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Infection and Immunity, July 2001, p. 4224-4231, Vol. 69, No. 7
Departments of
Medicine,1 Microbiology and
Immunology,2 and Pathology and
Laboratory Medicine,3 Indiana University,
Indianapolis, Indiana 46202
Received 5 February 2001/Returned for modification 6 March
2001/Accepted 27 March 2001
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- Haemophilus ducreyi
causes chancroid, a genital ulcer disease that facilitates human
immunodeficiency virus transmission (20, 56). Although
rare in the United States (14), chancroid is common in
Africa and Asia, where it accounts for up to 56% of genital ulcer
disease (9, 10, 16, 39, 45, 53). H. ducreyi
readily acquires antimicrobial resistance factors (38) and
remains uniformly susceptible only to macrolides, quinolones, and
broad-spectrum cephalosporins (33). Understanding the
immune response to H. ducreyi infection may facilitate the
development of alternative strategies to control chancroid.
Patients with chancroid do not seek medical attention until they have
painful ulcers, 3 to 6 weeks after initiation of infection (24,
38). Naturally occurring ulcers are characterized by an influx
of polymorphonuclear leukocytes (PMNs) which line the ulcer base and by
a perivascular and interstitial infiltrate of macrophages,
CD45RO+ T cells, and relatively few B cells (1, 27,
28, 32). Patients with ulcers of several weeks duration have
serum antibody and blastogenic responses to H. ducreyi
(15, 55), as well as increased levels of soluble
interleukin-2 (IL-2) receptors in their urine and serum
(2), suggesting the generation of a cell-mediated host
response. Although natural infection may not reliably induce protective
immunity on subsequent exposure, infection is confined to the skin,
mucous membranes, and regional lymph nodes (11, 24, 38,
54).
Our laboratory developed an experimental model of H. ducreyi
infection in human subjects that mimics the initial papular and pustular stages of natural infection (6, 48, 49). In the model, subjects are infected for up to 2 weeks and do not develop serum
antibody or blastogenic responses to H. ducreyi (6,
48) even on rechallenge (5), probably due to the
limited duration of infection. The cutaneous immune response to
experimental infection is similar to that seen in natural infection and
includes two major components: an infiltrate of PMNs that form
epidermal pustules and a dermal infiltrate that consists of
CD45RO+ T cells, macrophages, and some B cells (41,
48). The mononuclear cell infiltrate and the presence of mRNAs
for gamma interferon (IFN- Here we describe the isolation of T-cell lines harvested from lesions
of experimentally infected human volunteers. We characterized their
antigen responsiveness and cytokine production and examined their
cross-reactivity with related bacterial species and their response to
fractionated H. ducreyi whole cells.
Bacterial strains and culture conditions.
Actinobacillus actinomycetemcomitans (ATCC 29522) was kindly
provided by Dominique Galli of the Indiana University School of
Dentistry. Haemophilus parahaemolyticus (ATCC 10014),
Haemophilus paraphrohaemolyticus (ATCC 29237),
Haemophilus parainfluenzae (ATCC 33392), and
Haemophilus parainfluenzae (paraphrophilus) (ATCC
29242) were purchased from the American Type Culture Collection, Rockville, Md. Nontypeable Haemophilus influenzae 1479 and
H. ducreyi 35000 and 35000HP (where HP indicates human
passaged) were described previously (6, 46, 48). Bacterial
strains were maintained on chocolate agar plates or grown in brain
heart infusion broth containing hemin (50 µg/ml), 1% IsoVitaleX, and 5% fetal bovine serum as described previously (25).
Preparation of bacterial antigens.
Bacteria were grown to
mid-log phase, collected by centrifugation at 10,000 × g, and washed three times with sterile saline. Freeze-thawed whole
cells (FTWC) were suspended in 10 mM HEPES as described previously
(23). For some experiments, bacterial pellets were
suspended in 10 mM HEPES and lysed in a French pressure cell. A portion
of the lysate was centrifuged at 100,000 × g for 1 h at 4°C. The soluble fraction (So1) was harvested, and the total membrane pellet was suspended in HEPES buffer. The protein concentration was determined by a protein assay (Bio-Rad Laboratories, Hercules, Calif.) using bovine serum albumin as a standard. Each preparation was adjusted to a final concentration of 1 mg/ml. For some
experiments, the preparations were heat treated at 60°C for 45 min.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.7.4224-4231.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of Haemophilus
ducreyi-Specific T-Cell Lines from Lesions of Experimentally
Infected Human Subjects
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) alone or IFN- 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.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) and tumor necrosis factor alpha
(TNF-
) resembles a delayed-type hypersensitivity (DTH) response
(41, 48), even though the subjects have no history of
chancroid. H. ducreyi and other members of the
Pasteurellaceae family share multiple antigenic determinants on outer membrane proteins, heat shock proteins, and secreted products
(18, 19, 40, 42, 46, 47, 54). Thus, we have hypothesized
that previous colonization with the Pasteurellaceae may have
sensitized subjects so that cross-reactive memory cells are recruited
to the skin on their first exposure to H. ducreyi (41).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Human subjects.
Peripheral blood and biopsy specimens from
pustules were obtained from 24 subjects who participated in a
reinfection trial or mutant-parent comparison trials (Table
1) (3-5, 12, 21, 52, 57,
58) (unpublished observations). All specimens were obtained at
the clinical end point of infection, defined as the development of a
painful pustule or a pustule that was present 14 days after
inoculation, as described previously (3-5, 12, 21, 52, 57,
58). Informed consent was obtained from the subjects for
participation, in accordance with the human experimentation guidelines
of the U.S. Department of Health and Human Services and the
Institutional Review Board of Indiana University
Purdue University at
Indianapolis.
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Isolation of PBMCs and T-cell lines. Peripheral blood mononuclear cells (PBMCs) were enriched from whole blood by Ficoll-Hypaque density gradient centrifugation and diluted in RPMI 1640 supplemented with penicillin-streptomycin, L-glutamine (BioWhittaker, Walkersville, Md.), and 10% heat-inactivated human AB serum as described previously (48).
Mononuclear cells were obtained from biopsy specimens by mincing tissue with a scalpel, extruding it through a 70-µm mesh filter, and subjecting it to density gradient centrifugation as described previously (41). The usual cell yield was 105 to 106 cells per biopsy specimen. T-cell lines or clones were propagated from these mononuclear cells. In initial experiments, the T cells were grown in bulk in the presence of phytohemagglutinin (PHA; 2 µg/ml), 106-irradiated nonautologous PBMCs,
and IL-2 (50 U/ml) (Biotest Diagnostics Corp., Danville, N.J.) and then
cloned by limiting dilution as described by Koelle et al.
(30). Antigen-responsive clones were expanded by a
modification of a method utilizing autologous -irradiated PBMCs,
allogeneic B lymphoblastoid cells (a gift of Peter A. Sieling,
University of California Los Angeles), 30 ng of OKT3 (anti-CD3) per ml,
IL-2 (45 U/ml), and heat-treated Sol (5 µg/ml) (44;
P. A. Sieling, unpublished data). In all subsequent experiments,
T-cell lines were grown in the presence of heat-inactivated Sol or FTWC
(5 µg/ml), -irradiated autologous PBMCs, and IL-2 (50 U/ml) with
repetitive cycles of rest and stimulation as described previously
(17). Cells were cultivated in RPMI 1640 supplemented with
antibiotics, glutamine, and human serum as described above at 37°C in
a humid atmosphere containing 5% CO2.
Proliferation assays.
After being washed in RPMI 1640, 2 × 104 T cells and 105 -irradiated
autologous PBMCs were seeded in wells of a 96-well plate with different
amounts of bacterial preparations in triplicate. As controls for these
assays, T cells and PBMCs were incubated with tetanus toxoid (2 µg/ml) or PHA (2 µg/ml) and -irradiated autologous PBMCs were
incubated with H. ducreyi antigens in the absence of T
cells. The cells were incubated for 96 h, and
[3H]thymidine (0.5 µCi/well; Amersham Pharmacia
Biotech, Piscataway, N.J.) was added for the last 8 h of
incubation. The cells were harvested with a Filtermate Packard cell
harvester (Packard Instrument Co., Inc., Rockville, Md.), and
[3H]thymidine uptake was determined with a Direct Beta
Counter Matrix 9600 (Packard). A stimulation index (SI) of
proliferation was determined as E/C, where C (control) is defined as
the cpm in the absence of antigen or mitogen and E (experimental) is
defined as the cpm in the presence of antigen or mitogen. For each
value, a standard error was calculated as an approximate estimate of the variance for the ratio of two means (29). The
background cpm in the absence of antigen or mitogen was 43.0 ± 41.0 (mean ± standard deviation) for the proliferation assays
reported in Table 2.
Cytokine production.
T cells (106/ml) and
-irradiated autologous PBMCs (4 × 106/ml) were
cultured with or without the H. ducreyi preparations. As a
control, -irradiated autologous PBMCs alone were cultured under the
same conditions. The supernatants were harvested after a 24-h incubation, and cytokine production was determined by
enzyme-linked immunosorbent assay (PharMingen). The limits of
detection were 98 pg/ml for IFN- and 8 pg/ml for IL-4, IL-5, and
IL-10. Control supernatants did not contain any IFN-, but
-irradiated PBMCs from a few volunteers produced IL-10 in response
to H. ducreyi. Thus, IL-10 production is reported as the
difference between IL-10 produced by T cells plus feeder cells and
IL-10 produced by feeder cells alone in the presence of antigen (See
Table 2).
Flow cytometry. To determine the phenotype of the T-cell lines, approximately 105 cells were stained with fluorescent antibodies (BD Biosciences, San Jose, Calif.) as described previously (41). The following sets of antibodies were used: CD3-FITC plus CD16-PE plus CD56-PE, CD3-FITC plus CD19-PE or CD4-FITC plus CD8-PE. As controls, isotype-matched antibodies were used to detect nonspecific staining. Cells were washed, suspended in PBS plus 2% paraformaldehyde, and analyzed using a FACScan flow cytometer (BD Biosciences). A gate was placed around live cells, and percentages are reported as the proportion of gated cells.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation of an H. ducreyi antigen-specific human T-cell clone and lines. Twenty-nine punch biopsy specimens of pustules were obtained from 24 volunteers who participated in a reinfection trial or several mutant-parent comparison trials (Table 1). Samples were obtained from sites inoculated with the parent H. ducreyi strains 35000 or 35000HP or isogenic mutants derived from these strains. All samples were obtained at the clinical end point in the human challenge model, defined as development of a painful pustule or 14 days of infection.
H. ducreyi expresses a heat-labile cytolethal distending toxin that inhibits the growth of primary human T cells and causes apoptosis of Jurkat T cells in vitro (23). Therefore, we first attempted to propagate T cells in the absence of H. ducreyi antigens. Two biopsy specimens were obtained from a subject (H6R), who participated in a reinfection trial (5). One sample was obtained from an infected site, and another was obtained from a control site that was inoculated with heat-killed bacteria and appeared clinically normal. Cells were expanded in bulk in the presence of PHA, IL-2 and-irradiated
nonautologous PBMCs. No T cells were obtained in culture from the
control biopsy specimen, while T cells from the pustule expanded and
were cloned using a limiting-dilution technique (30). From
386 seeded wells, 64 clones were recovered and 1 responded to
heat-treated H. ducreyi Sol (optimal antigen concentration,
1 to 10 µg/ml; SI = 10) but did not respond to tetanus toxoid.
The clone was expanded in the presence of heat-treated antigen. The
expanded cells proliferated in response to heat-treated H. ducreyi Sol (optimal antigen concentration, 1 µg/ml; SI = 59) and FTWC (Table 2).
Fluorescence-activated cell sorter (FACS) analysis showed that the
clone consisted only of CD4+ cells (Fig.
1). By enzyme-linked immunosorbent assay,
the clone produced IFN- (500 pg/ml) and IL-10 (50 pg/ml) in response
to antigen stimulation (Table 2). The data suggested that primary T
cells could proliferate in the presence of heat-treated H. ducreyi antigens and that antigen-responsive T cells were
recruited to sites that were actively infected but were not present in
skin that was not infected.
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-irradiated autologous PBMCs, and IL-2. For two subjects (subjects 148 and 150), cells from parent and mutant inoculated sites were pooled. No lines were obtained from four biopsy
specimens; 21 biopsy specimens yielded T-cell lines. Of these, 12 repeatedly had SIs of >2 or produced cytokines in response to antigen
stimulation during in vitro expansion for a range of 15 days to 6 months (Table 2). These lines were further characterized.
Characterization of antigen-responsive lines.
Of the T cell
lines, 10 proliferated in response to H. ducreyi (Table 2).
In general, the proliferative responses were dose dependent (Fig.
2). The T-cell lines proliferated in
response to PHA and H. ducreyi FTWC but did not respond as
well to tetanus toxoid (Fig. 2 and Table 2), suggesting that the lines
were antigen specific. We also compared the proliferation of the T
cells in the presence of -irradiated allogeneic or autologous PBMCs.
Only autologous PBMCs supported the H. ducreyi-specific proliferation of T cells (data not shown),
suggesting that the proliferative response to H. ducreyi was
likely to be major histocompatibility complex restricted and not a
nonspecific response.
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) or not heat
treated. Results are expressed as mean and standard error of triplicate
wells. Similar results were obtained with clone H6R.
and IL-10 in response to H. ducreyi, while four cell lines (139/P,
155/P, 158/P, and 170/P) produced only IFN-. No IL-4 or IL-5 was
detected in 24-h culture supernatants (data not shown). Thus, some of
the lines had characteristics of the Th1 phenotype, while others may have contained regulatory T cells (Table 2).
Sufficient cell growth was obtained from 10 of the lines for flow
cytometry analysis; all values shown were obtained during the period
when the lines were antigen responsive. The cell lines were 89 to 99%
CD3+ (Table 2). After one to three rounds of antigen
stimulation, some cell lines consisted of both CD4+ and
CD8+ cells while others consisted primarily of
CD4+ cells. Lines that could be maintained for months in
culture (99/M, 105/P, 139/P, 155/P, 158/P, and 170/P) consisted
primarily of CD4+ cells (Table 2 and data not shown). With
the exception of 103/P, which had 9% CD16 and/or CD56+
cells, the cell lines had fewer than 0.5% CD16, CD56+, or
CD19+ cells.
Response to the Pasteurellaceae.
Four lines and
the clone were tested for proliferation in response to antigens
prepared from related bacterial species (Table 3). Two cell lines, 155/P and 158/P, were
strong responders to H. ducreyi (SIs = 109 and 85, respectively) and also responded to a few other species (10 > SI > 2). Other cell lines and the H6R/P clone showed no
cross-reactivity with other members of the Pasteurellaceae,
suggesting that these subjects developed an H. ducreyi-specific memory response during the course of experimental infection.
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Response to crude fractionated antigens.
To determine whether
an immunodominant protein was responsible for the antigen-specific
responses of T cells, we separated H. ducreyi cells by
preparative SDS-PAGE into nine fractions (data not shown). Proteins
were eluted from the fractions and coupled to latex microspheres. The
H6R/P clone proliferated only in response to a >83 kDa fraction (data
not shown). The cell lines from different subjects proliferated in
response to different and occasionally multiple fractions (Fig.
4), including those of >92 kDa (155/P and 158/P), 38 to 47 and 47 to 60 kDa (155/P), and 26 to 32 and 32 to
38 kDa (99/M and 158/P). Line 105/P produced IFN- in response to the
26- to 32-kDa and 32- to 38-kDa fractions (Fig. 4) but did not
proliferate well in response to any fractions (data not shown). These
data indicate that the cultured T cells did not respond to a single
immunodominant antigen and suggest that different subjects were
sensitized to different H. ducreyi antigens during the
course of experimental infection.
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), and
microspheres coupled to H. ducreyi whole cells (+).
Depending on the number of T cells available for an individual
experiment, the results of the proliferation assays were obtained from
single measurements or triplicate measurements for each fraction; error
bars are shown for the latter experiments. The results are
representative of two to four experiments done for each line at
different times. To display the data, the y axis for SI varies in the
figure.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we generated several H. ducreyi-specific T-cell lines and a clone from skin biopsy specimens from infected human volunteers. The T cells did not respond as well to related bacterial species as they did to H. ducreyi, and they responded to a variety of crude antigen fractions prepared from H. ducreyi. Taken together, the data suggest that subjects are specifically sensitized to H. ducreyi antigens and develop adaptive immunity during experimental infection.
The T cells were derived from biopsy specimens of sites infected with live H. ducreyi for 6 to 14 days. In the human model, sites inoculated with heat-killed bacteria do not contain an inflammatory infiltrate (51). PBMCs isolated on the day of infection, at end point, and 21 days after inoculation exhibit low levels of blastogenesis to H. ducreyi that do not vary over time, even in subjects who have been rechallenged (5, 48). In a reinfected subject (H6R), we did not recover T cells from a biopsy specimen of a site challenged with heat-killed control cells using PHA and IL-2 while a biopsy specimen of an infected site yielded an H. ducreyi-responsive clone. The remaining subjects who donated tissue to this study participated in mutant-parent comparison trials, and our protocols for these trials permit biopsies of infected sites only. Thus, although we attempted to grow T cells from noninfected skin in only one subject, it is likely that the antigen-responsive T-cell lines were cutaneous in origin and were not derived from peripheral blood, consistent with data reported for other cutaneous infections (30, 35).
The T-cell lines and clone were composed predominantly of
CD4+ cells that secreted IFN- only or IFN- plus IL-10
in response to H. ducreyi. In murine systems, IL-10
down-regulates IFN- and monokine responses to intracellular
infections and acts to prevent immunopathology (22). Thus,
some lines had characteristics of Th1 cells while others may have
contained regulatory T cells. The T-cell infiltrate in experimental
lesions consists primarily of CD45RO+ cells that are 60 to
80% CD4+, 20 to 40% CD8+, and of the 
lineage (41, 48). mRNAs for the Th1 cytokines IFN- and
IL-2 are detected in all experimental lesions, while mRNAs for the Th2
cytokines IL-4 and IL-5 are present in about half of the lesions; mRNA
for IL-10 was not measured in these studies (41). Some of
the lines initially contained CD8+ cells, but the methods
used to generate the lines are biased to expansion of CD4+
populations (30, 37), and eventually the lines became
predominantly CD4+.
Although the mononuclear cell infiltrate in experimental infection develops within 24 h of inoculation and resembles a DTH response, the volunteers have no prior exposure to H. ducreyi, a requirement for a classic DTH response. We had hypothesized previously that epitopes shared by H. ducreyi and the Pasteurellaceae elicit an infiltrate of cross-reactive memory cells in the initial stages of infection and that a limited number of immunodominant antigens elicit the stereotypic infiltrate of memory cells seen in all subjects who have been infected (41). As reported by others (36), human T-cell lines are difficult to maintain in culture and the number of lines or clones that could be characterized for antigen responsiveness was limited. However, the lack of strong cross-reactivity of the T-cell lines to related bacterial species and the response of the different lines to different antigen fractions suggest that the subjects were sensitized to H. ducreyi during the course of experimental infection.
Based on these data and the immunohistopathology and cytokine analysis
of experimental lesions (41, 48), we propose the following
working model for the recruitment of T cells into the skin by
H. ducreyi. Bacterial components such as lipoproteins and
lipooligosaccharide initiate innate immunity through activation of toll-like receptors on macrophages (13, 31, 34), which secrete TNF-, a potent inducer of E-selectin (ELAM-1) on the endothelium. In concert with other chemokines produced by the endothelial cells, macrophages, and CD1a cells, homing of
memory/effector cells to the skin occurs within 24 h of
inoculation (26, 43, 50). These memory effector cells may
not initially be antigen specific, which may explain why about half the
lines recovered from the biopsy specimens were not H. ducreyi responsive. During the course of infection in some
subjects, T cells are sensitized by macrophages and CD1a cells
presenting H. ducreyi antigens in the draining regional
lymph nodes. These sensitized memory/effector cells also home to the
lesion and expand, and the lesion becomes enriched in
antigen-responsive cells. Thus, the immune response to H. ducreyi has features of both innate and adaptive T-cell immunity.
Recently, we have shown that H. ducreyi colocalizes primarily with PMNs and macrophages but remains extracellular throughout experimental infection (7, 8). We have found no evidence for an intracellular niche for the organism. The antigen-specific CD4+ cells may eventually provide help for the development of antibody responses that usually occur late in the ulcerative stage of disease (15). The possible function of CD8+ cells, which account for 20 to 40% of the T-cell infiltrate in experimental infection, in response to this extracellular pathogen is less clear. Given that subjects who are experimentally infected with H. ducreyi are not protected against rechallenge and that natural infection does not seem to reliably confer protection against subsequent exposure to the organism (5, 11, 24, 38), the DTH-like response may contribute more to pathology than bacterial clearance. Future studies will include a determination of the time course of the development of the antigen-specific response, an investigation of whether CD1-restricted T cells are also recruited to lesions, an investigation of the cytokines and/or chemokines responsible for homing, and a study of how the bacteria are orchestrating the host response.
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ACKNOWLEDGMENTS |
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We thank Carol Schnizlein-Bick, Katy Palmer, Janet Arno, and Cong Ping Xie for their contributions to this work; Peter Sieling, Robert Modlin, and David Koelle for their advice on growing T cells; and Peter Sieling, Margaret Bauer, and Byron Batteiger for reviewing the manuscript. We also thank Jaffar Al-Tawfiq, Royden Young, and Clifton Bong for obtaining the clinical specimens, and we thank the volunteers who participated in the trials.
This work was supported by Public Health Service grants AI27863 and AI31494 from the National Institute of Allergy and Infectious Diseases (NIAID). The clinical samples used in this study were obtained from clinical trials supported by the Sexually Transmitted Diseases Clinical Trials Unit through contract NO1-AI175329 from the NIAID and by Public Health Service grant MO1RR00750 to the GCRC at Indiana University
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FOOTNOTES |
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* Corresponding author. Mailing address: 435 Emerson Hall, Indiana University, 545 Barnhill Dr., Indianapolis, IN 46202-5124. Phone: (317) 274-1427. Fax: (317) 274-1587. E-mail: sspinola{at}iupui.edu.
Editor: J. M. Mansfield
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, July 2001, p. 4224-4231, Vol. 69, No. 7
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.7.4224-4231.2001
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