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1.  Role of Complement in Mycobacterium avium Pathogenesis: In Vivo and In Vitro Analyses of the Host Response to Infection in the Absence of Complement Component C3 
Infection and Immunity  2001;69(12):7729-7735.
We investigated the importance of the host complement system in the pathogenesis of disease mediated by the intramacrophage pathogen Mycobacterium avium. Mycobacteria opsonized with complement are efficiently ingested by macrophages through various complement receptors. Furthermore, unlike other bacteria, mycobacteria can activate both the alternative and classical complement pathways in the absence of specific antibodies. Therefore, to examine the role of complement in the mycobacterial infection process in vivo, mice deficient in complement component C3 were infected with M. avium. Surprisingly, C3-deficient mice infected intravenously with M. avium displayed no difference in bacterial burden or granulomatous response compared to wild-type control mice. C3-sufficient mice and C3-deficient mice were equally susceptible to infection by M. avium regardless of the genotype at the bcg locus, a locus known to confer susceptibility to infection with intracellular pathogens. In vitro studies using mouse bone marrow-derived macrophages resulted in significant M. avium invasion of macrophages in the absence of C3; however, the kinetics of infection were delayed compared to complement-mediated invasion. The data indicate that complement does not play an essential role in mediating M. avium infections in the mouse and suggest either that other invasion mechanisms can compensate for the absence of complement-mediated entry or that complement is not a major mycobacterial opsonin in vivo.
doi:10.1128/IAI.69.12.7729-7735.2001
PMCID: PMC98868  PMID: 11705954
2.  Interleukin-4 Receptor–Stat6 Signaling in Murine Infections with a Tissue-Dwelling Nematode Parasite 
Infection and Immunity  2001;69(12):7743-7752.
Interleukin-4 (IL-4) has been shown to be crucial in parasite expulsion in several gastrointestinal nematode infection models. Data from both epidemiological studies with humans and experimental infections in animals imply a critical role for the type II helper response, dominated by IL-4, in host protection. Here we utilized inbred mice on two distinct backgrounds to document the involvement of IL-4 in the clearance of a primary infection of Brugia from the murine host. Our data from infections of IL-4 receptor−/− and Stat6−/− mice further indicate that IL-4 exerts its effects by activating the Stat6 molecule in host target cells, a finding which links clearance requirements of a gastrointestinal tract-dwelling nematode with those of a tissue-dwelling nematode. Additionally, we show that the requirements for IL-4 receptor binding and Stat6 activation extend to accelerated clearance of a secondary infection as well. The data shown here, including analysis of cell populations at the site of infection and infection of immunoglobulin E (IgE)−/− mice, lead us to suggest that deficiencies in eosinophil recruitment and isotype switching to IgE production may be at least partially responsible for slower parasite clearance in the absence of IL-4.
doi:10.1128/IAI.69.12.7743-7752.2001
PMCID: PMC98870  PMID: 11705956
3.  Role of Gamma Interferon in the Pathogenesis of Severe Schistosomiasis in Interleukin-4-Deficient Mice 
Infection and Immunity  2001;69(12):7445-7452.
In the absence of interleukin-4 (IL-4), infection with Schistosoma mansoni leads to a severe fatal disease rather than the chronic survivable condition that occurs in wild-type (WT) mice. Because the sustained production of NO most closely correlates to weight loss and fatality in infected IL-4−/− mice and because gamma interferon (IFN-γ) is an important inducer of inducible NO synthase, infected IL-4−/− mice were treated with anti-IFN-γ antibodies to determine the role of IFN-γ during schistosomiasis in WT and IL-4−/− animals. When IFN-γ was neutralized, Th2 responses were enhanced and NO production was reduced in both WT and IL-4−/− mice. The decreased NO production correlated with a rescue of proliferation in splenocytes from infected IL-4−/− mice. Furthermore, the neutralization of IFN-γ in vivo improved the gross appearance of the liver and led to a reduction in granuloma size in infected IL-4−/− but not WT mice. However, the neutralization of IFN-γ in vivo did not affect the development of severe disease in infected IL-4−/− mice. These results suggest that while the increased production of IFN-γ does lead to some of the pathology observed in infected IL-4−/− mice, it is not ultimately responsible for cachexia and death.
doi:10.1128/IAI.69.12.7445-7452.2001
PMCID: PMC98833  PMID: 11705919
4.  Dual Role of the Leishmania major Ribosomal Protein S3a Homologue in Regulation of T- and B-Cell Activation 
Infection and Immunity  2001;69(11):6588-6596.
We have recently characterized a novel Leishmania major gene encoding a polypeptide of 30 kDa that was homologous to mammalian ribosomal protein S3a and was named LmS3a-related protein (LmS3arp). The protein was found to be expressed by all the Leishmania species so far examined (L. infantum, L. amazonensis, and L. mexicana). In the present study we have extended our approach to the analysis of LmS3arp activity on T- and B-cell functions in a murine model. The results presented in this report show that LmS3arp plays a dual role in the regulation of T- and B-cell reactivity. Indeed, we found that injection of the LmS3arp recombinant protein (rLmS3arp) into BALB/c mice induces preferential activation of B cells, as shown by the following criteria: (i) increased expression of CD69 molecules on immunoglobulin M (IgM)-secreting spleen cells, (ii) a considerable increase of IgM-secreting B cells, and (iii) elevated levels of IgM antibodies in the sera of injected animals. Moreover, the IgM antibodies are not specific to the Leishmania antigens but preferentially recognize heterologous antigens like myosin, thyroglobulin, DNA, and keyhole limpet hemocyanin. Furthermore, the strong polyclonal expansion of nonspecific, non-parasite-directed B-cell clones induced by rLmS3arp is concomitant with a marked inhibition of T-cell proliferation. Analysis of cytokine production revealed a significant downregulation of gamma interferon, interleukin-2 (IL-2), and IL-12 secretion. Taken together, our data suggest that rLmS3arp, through direct or indirect action toward B and T cells and cytokine secretion, could participate in the immunoregulatory processes that play a role in the balance of the Th1 and Th2 immune response.
doi:10.1128/IAI.69.11.6588-6596.2001
PMCID: PMC100031  PMID: 11598026
5.  Biochemical and Biological Characterization of the Protective Leishmania pifanoi Amastigote Antigen P-8 
Infection and Immunity  2001;69(11):6776-6784.
The Leishmania pifanoi amastigote antigen P-8 has been previously shown to induce protective immunity in a murine model of cutaneous leishmaniasis (L. Soong, S. M. Duboise, P. Kima, and D. McMahon-Pratt, Infect. Immun. 63:3559–3566, 1995). As this antigen is of interest for further vaccine studies, the biochemical characterization of P-8 was undertaken. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot analysis, and gel filtration chromatography revealed that P-8 antigen consisted of two proteoglycolipid complexes. The P-8 epitope is associated with the L. pifanoi amastigote-specific glycolipid components found in the two complexes. The P-8 complex 1 (P-8c1) consists of a 56-kDa serine metalloproteinase, apolipoprotein E (derived from fetal bovine serum), and amastigote-specific glycolipids. The P-8 complex 2 (P-8c2) consists of a 31-kDa cysteine proteinase associated with amastigote glycolipids. Biochemical analyses suggest that the P-8 antigenic glycolipids may be distinct from previously described Leishmania glycolipids (glycosylinositolphospholipids and sphingoglycolipids). Protective immunity studies revealed that P-8c1 (serine metalloproteinase-glycolipid complex) confers comparable protection against infection as immunopurified P-8. The isolated P-8c2 (cysteine proteinase-glycolipid complex) does not provide significant protection, nor does stimulation with P-8c2 result in significant T-cell activation in P-8- or P-8c2-vaccinated mice. Consequently, the P-8c1 complex appears to be the immunodominant component of P-8.
doi:10.1128/IAI.69.11.6776-6784.2001
PMCID: PMC100055  PMID: 11598050
6.  HLA Class II Polymorphism Influences Onset and Severity of Pathology in Schistosoma mansoni-Infected Transgenic Mice 
Infection and Immunity  2001;69(9):5874-5882.
Genetic factors that might influence susceptibility or resistance in naive individuals and early-stage pathology in schistosomiasis are difficult to study in clinical trials, since in areas where the disease is endemic the first contact with the parasite occurs most often at very early ages. Therefore, four strains (DR1.Aβ°, DR2.Aβ°, DQ8.Aβ°, and DQ6.Aβ°) of major histocompatibility complex class II-deficient mice (Aβ°), transgenic for different HLA alleles, have been used to evaluate the potential role of HLA class II polymorphism in the onset of the infection by Schistosoma mansoni. The survival rates and parasitological and immunological parameters after infection were evaluated and compared against the control values obtained with Aβ° mice. All four mouse strains used in this study were able to generate a specific immune response against S. mansoni antigens (cytokine production and antibody production). However, only mice expressing DR alleles survived until the chronic stage of the infection and were able to mount protective granulomatous response avoiding hepatic damage, presenting predominant gamma interferon production. In contrast, strains expressing DQ alleles revealed an impairment in generating effective granulomas, resulting in earlier death, which was associated with an impaired hepatic granulomatous response and liquefactic necrosis, reflecting the influence of HLA polymorphism in the establishment of protective response in the early stage of infection.
doi:10.1128/IAI.69.9.5874-5882.2001
PMCID: PMC98706  PMID: 11500466
7.  Filarial Antigens Impair the Function of Human Dendritic Cells during Differentiation 
Infection and Immunity  2001;69(9):5813-5822.
The antigen-specific T-cell unresponsiveness seen in lymphatic filariasis is mediated, in part, by diminished antigen-presenting cell function and is most specific for microfilariae (MF), the parasite stage found in large numbers in the peripheral circulation. We investigated the effect of MF antigen (MFAg) on dendritic cells (DC) in both their differentiation process from monocyte precursors and also after they have developed into DC. When MFAg was added to cultures of monocytes during their differentiation process to immature DC, the production of interleukin 12 (IL-12) p40, p70 protein, and IL-10 was significantly (P < 0.03) inhibited in response to Staphylococcus aureus Cowan (SAC) and SAC-gamma interferon (IFN-γ) (60% to 80% inhibition). IL-10 was also inhibited (P = 0.04) in response to CD40 ligand–IFN-γ. Moreover, MFAg inhibited the mRNA expression of IL-12 p40 and IL-10 as assessed by RNA protection assays. This effect was antigen specific, as another parasite antigen (soluble Toxoplasma gondii antigen) did not inhibit the production of these cytokines. This effect was also not a result of diminished cell viability nor of an alteration in surface expression of most costimulatory surface molecules, including major histocompatibility complex class I and class II. In contrast to exposure throughout the differentiation process, MFAg added to immature DC had no effect on DC cytokine expression. Although MF-differentiated DC were capable of inducing an allogeneic mixed lymphocyte reaction, they did so to a significantly lesser degree than DC without antigen exposure. These data collectively suggest that once DC are differentiated from their precursor cells, they become resistant to changes by MFAg.
doi:10.1128/IAI.69.9.5813-5822.2001
PMCID: PMC98699  PMID: 11500459
8.  Cardiac Myosin Autoimmunity in Acute Chagas' Heart Disease 
Infection and Immunity  2001;69(9):5643-5649.
Infection with Trypanosoma cruzi, the agent of Chagas' disease, may induce antibodies and T cells reactive with self antigens (autoimmunity). Because autoimmunity is generally thought to develop during the chronic phase of infection, one hypothesis is that autoimmunity develops only after long-term, low-level stimulation of self-reactive cells. However, preliminary reports suggest that autoimmunity may begin during acute T. cruzi infection. The goal of the present study was to investigate whether cardiac autoimmunity could be observed during acute T. cruzi infection. A/J mice infected with the Brazil strain of T. cruzi for 21 days developed severe myocarditis, accompanied by humoral and cellular autoimmunity. Specifically, T. cruzi infection induced immunoglobulin G (IgG) autoantibodies and delayed type hypersensitivity (DTH) to cardiac myosin. This autoimmunity resembles that which develops in A/J mice immunized with myosin in complete Freund's adjuvant in that myosin-specific antibodies and DTH responses both develop by 21 days postinfection or postimmunization. While the levels of myosin IgG in T. cruzi-infected mice were slightly lower than those in myosin-immunized mice, the magnitude of myosin DTH in the two groups was statistically equivalent. In contrast, C57BL/6 mice, which are resistant to myosin-induced myocarditis and its associated autoimmunity, developed undetectable or low levels of myosin IgG and did not exhibit myosin DTH or myocarditis upon T. cruzi infection. Therefore, humoral and cellular cardiac autoimmunity can develop during acute T. cruzi infection in the genetically susceptible host.
doi:10.1128/IAI.69.9.5643-5649.2001
PMCID: PMC98680  PMID: 11500440
9.  Potent Stimulation of the Innate Immune System by a Leishmania brasiliensis Recombinant Protein 
Infection and Immunity  2001;69(9):5270-5277.
The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.
doi:10.1128/IAI.69.9.5270-5277.2001
PMCID: PMC98635  PMID: 11500395
10.  Dendritic Cells Can Initiate Protective Immune Responses against Malaria 
Infection and Immunity  2001;69(8):5173-5176.
An understanding of the antigen presentation mechanisms that mediate induction of protective immune responses against malaria is essential for the development of successful immunization approaches. Here we show that dendritic cells presenting Plasmodium yoelii sporozoite antigens are able to activate specific CD4+ and CD8+ T cells and initiate protective immune responses against malaria in mice.
doi:10.1128/IAI.69.8.5173-5176.2001
PMCID: PMC98615  PMID: 11447201
11.  Interleukin-6 Deficiency Influences Cytokine Expression in Susceptible BALB Mice Infected with Leishmania major but Does Not Alter the Outcome of Disease 
Infection and Immunity  2001;69(8):5189-5192.
Since interleukin-6 (IL-6) may promote Th2 responses, we infected BALB IL-6-deficient (IL-6−/−) mice with Leishmania major. There was not a significant difference between the courses of infection (lesion size and parasite burden) in IL-6−/− and wild-type mice, but IL-6−/− mice expressed lower levels of Th2- and Th1-associated cytokines.
doi:10.1128/IAI.69.8.5189-5192.2001
PMCID: PMC98619  PMID: 11447205
12.  Neutrophil Depletion during Toxoplasma gondii Infection Leads to Impaired Immunity and Lethal Systemic Pathology 
Infection and Immunity  2001;69(8):4898-4905.
The immunomodulatory role of neutrophils during infection with Toxoplasma gondii was investigated. Monoclonal antibody-mediated depletion revealed that neutrophils are essential for survival during the first few days of infection. Moreover, neutrophil depletion was associated with a weaker type 1 immune response as measured by decreased levels of gamma interferon, interleukin-12 (IL-12) and tumor necrosis factor alpha. IL-10 was also decreased in depleted animals. Additionally, splenic populations of CD4+ T cells, CD8+ T cells, and NK1.1+ cells were decreased in depleted mice. Neutrophil-depleted mice exhibited lesions of greater severity in tissues examined and a greater parasite burden as determined by histopathology and reverse transcription-PCR. We conclude that neutrophils are critical near the time of infection because they influence the character of the immune response and control tachyzoite replication.
doi:10.1128/IAI.69.8.4898-4905.2001
PMCID: PMC98580  PMID: 11447166
13.  Tapeworm Infection Reduces Epithelial Ion Transport Abnormalities in Murine Dextran Sulfate Sodium-Induced Colitis 
Infection and Immunity  2001;69(7):4417-4423.
The rat tapeworm Hymenolepis diminuta was used to test the hypothesis that helminth infection could modulate murine colitis. Mice were infected with five H. diminuta cysticercoids, and colitis was evoked via free access to 4% (wt/vol) dextran sulfate sodium (DSS)-containing drinking water for 5 days. BALB/c mice were either infected with H. diminuta and 7 days later exposed to DSS (prophylactic strategy) or started on DSS and infected with H. diminuta 48 h later (treatment strategy). Naive and H. diminuta-only-infected mice served as controls. On autopsy, colonic segments were processed for histological examination and myeloperoxidase (MPO) measurement or mounted in Ussing chambers for assessment of epithelial ion transport. Cytokines (gamma interferon [IFN-γ], interleukin 12 [IL-12], and IL-10) were measured in serum and colonic tissue homogenates. DSS treatment resulted in reduced ion responses (indicated by short-circuit current [Isc]) to electrical nerve stimulation, the cholinergic agonist carbachol, and the adenylate cyclase activator forskolin compared to controls. H. diminuta infection, either prophylactic or therapeutic, caused a significant (P < 0.05) amelioration of these DSS-induced irregularities in stimulated ion transport. In contrast, the histopathology (i.e., mixed immune cell infiltrate, edema, and ulcerative damage) and elevated MPO levels that accompany DSS colitis were unaffected by concomitant H. diminuta infection. Similarly, there were no significant differences in levels of IFN-γ, IL-12, or IL-10 in serum or tissue from any of the treatment groups at the time of autopsy. We suggest that abolishment of colitis-induced epithelial ion transport abnormalities by H. diminuta infection provides proof-of-principle data and speculate that helminth therapy may provide relief of disease symptoms in colitis.
doi:10.1128/IAI.69.7.4417-4423.2001
PMCID: PMC98514  PMID: 11401981
14.  Down-Regulated Lymphoproliferation Coincides with Parasite Maturation and with the Collapse of Both Gamma Interferon and Interleukin-4 Responses in a Bovine Model of Onchocerciasis† 
Infection and Immunity  2001;69(7):4313-4319.
Onchocerciasis is a debilitating parasitic infection caused by the filarial nematode Onchocerca volvulus. Infections are chronic, and persistence of the parasites for several years argues for highly adapted mechanisms of immune evasion. Due to the restricted host repertoire of O. volvulus, we have used the cattle parasite Onchocerca ochengi to investigate the nature of immunomodulation underpinning these long-term infections. Cattle were infected with a single inoculation of 350 infective-stage larvae under laboratory conditions (n = 6). Intradermal nodules containing immature adult worms were detected from 110 days postinfection, and microfilariae in skin were detected from day 280 postinfection. Parasite-specific responses during early infection were nonpolarized with respect to the major Th cytokines (interleukin-4 [IL-4], IL-2, and gamma interferon [IFN-γ]) produced by antigen-stimulated peripheral blood mononuclear cells (PBMC) or serum antibody isotypes. Antigen-induced proliferation of PBMC peaked shortly after exposure and remained high during the prepatent infection. As the parasites matured and animals developed patent infections, there was a profound down-regulation of lymphoproliferation, accompanied by sharp falls in the expression of both IL-4 and IFN-γ and a gradual decline in IL-2. Levels of immunoglobulin G2 (IgG2) fell, while those of IgG1 remained high. We conclude that neither a classical Th2 response nor a simple Th1-to-Th2 switch is sufficient to explain the immunomodulation associated with patent Onchocerca infections. Instead, there is an initial Th0 response, which matures into a response with some, but not all of the features of a Th2 response. The natural host-parasite relationship of O. ochengi in cattle may be useful as both a descriptive and predictive tool to test more refined models of immunomodulation in onchocerciasis.
doi:10.1128/IAI.69.7.4313-4319.2001
PMCID: PMC98501  PMID: 11401968
15.  Cloning and Expression of Two Novel Hemin Binding Protein Genes from Treponema denticola 
Infection and Immunity  2001;69(7):4465-4472.
Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed, T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of the hbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in several T. denticola ATCC strains and clinical isolates, but not in Treponema pectinovorum, Treponema socranskii, or Escherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, called hbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. The hbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.
doi:10.1128/IAI.69.7.4465-4472.2001
PMCID: PMC98520  PMID: 11401987
16.  CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis 
Infection and Immunity  2001;69(7):4320-4328.
The contribution of CD8+ T cells to the control of tuberculosis has been studied primarily during acute infection in mouse models. Memory or recall responses in tuberculosis are less well characterized, particularly with respect to the CD8 T-cell subset. In fact, there are published reports that CD8+ T cells do not participate in the memory immune response to Mycobacterium tuberculosis. We examined the CD8+ T-cell memory and local recall response to M. tuberculosis. To establish a memory immunity model, C57BL/6 mice were infected with M. tuberculosis, followed by treatment with anti-mycobacterial drugs and prolonged rest. The lungs of memory immune mice contained CD4+ and CD8+ T cells with the cell surface phenotype characteristic of memory cells (CD69low CD25low CD44high). At 1 week postchallenge with M. tuberculosis via aerosol, ≥30% of both CD4+ and CD8+ T cells in the lungs of immune mice expressed the activation marker CD69 and could be restimulated to produce gamma interferon (IFN-γ). In contrast, <6% of T cells in the lungs of naive challenged mice were CD69+ at 1 week postchallenge, and IFN-γ production was not observed at this time point. CD8+ T cells from the lungs of both naive and memory mice after challenge were cytotoxic toward M. tuberculosis-infected macrophages. Our data indicate that memory and recall immunity to M. tuberculosis is comprised of both CD4+ and CD8+ T lymphocytes and that there is a rapid response of both subsets in the lungs following challenge.
doi:10.1128/IAI.69.7.4320-4328.2001
PMCID: PMC98502  PMID: 11401969
17.  Characterization of Haemophilus ducreyi-Specific T-Cell Lines from Lesions of Experimentally Infected Human Subjects 
Infection and Immunity  2001;69(7):4224-4231.
Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease that facilitates the transmission of human immunodeficiency virus. In the human model of infection, the histopathology of infected sites in part resembles a delayed-type hypersensitivity (DTH) response. In this study, T cells were isolated from skin biopsy specimens obtained from 24 subjects who were infected for 7 to 14 days. One clone and 12 lines that responded to H. ducreyi antigens were obtained from 12 of the subjects. Fluorescence-activated cell sorter analysis showed that the antigen-responsive lines and clone were predominantly CD3+ and CD4+. The lines and clone responded to H. ducreyi antigen in a dose-dependent manner and produced gamma interferon (IFN-γ) alone or IFN-γ and interleukin-10 (IL-10) but no IL-4 or IL-5 in response to H. ducreyi. Proliferation of T cells was dependent on the presence of autologous antigen-presenting cells. The lines showed little response to antigens prepared from other members of the Pasteurellaceae and responded to different fractions of H. ducreyi separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that T cells that recognize H. ducreyi antigens are recruited to sites experimentally infected with the organism. The lack of cross-reactivity to the Pasteurellaceae and the response of the lines to different antigen fractions suggest that subjects are sensitized to H. ducreyi during the course of infection.
doi:10.1128/IAI.69.7.4224-4231.2001
PMCID: PMC98455  PMID: 11401958
18.  Differential Vβ T-Cell Receptor Usage during Chronic Experimental Schistosomiasis Corresponds with Distinct Pathological Presentations 
Infection and Immunity  2001;69(6):4177-4179.
CBA/J male mice with chronic Schistosoma mansoni infections display either moderate splenomegaly syndrome (MSS) or hypersplenomegaly syndrome (HSS). As MSS and HSS mice differ in several immunologic characteristics, we investigated T-cell receptor Vβ usage. The groups had significantly different expression of several Vβs, suggesting a relationship between the T-cell repertoire and schistosomiasis pathology.
doi:10.1128/IAI.69.6.4177-4179.2001
PMCID: PMC98489  PMID: 11349096
19.  Molecular Basis for Variable Expression of Merozoite Surface Antigen gp45 among American Isolates of Babesia bigemina 
Infection and Immunity  2001;69(6):3782-3790.
Immunization with the merozoite surface glycoprotein gp45 induces protection against challenge using the homologous Babesia bigemina strain. However, gp45 B-cell epitopes are highly polymorphic among B. bigemina strains isolated from different geographical locations within North and South America. The molecular basis for this polymorphism was investigated using the JG-29 biological clone of a Mexico strain of B. bigemina and comparison with the Puerto Rico, St. Croix, and Texcoco strains. The molecular size and antibody reactivity of gp45 expressed by the JG-29 clone were identical to those of the parental Mexico strain. gp45 cDNA and the genomic locus encompassing gp45 were cloned and sequenced from JG-29. The locus sequence and Southern blot data were consistent with a single gp45 copy in the JG-29 genome. The JG-29 cDNA expressed the full-length protein recognized by the gp45-specific monoclonal antibody 14/1.3.2. The genomes of the Puerto Rico and St. Croix strains of B. bigemina were shown to lack a closely related gp45-like gene by PCR using multiple primer sets and by Southern blots using both full-length and region-specific gp45 probes. This genomic difference was confirmed using unpassaged isolates from a 1999 disease outbreak in Puerto Rico. In contrast, the Texcoco strain retains a gp45 gene, encoding an open reading frame identical to that of JG-29. However, the Texcoco gp45 gene is not transcribed. These two mechanisms, lack of a closely related gp45-like gene and failure to transcribe gp45, result in generation of antigenic polymorphism among B. bigemina strains, and the latter mechanism is unique compared to prior mechanisms of antigenic polymorphism identified in babesial parasites.
doi:10.1128/IAI.69.6.3782-3790.2001
PMCID: PMC98392  PMID: 11349043
20.  Antigenic Variation of Anaplasma Marginale: Major Surface Protein 2 Diversity during Cyclic Transmission between Ticks and Cattle 
Infection and Immunity  2001;69(5):3057-3066.
The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginale and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginale infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginale transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each A. marginale population. The major sequence variants did not change on passage of A. marginale between culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.
doi:10.1128/IAI.69.5.3057-3066.2001
PMCID: PMC98260  PMID: 11292724
21.  Exacerbation of Acanthamoeba Keratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies 
Infection and Immunity  2001;69(5):2988-2995.
Neutrophils are thought to be involved in many infectious diseases and have been found in high numbers in the corneas of patients with Acanthamoeba keratitis. Using a Chinese hamster model of keratitis, conjunctival neutrophil migration was manipulated to determine the importance of neutrophils in this disease. Inhibition of neutrophil recruitment was achieved by subconjunctival injection with an antibody against macrophage inflammatory protein 2 (MIP-2), a powerful chemotactic factor for neutrophils which is secreted by the cornea. In other experiments, neutrophils were depleted by intraperitoneal injection of anti-Chinese hamster neutrophil antibody. The inhibition of neutrophils to the cornea resulted in an earlier onset and more severe infection compared to controls. Anti-MIP-2 antibody treatment produced an almost 35% reduction of myeloperoxidase activity in the cornea 6 days postinfection, while levels of endogenous MIP-2 secretion increased significantly. Recruitment of neutrophils into the cornea via intrastromal injections of recombinant MIP-2 generated an initially intense inflammation that resulted in the rapid resolution of the corneal infection. The profound exacerbation of Acanthamoeba keratitis seen when neutrophil migration was inhibited, combined with the rapid clearing of the disease in the presence of increased neutrophils, strongly suggests that neutrophils play an important role in combating Acanthamoeba infections in the cornea.
doi:10.1128/IAI.69.5.2988-2995.2001
PMCID: PMC98252  PMID: 11292716
22.  Saccharomyces boulardii Stimulates Intestinal Immunoglobulin A Immune Response to Clostridium difficile Toxin A in Mice 
Infection and Immunity  2001;69(4):2762-2765.
Saccharomyces boulardii is a nonpathogenic yeast that protects against antibiotic-associated diarrhea and recurrent Clostridium difficile colitis. The administration of C. difficile toxoid A by gavage to S. boulardii-fed BALB/c mice caused a 1.8-fold increase in total small intestinal immunoglobulin A levels (P = 0.003) and a 4.4-fold increase in specific intestinal anti-toxin A levels (P < 0.001). Enhancing host intestinal immune responses may be an important mechanism for S. boulardii-mediated protection against diarrheal illnesses.
doi:10.1128/IAI.69.4.2762-2765.2001
PMCID: PMC98222  PMID: 11254650
23.  Antibody Recognition of Rodent Malaria Parasite Antigens Exposed at the Infected Erythrocyte Surface: Specificity of Immunity Generated in Hyperimmune Mice 
Infection and Immunity  2001;69(4):2535-2541.
In regions where malaria is endemic, inhabitants remain susceptible to repeated reinfection as they develop and maintain clinical immunity. This immunity includes responses to surface-exposed antigens on Plasmodium sp.-infected erythrocytes. Some of these parasite-encoded antigens may be diverse and phenotypically variable, and the ability to respond to this diversity and variability is an important component of acquired immunity. Characterizing the relative specificities of antibody responses during the acquisition of immunity and in hyperimmune individuals is thus an important adjunct to vaccine research. This is logistically difficult to do in the field but is relatively easily carried out in animal models. Infections in inbred mice with rodent malaria parasite Plasmodium chabaudi chabaudi AS represent a good model for Plasmodium falciparum in humans. This model has been used in the present study in a comparative analysis of cross-reactive and specific immune responses in rodent malaria. CBA/Ca mice were rendered hyperimmune to P. chabaudi chabaudi (AS or CB lines) or Plasmodium berghei (KSP-11 line) by repeated infection with homologous parasites. Serum from P. chabaudi chabaudi AS hyperimmune mice reacted with antigens released from disrupted P. chabaudi chabaudi AS-infected erythrocytes, but P. chabaudi chabaudi CB and P. berghei KSP-11 hyperimmune serum also contained cross-reactive antibodies to these antigens. However, antibody activity directed against antigens exposed at the surfaces of intact P. chabaudi chabaudi-infected erythrocytes was mainly parasite species specific and, to a lesser extent, parasite line specific. Importantly, this response included opsonizing antibodies, which bound to infected erythrocytes, leading to their phagocytosis and destruction by macrophages. The results are discussed in the context of the role that antibodies to both variable and invariant antigens may play in protective immunity in the face of continuous susceptibility to reinfection.
doi:10.1128/IAI.69.4.2535-2541.2001
PMCID: PMC98189  PMID: 11254617
24.  Analysis of Chicken Mucosal Immune Response to Eimeria tenella and Eimeria maxima Infection by Quantitative Reverse Transcription-PCR 
Infection and Immunity  2001;69(4):2527-2534.
The recent cloning of chicken genes coding for interleukins, chemokines, and other proteins involved in immune regulation and inflammation allowed us to analyze their expression during infection with Eimeria. The expression levels of different genes in jejunal and cecal RNA extracts isolated from uninfected chickens and chickens infected with Eimeria maxima or E. tenella were measured using a precise quantitative reverse transcription-PCR technique. Seven days after E. tenella infection, expression of the proinflammatory cytokine interleukin-1β (IL-1β) mRNA was increased 80-fold. Among the chemokines analyzed, the CC chemokines K203 (200-fold) and macrophage inflammatory factor 1β (MIP-1β) (80-fold) were strongly upregulated in the infected ceca, but the CXC chemokines IL-8 and K60 were not. However, the CXC chemokines were expressed at very high levels in uninfected cecal extracts. The levels of gamma interferon (IFN-γ) (300-fold), inducible nitric oxide synthase (iNOS) (200-fold), and myelomonocytic growth factor (MGF) (50-fold) were also highly upregulated during infection with E. tenella, whereas cyclooxygenase 2 showed a more modest (13-fold) increase. The genes upregulated during E. tenella infection were generally also upregulated during E. maxima infection but at a lower magnitude except for those encoding MIP-1β and MGF. For these two cytokines, no significant change in expression levels was observed after E. maxima infection. CD3+ intraepithelial lymphocytes may participate in the IFN-γ upregulation observed after infection, since both recruitment and upregulation of the IFN-γ mRNA level were observed in the infected jejunal mucosa. Moreover, in the chicken macrophage cell line HD-11, CC chemokines, MGF, IL-1β, and iNOS were inducible by IFN-γ, suggesting that macrophages may be one of the cell populations involved in the upregulation of these cytokines observed in vivo during infection with Eimeria.
doi:10.1128/IAI.69.4.2527-2534.2001
PMCID: PMC98188  PMID: 11254616
25.  Down-Regulation of Interleukin-8 Secretion from Mycobacterium tuberculosis-Infected Monocytes by Interleukin-4 and -10 but Not by Interleukin-13 
Infection and Immunity  2001;69(4):2470-2476.
Interleukin-8 (IL-8), a CXC chemokine, has a central role in leukocyte recruitment to areas of granuloma formation in tuberculosis. In the present studies, we investigated the effect of the TH2-derived cytokines IL-4, IL-10, and IL-13 on Mycobacterium tuberculosis-induced IL-8 secretion from purified human monocytes. Our results demonstrate that IL-4 and IL-10 have a down-regulatory effect on IL-8 secretion and that this effect is dose dependent. IL-10 has a greater effect than IL-4 on secretion, and autologous IL-10 secreted from M. tuberculosis-infected monocytes also down-regulates IL-8 secretion. The down-regulatory effect is partly a result of reduced IL-8 mRNA accumulation analyzed by reverse transcription-PCR. When combined, 1 μM IL-4 and IL-10 had an additive effect in decreasing IL-8 secretion and transcription; there was no synergy of action. IL-13 did not have any significant effect on IL-8 gene expression or secretion. The inhibitory effect of IL-10 but not of IL-4 is associated with decreased nuclear binding of the key activating transcription factor NF-κB. We show for the first time that M. tuberculosis causes up-regulation of nuclear binding of Oct-1 detected by electromobility gel shift assay. However, neither AP-1 nor Oct-1 nuclear binding was altered by IL-4 or IL-10. In summary, this study demonstrates that type 2 responses have an important role in the regulation of M. tuberculosis-induced IL-8 expression but that the mechanisms by which the different cytokines act are distinct.
doi:10.1128/IAI.69.4.2470-2476.2001
PMCID: PMC98181  PMID: 11254609

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