix Metalloproteinases Contribute to Brain Damage in Experimental Pneumococcal Meningitis
Authors: Leib Stephen L. , Leppert David , Clements John , Täuber Martin G.
Published: July 2002

The present study was performed to evaluate the role of matrix metalloproteinases (MMP) in the pathogenesis of the inflammatory reaction and the development of neuronal injury in a rat model of bacterial meningitis. mRNA encoding specific MMPs (MMP-3, MMP-7, MMP-8, and MMP-9) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α) were significantly (P< 0.04) upregulated, compared to the β-actin housekeeping gene, in cortical homogenates at 20 h after infection. In parallel, concentrations of MMP-9 and TNF-α in cerebrospinal fluid (CSF) were significantly increased in rats with bacterial meningitis compared to uninfected animals (P= 0.002) and showed a close correlation (r= 0.76;P< 0.001). Treatment with a hydroxamic acid-type MMP inhibitor (GM6001; 65 mg/kg intraperitoneally every 12 h) beginning at the time of infection significantly lowered the MMP-9 (P< 0.02) and TNF-α (P< 0.02) levels in CSF. Histopathology at 25.5 ± 5.7 h after infection showed neuronal injury (median [range], 3.5% [0 to 17.5%] of the cortex), which was significantly (P< 0.01) reduced to 0% (0 to 10.8%) by GM6001. This is the first report to demonstrate that MMPs contribute to the development of neuronal injury in bacterial meningitis and that inhibition of MMPs may be an effective approach to prevent brain damage as a consequence of the disease.
The Cytotoxic T-Lymphocyte Epitope of thePlasmodium falciparumCircumsporozoite Protein Also Modulates the Efficiency of Receptor-Ligand Interaction with Hepatocytes
Authors: Rathore Dharmendar , McCutchan Thomas F.
Published: July 2002

Malaria sporozoites are transmitted from the mosquito salivary gland to host hepatocytes within minutes of an infectious bite. The circumsporozoite protein (CS), which covers the surface ofPlasmodiumsporozoites, functions during these minutes in the targeting of host liver cells. The protein's potentially important role in an antimalaria vaccine has spawned interest in both the host immune responses to the parasite's presence and the actual functional role of the protein in the targeting of host liver cells. Here we show that the region of CS known to elicit a cytotoxic T-lymphocyte (CTL) response to irradiated sporozoites also, somewhat ironically, mediates the receptor-ligand interaction essential to parasite invasion of the host. Hence, the structure of CS represents a balance of potentially counterdirectional forces. Polymorphism in the CTL epitope appears to be a product of this balanced state as opposed to an “arms race” as it is so often portrayed. The conceptual difference between the theories regarding the maintainance of polymorphism in CTL epitopes may have significant implication for vaccine design.
Alkyl Hydroperoxide Reductases C and D Are Major Antigens Constitutively Expressed by
Mycobacterium avium
subsp.
paratuberculosis
Authors: Olsen Ingrid , Reitan Liv J. , Holstad Gudmund , Wiker Harald G.
Published: July 2002

Antigens characteristic for
Mycobacterium avium
subspecies
paratuberculosis
were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent
Mycobacterium avium
subspecies
avium
antiserum. Two antigens were present in
M. avium
subsp.
paratuberculosis
and not detected in
Mycobacterium avium
subsp.
avium
. They were identified as antigens 17 and 20 in a CIE reference system for
M. avium
subsp.
paratuberculosis
antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of
Mycobacterium tuberculosis
. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of
M. avium
subsp.
paratuberculosis
but not with 20 other mycobacterial strains except for a
Mycobacterium gordonae
strain, against which a weak cross-reactive band was produced. Goats experimentally infected with
M. avium
subsp.
paratuberculosis
had strong gamma interferon (IFN-γ) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-γ production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in
M. avium
subsp.
paratuberculosis
without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences.
Allele Substitution of the Streptokinase Gene Reduces the Nephritogenic Capacity of Group A Streptococcal Strain NZ131
Authors: Nordstrand Annika , McShan W. Michael , Ferretti Joseph J. , Holm Stig E. , Norgren Mari
Published: July 2002

To investigate the role of allelic variants of streptokinase in the pathogenesis of acute poststreptococcal glomerulonephritis (APSGN), site-specific integration plasmids were constructed, which contained either the non-nephritis-associated streptokinase gene (skc5) from the group C streptococcal strainStreptococcus equisimilisH46A or the nephritis-associated streptokinase gene (ska1) from the group A streptococcal nephritogenic strain NZ131. The plasmids were introduced by electroporation and homologous recombination into the chromosome of an isogenic derivative of strain NZ131, in which the streptokinase gene had been deleted and which had thereby lost its nephritogenic capacity in a mouse model of APSGN. The introduction of a non-nephritis-associated allelic variant of streptokinase did not rescue the nephritogenic capacity of the strain. The mutant and the wild-type strains produced equivalent amounts of streptokinase. Complementation of theskadeletion derivative with the originalskaallele reconstituted the nephritogenicity of wild-type NZ131. The findings support the hypothesis that the role of streptokinase in the pathogenesis of APSGN is related to the allelic variant of the protein.
Eukaryotic Cell Uptake of Heparin-Coated Microspheres: a Model of Host Cell Invasion by
Chlamydia trachomatis
Authors: Stephens Richard S. , Fawaz Farah S. , Kennedy Kathleen A. , Koshiyama Kelly , Nichols Barbara , van Ooij Christiaan , Engel Joanne N.
Published: July 2002

Using polystyrene microspheres coated with heparin or heparan sulfate, it was shown that coated microspheres specifically bound eukaryotic cells and were endocytosed by nonprofessional phagocytic cells. Coated microspheres displayed properties of binding to eukaryotic cells that were similar to those of chlamydiae, and the microspheres were competitively inhibited by chlamydial organisms. Endocytosis of heparin-coated beads resulted in the tyrosine phosphorylation of a similar set of host proteins as did endocytosis of chlamydiae; however, unlike viable chlamydial organisms, which prevent phagolysosomal fusion, endocytosed beads were trafficked to a lysosomal compartment. These findings suggest that heparin-coated beads and
Chlamydia trachomatis
enter eukaryotic cells by similar pathways.
Cloning of PorcineNRAMP1and Its Induction by Lipopolysaccharide, Tumor Necrosis Factor Alpha, and Interleukin-1β: Role of CD14 and Mitogen-Activated Protein Kinases
Authors: Zhang Guolong , Wu Hua , Ross Christopher R. , Minton J. Ernest , Blecha Frank
Published: July 2002

The gene for natural resistance-associated macrophage protein 1 (NRAMP1) plays a dominant role in controlling the resistance of inbred mice to infection with intracellular bacteria, such asMycobacteria,Salmonella, andLeishmania. NRAMP1 is a membrane protein with a consensus transport motif present in one of the intracellular loops. Although its functions remain unclear, recent clues suggest that NRAMP1 protein plays a potential role in ion transport, which presumably accounts for the ability of this single protein to regulate the intraphagosomal replication of several species of antigenically unrelated intracellular pathogens. Expression ofNRAMP1in mice can be induced by lipopolysaccharide (LPS) or bacterial infection; however, little is known about the mechanisms of induction. Here, we report the cloning of the full-length cDNA for porcineNRAMP1, which had over 85% identity in amino acid sequence to its congeners from humans, mice, cattle, and sheep. As for its mammalian congeners, expression of porcineNRAMP1mRNA was cell and tissue specific and was highest in macrophages. Investigation of the molecular mechanisms by whichNRAMP1is induced showed that LPS-induced expression in macrophages, neutrophils, and peripheral blood mononuclear cells was time and dose dependent and was mediated primarily through CD14. Induction ofNRAMP1required de novo protein synthesis, and mitogen-activated protein kinases (MAPK) were essential. Blockage of either p38 or p42/44 MAPK pathways suppressed the expression ofNRAMP1to basal levels. These findings suggest that bacterial infection and proinflammatory mediators induceNRAMP1expression via activation of MAPK pathways.
Iron Acquisition fromPseudomonas aeruginosaSiderophores by Human Phagocytes: an Additional Mechanism of Host Defense through Iron Sequestration?
Authors: Britigan Bradley E. , Rasmussen George T. , Olakanmi Oyebode , Cox Charles D.
Published: July 2002

Chelation of iron to iron-binding proteins is a strategy of host defense. Some pathogens counter this via the secretion of low-molecular-weight iron-chelating agents (siderophores). Human phagocytes possess a high-capacity mechanism for iron acquisition from low-molecular-weight iron chelates. Efficient acquisition and sequestration of iron bound to bacterial siderophores by host phagocytes could provide a secondary mechanism to limit microbial access to iron. In the present work we report that human neutrophils, macrophages, and myeloid cell lines can acquire iron from the twoPseudomonas aeruginosasiderophores. Analogous to iron acquisition from other low-molecular-weight chelates, iron acquisition from the siderophores is ATP independent, induced by multivalent cationic metals, and unaffected by inhibitors of endocytosis and pinocytosis. In vivo, this process could serve as an additional mechanism of host defense to limit iron availability to invading siderophore-producing microbes.
Polymorphonuclear Neutrophils Are Necessary for the Recruitment of CD8+T Cells in the Liver in a Pregnant Mouse Model ofChlamydophila abortus(Chlamydia psittaciSerotype 1) Infection
Authors: de Oca Roberto Montes , Buendía Antonio J. , Del Río Laura , Sánchez Joaquín , Salinas Jesús , Navarro Jose A.
Published: July 2002

The role of polymorphonuclear neutrophils (PMNs) in the development of the specific immune response againstChlamydophila abortus(Chlamydia psittaciserotype 1) infection was studied in a pregnant mouse model involving treatment with RB6-8C5 monoclonal antibody. PMN depletion significantly affected the immune response in the liver, in which the T-lymphocyte and F4/80+cell populations decreased, particularly the CD8+T-cell population. A Th1-like response, characterized by high levels of gamma interferon without detectable levels of interleukin 4 (IL-4) in serum, was observed in both depleted and nondepleted mice, although an increased production of IL-10 was detected in the depleted group. Our results suggest that PMNs play a very important role in the recruitment of other leukocyte populations to the inflammatory foci but have little influence in the polarization of the immune specific response toward a Th1-like response.
In Situ Analysis of the Evolution of the Primary Immune Response in MurineChlamydia trachomatisGenital Tract Infection
Authors: Morrison Sandra G. , Morrison Richard P.
Published: July 2002

Adaptive immune responses contribute to the resolution ofChlamydia trachomatisgenital tract infection and protect against reinfection, but our understanding of the mechanisms of those protective responses is incomplete. In this study, we analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice vaginally infected withC. trachomatisstrain mouse pneumonitis. The cellular inflammatory response was characterized by an initial elevation in myeloid cells in the vagina (day 3) and uterine horns (day 7), followed by a marked rise in the number of T cells, predominantly CD4+cells. CD8+T cells and CD45R+B cells were also detected but were much less numerous. Perivascular clusters of CD4+T cells, which resembled clusters of T cells seen in delayed-type hypersensitivity responses, were evident by 2 weeks postinfection. Following the resolution of infection, few CD8+T cells and CD45R+B cells remained, whereas numerous CD4+T cells and perivascular clusters of CD4+T cells persisted in genital tract tissues. Interleukin-12 (IL-12)- and tumor necrosis factor alpha (TNF-α)-producing cells were observed in vaginal tissue by day 3 of infection and in uterine tissues by day 7. Cells producing IL-4 or IL-10 were absent from vaginal tissues at day 3 of infection but were present in uterine tissues by day 7 and were consistently more numerous than IL-12- and TNF-α-producing cells. Thus, the evolution of the local inflammatory response was characterized by the accumulation of CD4+T cells into perivascular clusters and the presence of cells secreting both Th1- and Th2-type cytokines. The persistence of CD4+-T-cell clusters long after infection had resolved (day 70) may provide for a readily mobilizable T-cell response by which previously infected animals can quickly respond to and control a secondary infectious challenge.
Immunization with Parasite-Derived Apical Membrane Antigen 1 or Passive Immunization with a Specific Monoclonal Antibody Protects BALB/c Mice against Lethal
Plasmodium yoelii yoelii
YM Blood-Stage Infection
Authors: Narum David L. , Ogun Solabomi A. , Thomas Alan W. , Holder Anthony A.
Published: July 2002

We have purified apical merozoite antigen 1 (AMA-1) from extracts of red blood cells infected with the rodent malaria parasite
Plasmodium yoelii yoelii
YM. When used to immunize mice, the protein induced a strong protective response against a challenge with the parasite. Monoclonal antibodies specific for
P. yoelii yoelii
AMA-1 were prepared, and one was very effective against the parasite on passive immunization. A second protein that appears to be located in the apical rhoptry organelles and associated with AMA-1 was identified.
Interleukin-7 or Interleukin-15 Enhances Survival ofMycobacterium tuberculosis-Infected Mice
Authors: Maeurer Markus J. , Trinder Peter , Hommel Gerhard , Walter Wolfgang , Freitag Kirsten , Atkins Derek , Störkel Stefan
Published: July 2002

Both antigen-presenting cells and immune effector cells are required to effectively eradicate or containMycobacterium tuberculosis-infected cells. A variety of cytokines are involved to ensure productive “cross talk” between macrophages and T lymphocytes. For instance, infection of macrophages with mycobacteria leads to effective interleukin-7 (IL-7) and IL-15 secretion, and both cytokines are able to maintain strong cellular immune responses of α/β and γ/δ T cells. Here we show that either cytokine is able to enhance survival ofM. tuberculosis-infected BALB/c mice significantly compared to application of IL-2, IL-4, or phosphate-buffered saline (as a control). Enhanced survival could be achieved only when IL-7 or IL-15 was delivered as a treatment (i.e., 3 weeks postinfection), not when it was administered at the time of infection. Increased survival ofM. tuberculosis-infected animals was observed following passive transfer of spleen cells harvested fromM. tuberculosis-infected, IL-7- or IL-15-treated animals, but not after transfer of spleen cells obtained from mice which received either cytokine alone. Histological examination revealed that IL-7 and IL-15 failed to significantly impact on the number and composition of granulomas formed or the bacterial load. Our data indicated that administration of IL-7 or IL-15 toM. tuberculosis-treated animals resulted in a qualitatively different cellular immune response in spleen cells as reflected by increased tumor necrosis factor alpha and decreased gamma interferon secretion in response toM. tuberculosis-infected antigen-presenting cells.
Immunization of Mice with Combinations of Pneumococcal Virulence Proteins Elicits Enhanced Protection against Challenge with
Streptococcus pneumoniae
Authors: Ogunniyi A. David , Folland Rebekah L. , Briles David E. , Hollingshead Susan K. , Paton James C.
Published: July 2002

The vaccine potential of a combination of three pneumococcal virulence proteins was evaluated in an active-immunization–intraperitoneal-challenge model in BALB/c mice, using very high challenge doses of
Streptococcus pneumoniae
. The proteins evaluated were a genetic toxoid derivative of pneumolysin (PdB), pneumococcal surface protein A (PspA), and a 37-kDa metal-binding lipoprotein referred to as PsaA. Mice immunized with individual proteins or combinations thereof were challenged with high doses of virulent type 2 or type 4 pneumococci. The median survival times for mice immunized with combinations of proteins, particularly PdB and PspA, were significantly longer than those for mice immunized with any of the antigens alone. A similar effect was seen in a passive protection model. Thus, combinations of pneumococcal proteins may provide the best non-serotype-dependent protection against
S. pneumoniae
.
Priming withChlamydia trachomatisMajor Outer Membrane Protein (MOMP) DNA followed by MOMP ISCOM Boosting Enhances Protection and Is Associated with Increased Immunoglobulin A and Th1 Cellular Immune Responses
Authors: Dong-Ji Zhang , Yang Xi , Shen Caixia , Lu Hong , Murdin Andrew , Brunham Robert C.
Published: July 2002

We previously reported that DNA vaccination was able to elicit cellular immune responses and partial protection againstChlamydia trachomatisinfection. However, DNA immunization alone did not generate immune responses or protection as great as that induced by using live organisms. In this study, we evaluated the immunologic effects of a combinational vaccination approach usingC. trachomatismouse pneumonitis (MoPn) major outer membrane protein (MOMP) DNA priming followed by boosting with immune-stimulating complexes (ISCOM) of MOMP protein (MOMP ISCOM) for protection of BALB/c mice against MoPn lung infection. Substantially better protection to challenge infection was observed in mice given combinational vaccination compared with mice given MOMP ISCOM immunization alone, and the protection approximated that induced by live organisms. Enhanced protection was correlated with stronger delayed-type hypersensitivity, higher levels of gamma interferon production, and increased immunoglobulin A antibody responses in lung homogenates. The results indicate that DNA priming followed by ISCOM protein boosting may be useful in designing a fully protective chlamydial vaccine.
The Abundant Larval Transcript-1 and -2 Genes ofBrugia malayiEncode Stage-Specific Candidate Vaccine Antigens for Filariasis
Authors: Gregory William F. , Atmadja Agnes K. , Allen Judith E. , Maizels Rick M.
Published: July 2002

Lymphatic filariasis is a major tropical disease caused by the mosquito-borne nematodesBrugiaandWuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. Vaccines against filariasis must generate immunity to the infective mosquito-derived third-stage larva (L3) without accentuating immunopathogenic responses to lymphatic-dwelling adult parasites. We have identified two highly expressed genes, designated abundant larval transcript-1 and -2 (alt-1andalt-2), from each of which mRNAs account for >1% of L3 cDNAs. ALT-1 and ALT-2 share 79% amino acid identity across 125 residues, including a putative signal sequence and a prominent acidic tract. Expression ofalt-1andalt-2is initiated midway through development in the mosquito, peaking in the infective larva and declining sharply following entry into the host. Humans exposed toBrugia malayishow a high frequency of immunoglobulin G1 (IgG1) and IgG3 antibodies to ALT-1 and -2, distinguishing them from adult-stage antigens, which are targeted by the IgG4 isotype. Immunization of susceptible rodents (jirds) with ALT-1 elicited a 76% reduction in parasite survival, the highest reported for a single antigen from any filarial parasite. ALT-1 and the closely related ALT-2 are therefore strong candidates for a future vaccine against human filariasis.
Toll-Like Receptor 4-Deficient Mice Have Reduced Bone Destruction following Mixed Anaerobic Infection
Authors: Hou Linda , Sasaki Hajime , Stashenko Philip
Published: July 2002

C3H/HeJ mice have an impaired ability to respond to lipopolysaccharide (LPS) due to a mutation in the gene that encodesToll-like receptor 4 (TLR4). The effect of TLR4 deficiency on host responses to endodontic infections is unknown. In the present study, we compared periapical bone destruction, sepsis, and inflammatory cytokine production in LPS-hyporesponsive C3H/HeJ and wild-type control C3H/HeOuJ mice. The mandibular first molars of both strains were subjected to pulpal exposure and infection with a mixture of four anaerobic pathogens,Prevotella intermedia,Fusobacterium nucleatum,Streptococcus intermedius, andPeptostreptococcus micros. At sacrifice on day 21, TLR4-deficient C3H/HeJ mice had significantly reduced periapical bone destruction compared to wild-type C3H/HeOuJ mice (P< 0.001). The decreased bone destruction in C3H/HeJ correlated with reduced expression of the bone resorptive cytokines interleukin 1α (IL-1α) (P< 0.01) and IL-1β (P< 0.05) as well as the proinflammatory cytokine IL-12 (P< 0.05). No significant differences were seen in the levels of gamma interferon, tumor necrosis factor alpha (TNF-α), or IL-10 between the two strains. The expression of IL-1α, IL-1β, TNF-α, IL-10, and IL-12 were all significantly reduced in vitro in macrophages from both TLR4-deficient C3H/HeJ and C57BL/10ScNCr strains, compared to wild-type controls. Notably, the responses of TLR4-deficient macrophages to both gram-positive and gram-negative bacteria were similarly reduced. Neither C3H/HeJ nor C3H/HeOuJ mice exhibited orofacial abscess development or infection dissemination as determined by splenomegaly or cachexia. We conclude that intact TLR function mediates increased proinflammatory responses and bone destruction in response to mixed anaerobic infections.
Mucosal Defense against Gastrointestinal Nematodes: Responses of Mucosal Mast Cells and Mouse Mast Cell Protease 1 during PrimaryStrongyloides venezuelensisInfection in FcRγ-Knockout Mice
Authors: Onah Denis N. , Uchiyama Fukumi , Nagakui Yuuko , Ono Masao , Takai Toshiyuki , Nawa Yukifumi
Published: July 2002

A possible role for the γ subunit of immunoglobulin Fc receptors (FcR) in mucosal defenses against intestinal nematode parasites was studied using age-matched FcRγ-knockout (FcRγ−/−) and wild-type (FcRγ+/+) C57BL/6 mice. Mice were infected subcutaneously with 3,000 infective larvae ofStrongyloides venezuelensis, and the degree of infection was monitored by daily fecal egg counts and adult worm recovery on days 8 and 13 postinfection. Mucosal mast cell (MMC) responses were assayed by in situ intestinal mast cell counts in stained histological sections of the jejunum and by measuring mouse mast cell protease 1 (MMCP-1) release in serum using sandwich enzyme-linked immunosorbent assay. FcRγ−/−mice had significantly higher egg counts (P< 0.01) and numbers of adult worms (P< 0.05) than FcRγ+/+mice, but mastocytosis and serum MMCP-1 release were comparable. It was concluded that MMCP-1 release may be spontaneous, does not depend on mast cell degranulation via the FcRγ signaling system, and appears to play no role in the expulsion ofS. venezuelensis. The delay in worm expulsion in the FcRγ−/−mice might be related to inability of the MMC to degranulate and release effector molecules other than MMCP-1, since FcRγ deletion abrogates mast cell degranulative responses.
Shigella flexneri
LuxS Quorum-Sensing System Modulates
virB
Expression but Is Not Essential for Virulence
Authors: Day William A. , Maurelli Anthony T.
Published: July 2002

Quorum-sensing systems regulate the expression of virulence factors in a wide variety of plant and animal pathogens, including members of the
Enterobacteriaceae
. Studies of
Shigella
virulence gene expression have demonstrated that maximal expression of genes encoding the type III secretion system and its substrates and maximal activity of this virulence organelle occur at high cell density. In these studies, we demonstrate that the expression of
ipa
,
mxi
, and
spa
invasion operons is maximal in stationary-phase bacteria and that conditioned media derived from stationary-phase cultures enhance the expression of these loci. In contrast, expression of
virB
, a transcription factor essential for the expression of invasion loci, peaks in late log phase; accordingly,
virB
expression is enhanced by a signal(s) present in conditioned media derived from late-log-phase cultures. Autoinducer 2 (AI-2), a quorum signaling molecule active in late log phase, was synthesized by
Shigella
species and enteroinvasive
Escherichia coli
and shown to be responsible for the observed peak of
virB
expression. However, AI-2 does not influence invasion operon expression and is not required for
Shigella
virulence, as mutants deficient in AI-2 synthesis are fully virulent. The implications of these findings with regard to both
virB
and invasion operon expression and the evolution of circuitries governing virulence gene expression are discussed.
Role of Genetic Resistance in Invasive Pneumococcal Infection: Identification and Study of Susceptibility and Resistance in Inbred Mouse Strains
Authors: Gingles Neill A. , Alexander Janet E. , Kadioglu Aras , Andrew Peter W. , Kerr Alison , Mitchell Timothy J. , Hopes Elaine , Denny Paul , Brown Steve , Jones Huw B. , Little Steve , Booth George C. , McPheat William L.
Published: July 2002

From a panel of nine inbred mice strains intranasally infected withStreptococcus pneumoniaetype 2 strain, BALB/c mice were resistant and CBA/Ca and SJL mice were susceptible to infection. Further investigation revealed that BALB/c mice were able to prevent proliferation of pneumococci in the lungs and blood, whereas CBA/Ca mice showed no bacterial clearance. Rapidly increasing numbers of bacteria in the blood was a feature of CBA/Ca but not BALB/c mice. In the lungs, BALB/c mice recruited significantly more neutrophils than CBA/Ca mice at 12 and 24 h postinfection. Inflammatory lesions in BALB/c mice were visible much earlier than in CBA/Ca mice, and there was a greater cellular infiltration into the lung tissue of BALB/c mice at the earlier time points. Our data suggest that resistance or susceptibility to intranasal pneumococci may have an association with recruitment and/or function of neutrophils.
Increase of γδ T Lymphocytes in Murine Lungs Occurs during Recovery from Pulmonary Infection byNocardia asteroides
Authors: Tam Stanley , King Donald P. , Beaman Blaine L.
Published: July 2002

Previous studies have demonstrated that γδ T lymphocytes are important for host resistance to pulmonary infection of the murine lung by log-phase cells ofNocardia asteroides. To study the role of γδ T cells in nocardial interactions in the murine lung, C57BL/6J wild type and C57BL/6J-Tcrd (γδ T-cell knockout mice) were infected intranasally with log-phase cells ofN. asteroidesGUH-2. At 3, 5, and 7 days after infection, the γδ T cells were quantified by multiparameter flow cytometry. At the same time, Gram and hematoxylin-eosin stains of paraffin sections were performed to monitor the host responses. The data showed that γδ T lymphocytes increased significantly within the lungs after intranasal infection, and the peak of this cellular increase occurred at 5 days. Furthermore, at this time, greater than 50% of the CD3 T-cell receptor (TCR)-positive (CD3+) cells were γδ TCR positive. Histological examination clearly showed divergent inflammatory responses in the lungs of wild-type mice compared to γδ T-cell knockout mice. The C57BL/6J-Tcrd mice were less capable of clearing the organism, and the polymorphonuclear leukocyte response lasted longer than in wild-type C57BL/6J mice. These results showed that γδ T cells were actively involved in modulating the innate host responses to murine pulmonary infection byN. asteroides.
Tuberculosis Contacts but Not Patients Have Higher Gamma Interferon Responses to ESAT-6 than Do Community Controls in The Gambia
Authors: Vekemans Johan , Lienhardt Christian , Sillah Jackson S. , Wheeler Jeremy G. , Lahai George P. , Doherty Mark T. , Corrah Tumani , Andersen Peter , McAdam Keith P. W. J. , Marchant Arnaud
Published: July 2002

The
Mycobacterium tuberculosis
antigen ESAT-6 has been proposed for tuberculosis immunodiagnosis. In The Gambia, 30% of community controls produced gamma interferon (IFN-γ) in response to ESAT-6. Increased proportions of responders and intensities of responses were found in household contacts. Responses that were initially low in tuberculosis patients increased after treatment. An ESAT-6 IFN-γ assay will be of limited use in the diagnosis of tuberculosis in countries where tuberculosis is endemic. Its role in contact tracing should be evaluated further.
Pathoadaptive Mutations That Enhance Virulence: Genetic Organization of the
cadA
Regions of
Shigella
spp
Authors: Day William A. , Fernández Reinaldo E. , Maurelli Anthony T.
Published: July 2002

Pathoadaptive mutations improve the fitness of pathogenic species by modification of traits that interfere with factors (virulence and ancestral) required for survival in host tissues. A demonstrated pathoadaptive mutation is the loss of lysine decarboxylase (LDC) expression in
Shigella
species that have evolved from LDC-expressing
Escherichia coli
. Previous studies demonstrated that the product of LDC activity, cadaverine, blocks the action of
Shigella
enterotoxins and that the gene encoding LDC,
cadA
, was abolished by large chromosomal deletions in each
Shigella
species. To better understand the nature and evolution of these pathoadaptive mutations, remnants of the
cad
region were sequenced from the four
Shigella
species. These analyses reveal novel gene arrangements in this region of the pathogens' chromosomes. Insertion sequences, a phage genome, and/or loci from different positions on the ancestral
E. coli
chromosome displaced the
cadA
locus to form distinct genetic linkages that are unique to each
Shigella
species. Hybridization studies, using an
E. coli
K-12 microarray, indicated that the genes displaced to form the novel linkages still remain in the
Shigella
genomes. None of these novel gene arrangements were observed in representatives of all
E. coli
phylogenies. Collectively, these observations indicate that inactivation of the
cadA
antivirulence gene occurred independently in each
Shigella
species. The convergent evolution of these pathoadaptive mutations demonstrates that, following evolution from commensal
E. coli
, strong pressures in host tissues selected
Shigella
clones with increased fitness and virulence through the loss of an ancestral trait (LDC). These observations strongly support the role of pathoadaptive mutation as an important pathway in the evolution of pathogenic organisms.
Synergistic Effect of Muramyldipeptide with Lipopolysaccharide or Lipoteichoic Acid To Induce Inflammatory Cytokines in Human Monocytic Cells in Culture
Authors: Yang Shuhua , Tamai Riyoko , Akashi Sachiko , Takeuchi Osamu , Akira Shizuo , Sugawara Shunji , Takada Haruhiko
Published: July 2002

An analog of 1α,25-dihydroxyvitamin D3, 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components. Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects withSalmonellalipopolysaccharide (LPS) or lipoteichoic acid (LTA) fromStaphylococcus aureus, both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti-CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed with MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed with LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic cells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.
Protection of Mice with a Tuberculosis Subunit Vaccine Based on a Fusion Protein of Antigen 85B and ESAT-6
Authors: Olsen Anja Weinreich , van Pinxteren Laurens A. H. , Okkels Limei Meng , Rasmussen Peter Birk , Andersen Peter
Published: July 2002

In this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dose-dependent immune response was induced to both single components as well as to the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level ofMycobacterium bovisBCG-induced protection over a broad dose range. The vaccine induced efficient immunological memory, which remained stable 30 weeks postvaccination.
Distribution and Kinetics of Lipoprotein-Bound Endotoxin
Authors: Levels J. H. M. , Abraham P. R. , van den Ende A. , van Deventer S. J. H.
Published: July 2002

Lipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent endotoxin responsible for pathophysiological symptoms characteristic of infection. The observation that the majority of LPS is found in association with plasma lipoproteins has prompted the suggestion that sequestering of LPS by lipid particles may form an integral part of a humoral detoxification mechanism. Previous studies on the biological properties of isolated lipoproteins used differential ultracentrifugation to separate the major subclasses. To preserve the integrity of the lipoproteins, we have analyzed the LPS distribution, specificity, binding capacity, and kinetics of binding to lipoproteins in human whole blood or plasma by using high-performance gel permeation chromatography and fluorescent LPS of three different chemotypes. The average distribution of O111:B4, J5, or Re595 LPS in whole blood from 10 human volunteers was 60% (±8%) high-density lipoprotein (HDL), 25% (±7%) low-density lipoprotein, and 12% (±5%) very low density lipoprotein. The saturation capacity of lipoproteins for all three LPS chemotypes was in excess of 200 μg/ml. Kinetic analysis however, revealed a strict chemotype dependence. The binding of Re595 or J5 LPS was essentially complete within 10 min, and subsequent redistribution among the lipoprotein subclasses occurred to attain similar distributions as O111:B4 LPS at 40 min. We conclude that under simulated physiological conditions, the binding of LPS to lipoproteins is highly specific, HDL has the highest binding capacity for LPS, the saturation capacity of lipoproteins for endotoxin far exceeds the LPS concentrations measured in clinical situations, and the kinetics of LPS association with lipoproteins display chemotype-dependent differences.
Molecular Basis for Immunoglobulin M Specificity to Epitopes inCryptococcus neoformansPolysaccharide That Elicit Protective and Nonprotective Antibodies
Authors: Nakouzi Antonio , Valadon Philippe , Nosanchuk Joshua , Green Nancy , Casadevall Arturo
Published: July 2002

The protective efficacy of antibodies (Abs) toCryptococcus neoformansglucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype DC. neoformans,while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (VH) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 VHbinding cavity to the corresponding residues in the 13F1 VHaltered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy againstC. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.
PspC, a Pneumococcal Surface Protein, Binds Human Factor H
Authors: Dave Sandhya , Brooks-Walter Alexis , Pangburn Michael K. , McDaniel Larry S.
Published: July 2002

PspC was found to bind human complement factor H (FH) by Western blot analysis of D39 (
pspC
+
) and an isogenic mutant TRE108 (
pspC
). We confirmed that PspA does not bind FH, while purified PspC binds FH very strongly. The binding of FH to exponentially growing pneumococci varied among different isolates when analyzed by fluorescence activated cell sorting analysis.
Normal Human Fibroblasts Express Pattern Recognition Receptors for Fungal (1→3)-β-d-Glucans
Authors: Kougias Panagiotis , Wei Duo , Rice Peter J. , Ensley Harry E. , Kalbfleisch John , Williams David L. , Browder I. William
Published: July 2002

Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity. Glucans are thought to mediate their effects via interaction with membrane receptors on macrophages, neutrophils, and NK cells. There have been no reports of glucan receptors on nonimmune cells. We investigated the binding of a water-soluble glucan in primary cultures of normal human dermal fibroblasts (NHDF). Membranes from NHDF exhibited saturable binding with an apparent dissociation constant (KD) of 8.9 ± 1.9 μg of protein per ml and a maximum binding of 100 ± 8 resonance units. Competition studies demonstrated the presence of at least two glucan binding sites on NHDF. Glucan phosphate competed for all binding sites, with aKDof 5.6 μM (95% confidence interval [CI], 3.0 to 11 μM), while laminarin competed for 69% ± 6% of binding sites, with aKDof 3.7 μM (95% CI, 1.9 to 7.3 μM). Glucan (1 μg/ml) stimulated fibroblast NF-κB nuclear binding activity and interleukin 6 (IL-6) gene expression in a time-dependent manner. NF-κB was activated at 4, 8, and 12 h, while IL-6 mRNA levels were increased by 48% at 8 h. This is the first report of pattern recognition receptors for glucan on human fibroblasts and the first demonstration of glucan binding sites on cells other than leukocytes. It also provides the first evidence that glucans can directly modulate the functional activity of NHDF. These results provide new insights into the mechanisms by which the host recognizes and responds to fungal (1→3)-β-d-glucans and suggests that the response to glucans may not be confined to cells of the immune system.