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*
Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom;
Department of Cell Biology and Development, University "La Sapienza", Rome, Italy;
Royal Brompton Hospital, London, United Kingdom;
Department of Medicine, University of Wales College of Medicine, Cardiff, United Kingdom; and
¶ Division of Respiratory Diseases of the University of "Tor Vergata" at the L. Spallanzani Hospital, Institute for Research and Clinical Care, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3% of individuals
exposed to this element. Immunogenetic studies have demonstrated a
strong association between CBD and possession of alleles of HLA-DP
containing glutamic acid (Glu) at position 69 in the HLA-DP
-chain. T
cell clones were raised from three patients with CBD in whom exposure
occurred 10 and 30 years previously. Of 25 Be-specific clones that were
obtained, all were restricted by HLA-DP alleles with Glu at DP69.
Furthermore, the proliferative responses of the clones were absolutely
dependent upon DP Glu69 in that a single amino acid
substitution at this position abolished the response. As befits a
disease whose pathogenesis involves a delayed type hypersensitivity
response, the large majority of Be-specific clones secreted IFN-
(Th1) and little or no IL-4 (Th2) cytokines. This study provides
insights into the molecular basis of DP2-associated susceptibility to
CBD. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 1970 Hafin et al. showed that mononuclear cells from CBD patients react against Be in vitro (7). In 1990 we demonstrated the involvement of CD4+ T cells in CBD by showing an accumulation of Be-specific CD4+ T cells in the lung of CBD patients (8, 9). The specificity of these CD4+ T cells was further confirmed in vitro by showing their reactivity to Be and the lack of responses to other metals (8). Similar results were obtained in different contexts with nickel (Ni) and gold (Au) by showing T cell reactivity to these metals from PBMC of patients with contact sensitivity reactions (10, 11).
It has long been known that allelic polymorphism in MHC genes
determines immune response variation to individual Ags including
autoantigens (12). Recently, we have investigated the
involvement of MHC class II genes in the susceptibility to Be disease.
Analysis of MHC class II genes in patients with CBD has shown a
positive association with HLA-DPB1*0201 and a negative
association with HLA-DPB1*0401 (13).
Furthermore, sequence comparison of DP-chains suggested that
susceptibility to CBD was conferred by expression of DP alleles with
glutamic acid (Glu) at position 69. A subsequent population study
demonstrated that the carriage of Glu69 was
associated with an almost 10-fold increase of disease risk in exposed
individuals (14). In this regard, HLA-DPB1*0201
is the most common allele with Glu at this position; however, 15 of 32
HLA-DP alleles carry this polymorphism. Interestingly, we have recently
shown an identical HLA-DP association in patients with hard metal
disease. All patients in that study possessed a DP-chain with Glu at
position 69 as compared with 17 of 35 exposed unaffected individuals
(15).
In this study, we have investigated the functional basis of HLA-DP
susceptibility in Be disease by generating T cell clones from patients
with CBD. We found that all the Be-specific T cell clones obtained were
restricted by HLA-DP alleles with Glu at DP69, and that a single
amino acid substitution at residue 69 in the -chain of the HLA-DP
molecule completely abrogated their responses. These data elucidate the
molecular basis of DP-associated susceptibility to CBD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three male patients named, respectively, TDC, FC, and NG, with
CBD were identified through the University of Wales College of Medicine
and the Royal Brompton Hospital (London, U.K.). The HLA-DR, -DQ, and
-DP typing for the three patients is indicated in Table I
.
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The beryllium sulfate tetrahydrate (BeSO4·4H2O), cobalt chloride hexahydrate (CoCl2·6H2O), and nickel (II) sulfate 7-hydrate (NiSO4·7H2O) were purchased from Sigma-Aldrich (Dorset, U.K.).
Monoclonal Abs
The B7/21 (anti-HLA-DP; American Type Culture Collection (ATCC), Manassas, VA), L2 (anti-HLA-DQ, ATCC), and L243 (anti-HLA-DR, ATCC) mAbs were used after purification on protein A-Sepharose beads by standard methods. Eluted Ab was dialyzed against three changes of PBS.
Cell lines
EBV-transformed B lymphoblastoid cell lines (B-LCLs) were obtained from the Xth International Histocompatibility Workshop (Xth IHW). HLA-DP2+ B-LCLs (9045: DRB1*1104/1201, DPA1*01, DPB1*02012/0402; 9038: DRB1*1201, DPA1*01, DPB1*02012; 9039: DRB1*1102, DPA1*01, DPB1*02012; 9036: DRB1*1101, DPA1*01, DPB1*02012) and HLA-DP2- B-LCLs (9037: DRB1*1101, DPA1*-, DPB1*0402; 9043: DRB1*1101, DPA1*0201, DPB1*1001; 9001: DRB1*0101, DPA1*-, DPB1*0402; 9013: DRB1*1501, DPA1*01, DPB1*0402) were used for this study. B-LCLs were cultured in RPMI 1640 tissue culture medium (Life Technologies, Paisley, U.K.) supplemented with 10% FCS, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin in 25-cm2 flasks, and were regularly passaged.
Murine DAP.3 cell transfectants expressing either HLA-DPB1*0201 (workshop number 8301) or HLA-DPB1*0402 (workshop number 8305) were obtained from the Xth IHW. They were maintained in DMEM supplemented with 10% FCS, 0.2% sodium bicarbonate, 2 µM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and MXH (6 µg/ml mycophenolic acid, 250 µg/ml xanthine, and 15 µg/ml hypoxanthine) to maintain expression of the transfected genes. Cells were grown in 25-cm2 flasks and passaged, following trypsinization, twice weekly.
Generation and maintenance of Be-specific T cell clones
PBMC were isolated from heparinized whole blood from the three patients by density centrifugation on a Lymphoprep gradient (Nycomed, Birmingham, U.K.). PBMC were cultured with BeSO4·4H2O in 24-well plates (Costar, High Wycombe, U.K.). After 6 days, the cultures were enriched for lymphoblasts by centrifugation on Ficoll-Hypaque and cultured for two more weeks in the presence of Be-pulsed autologous irradiated PBMC and 20 U/ml rIL-2 (Boehringer Mannheim, East Sussex, U.K.). The T cells were then cloned by limiting dilution at 0.3–1 cell/well in Terasaki trays (Greiner, Gloucester shine, U.K.) in the presence of PHA (2 µg/ml) and 104 allogeneic irradiated PBMC and rIL-2. After 10 days, cell growth was detected microscopically, and the contents of wells with growing cells were expanded further in medium with PHA, allogeneic PBMC, and rIL-2. The clones were maintained in culture by weekly stimulation with PHA, allogeneic PBMC, and rIL-2, in RPMI 1640 medium supplemented with 10% human serum, 2 µM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. T cell clones were used for functional assays between 1 and 2 wk after their last stimulation.
Proliferation assay
PBMC (105 cells/well) were cultured with different doses of Be. T cell clones (104 cells/well) were cultured with either B-LCL (3 x 104 cells/well) or DP-expressing murine DAP.3 transfectants (3 x 104 cells/well) pulsed with BeSO4·4H2O for 4 h and then treated with 120 Gy X-irradiation or with mitomycin C (50 µg/ml), respectively. The cells were plated out in flat-bottom microtiter plates, in a total volume of 200 µl, in RPMI 1640 medium supplemented with 10% human serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. Wells were pulsed with 1 µCi of [3H]TdR (Amersham International, Amersham, U.K.), after either 5 days (PBMC) or 48 h (T cell clones), and the cultures were harvested onto glass fiber filters 18 h later. Proliferation was measured as [3H]TdR incorporation by liquid scintillation spectroscopy. The results are expressed as the mean of triplicate cultures.
Annexin V staining
The B-LCL 9036 was pulsed with BeSO4.4H2O for 4 h and irradiated. The B-LCL were then incubated either in the absence or in the presence of anti-HLA-DP mAb (B7/21) or anti-HLA-DR mAb (L243) at a concentration of 10 µg/ml. The cells were then stained at various time points (45 min, and 1.5 and 3 h) with annexin V-FITC and the vital dye propidium iodide (PI) to assess apoptosis. The cells were then analyzed by flow cytometry on a Becton Dickinson (Mountain View, CA) FACSCalibur.
Lymphokine production
Supernatants were collected after 24 h of T cell culture.
Th1 and Th2 cytokines were measured using a standard ELISA. IFN-
primary and secondary Abs were purchased from AMS Biotechnology (Oxon,
U.K.), and IL-4 Abs were obtained from BioSource (Hertfordshine, U.K.).
Briefly, primary Abs were coated overnight onto 96-well plates. The
plate was then washed, and 1% BSA (Sigma-Aldrich) was used as the
blocking reagent for 2 h. After washing, samples for testing were
added to the wells along with biotinylated secondary Ab. After 2
h, wells were washed and streptavidin peroxidase conjugate (BioSource)
was added for 45 min. Wells were washed thoroughly, and
tetramethylbenzidine substrate was added. This led to color
development, which was stopped using
H2SO4. The plates were then
read at 540 nm.
RNA extraction and cDNA synthesis
Cells (5 x 105) were placed in a sterile Eppendorf tube for RNA extraction. RNA was extracted using the method of Chomczynski and Sacchi (16). To each tube, 1 ml of cold RNAzol was added and the tube was vortexed. After a 5-min incubation on ice, 100 µl of chloroform was added, and the samples were centrifuged for 15 min at 4°C. The upper phase was then transferred to a clean tube containing an equal volume of isopropanol and 10 µg of transfer RNA as a carrier. After a 15-min incubation on ice, the sample was again centrifuged at 4°C for 15 min, the supernatant was removed, and the RNA pellet was washed with 1 ml of cold ethanol. The air-dried pellet was resuspended in 10 µl of autoclaved distilled water and stored at -70°C. cDNA was synthesized using oligo-dT-primed, Moloney murine leukemia virus-derived reverse transcriptase (Life Technologies) and 5 µl of RNA sample in 20-µl reactions. cDNA reactions were diluted to 100 µl and stored at -70°C; 2 µl of diluted cDNA was used for each PCR.
PCR amplification and sequencing
For analysis of TCR V-gene usage cDNAs were amplified
using a panel of primers specific for TCR V
or V families in
combination with a C or C primer. TCR primer sequences were as in
Reference (17). Samples were amplified by PCR for 30
cycles (30 s at 94°C, 30 s at 64°C, and 30 s at 72°C)
for both TCR V and TCR V primers. The TCR C reverse primer
sequence was: 5'-GGCAGACAGGACCCCTTGCTGGTAGGACAC-3'. The TCR C
reverse primer sequence was: 5'-ACTTTGTGACACATTTGTTTGAG-3'.
The amplified fragments were separated by agarose gel
electrophoresis and stained with ethidium bromide. The specific bands
were cut, and the amplified products were purified using a gel band
purification kit (Pharmacia Biotech, Uppsala, Sweden). Direct
sequencing was performed using a primer upstream of the TCR C
reverse primer: 5'-TGTGCACCTCCTTCCCATTCA-3' and a primer upstream of
the TCR C reverse primer: 5'-AGGCAGACAGACTTGTCACTG-3'. A
PCR-based sequence kit (Perkin-Elmer, Norwalk, CT) was used following
the manufacturer’s instructions and using
[32P]dATP as isotope; 1 µl of each sequencing
reaction was loaded onto a 6% urea-polyacrylamide gel, and the bands
were separated by electrophoresis. The gels were dried and analyzed by
autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three patients with CBD were selected for this study. They all
expressed the susceptibility-conferring Glu at position 69 in one or
both of their DP-chains. PBMC were separated and cultured with
different doses of Be. Dose-dependent T cell proliferation was seen in
all three patients. In Table I
is shown the proliferation of PBMC to
the optimal dose of Be. PBMC from healthy controls did not show any
significant response to Be, although three of six control individuals
carried HLA-DP alleles with glutamic acid at position 69 in the
DP-chain (data not shown).
T cell lines and clones were generated from CBD patients by culturing
PBMC with BeSO4·4H2O, as
described in Materials and Methods. The T cell clones
obtained were CD4 positive and, first, they were tested against
BeSO4·4H2O. Of the T cell
clones obtained, 65% (11/17) from patient TDC, 35% (13/37) from
patient FC, and 30% (3/10) from patient NG were specific for Be. Their
reactivity to different doses of Be was then investigated using
homozygous B-LCLs from the Xth IHW as APCs. In Table II is shown the summary of the
proliferative responses to the optimal concentration of Be of 11 T cell
clones from patient TDC, 12 T cell clones from patient FC, and two
clones from patient NG. The dose response of four T cell clones, two
from TDC and two from FC, are shown in Fig. 1, a–d.
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, e and
f). T cell clones specific for Be are restricted by HLA-DP
The restriction element used by the Be-specific T cell clones was
first examined by using a panel of homozygous B-LCLs from the Xth IHW
as APCs. In Fig. 2 is shown the
proliferation of three representative Be-specific T cell clones. All
the T cell clones tested responded to Be only when it was presented by
B-LCLs expressing HLA-DPB1*0201. No response was detected to
DPB1*0201-negative B-LCLs expressing either HLA-DRB1*1101 (patient TDC)
or HLA-DRB1*0301 (patient FC) (Fig. 2 and data not shown).
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, a–c. All the T cell clones
tested were completely inhibited by the presence of anti-HLA-DP
mAb. In contrast, no inhibition was observed with the mAb against
HLA-DQ molecules. In all the experiments performed, the presence of the
anti-HLA-DR mAb (L243) consistently led to 30–50% inhibition.
First, to exclude the possibility that the inhibition with the
anti-DR mAb reflected nonclonality in the Be-specific T cell
populations, T cell clones were further subcloned by limiting dilution.
No differences were observed in the degree of anti-HLA-DR
inhibition of the subclones when compared with the original T cell
population (data not shown). The alternative hypothesis tested was that
the inhibition observed with the anti-DR mAb was due to cell death
of the B-LCL presenting Be. This hypothesis is in agreement with the
work of Drenou et al. (18). They showed, in a slightly
different system, that cross-linking of MHC-class II molecules on a B
cell lymphoma led to apoptosis (18). To address this
possibility, B-LCL were incubated with the anti-DR mAb (L243) for
different lengths of time as described in Materials and
Methods, and the percentage of apoptotic and necrotic cells was
measured using PI and annexin V staining. After 45 min nearly 10% of
the cells were undergoing apoptosis and close to 20% had already died
(data not shown). After 3 h the addition of anti-DR (L243) mAb
induced >30% cell death and another 14% undergoing apoptosis (Fig. 4, e and f). In
contrast, anti-DP mAb (Fig. 4, c and d),
which inhibited T cell proliferation, did not cause any cell death
compared with untreated cells (Fig. 4, a and
b).
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-chain is critically important in T cell
recognition of Be
The genetic analysis of patients with CBD has revealed an
association with alleles of HLA-DPB1 encoding a DP-chain with
glutamic acid at residue 69. To investigate the functional relevance of
Glu at residue 69 in the -chain, we tested the response of
Be-specific T cell clones to B-LCLs expressing either HLA-DPB1*0201 or
HLA-DPB1*0402 that shared the same DP-chain and differ only at
position 69 in the -chain. All the Be-specific T cell clones
obtained responded only to the B-LCL with Glu at residue 69. The
dose-dependent proliferation for four representative T cell clones is
shown in Fig. 5, a–d.
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are shown the proliferative responses of Be 23
(e) and Be41 (f) to different doses of Be
presented by DAP.3-DP2 transfectants. No proliferation was seen to
DAP.3 cells expressing comparable levels of DPB1*0402 (Fig. 5).
Altogether, these data suggest that HLA-DP is the restriction element
for Be-specific T cells and that the presence of Glu at residue 69 is
essential for the reactivity of these T cell clones to Be. The Be-specific T cell clones are predominantly Th1 cells with biased TCR usage
It has been shown previously that delayed type hypersensitivity
reactions are mediated by T cells of the Th1 phenotype. In this
context, we have analyzed the cytokine production profile of
Be-specific T cell clones. As shown in Table III, all the Be-specific T cell clones
tested produced IFN- and little or no IL-4. These results confirm
similar data obtained using bronchoalveolar lavage (BAL) cells derived
from patients with CBD (19) and suggest that the
Be-specific T cell clones raised from PBMC of CBD patients are likely
to be derived from pathogenic T cells. Further data supportive of this
suggestion were provided by TCR gene analysis on four of the TDC clones
(Table IV). Two distinguishable patterns
were recognizable. T cell clones TDC1 and 30 shared a
"hydrophobic-Ser-negatively charged amino acid" sequence motif at
the N-terminal of CDR3 region as well as a Thr at position 94 of
the CDR3 region, whereas TDC17 and 38 showed an identical motif
"Ser-Gly-Gly-Ser" in the CDR3 region of the -chain as well as a
Ser at position 97 of the CDR3 region. However, the most striking
observation is that TDC30 displayed a CDR3 region identical, besides a
conservative substitution (Val-Leu) at position 92, to a sequence
previously reported as present at high frequency in the BAL of a
patient with CBD (20). The associated -chains also
showed a common motif "Gly-Asp", also overrepresented in the CDR3
region of all patients with CBD reported in the same paper
(20). Moreover, the T cell clone TDC38 used the V22
chain as reported for the patients analyzed by Fontenot et al.
(20).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The DP
restriction of the Be-specific clones observed here suggests strongly
that the DP association in CBD reflects the role of certain alleles of
DP in presentation of this metal to T cells in vivo.
There are many examples of HLA-DR and -DQ associations with resistance
or susceptibility to a variety of diseases. In particular, celiac
disease (CD) has been shown to be primarily associated with a pair of
HLA genes: DQA1*0501 and DQB1*0201
(21). More recently, the same group has demonstrated that
the CD-associated heterodimer is used as a restriction element in the
recognition of gliadin peptides by T cells derived from the gut of CD
patients (22). HLA-DP associations have also been
described in autoimmune diseases, such as CD (23),
insulin-dependent diabetes mellitus (24), pauciarticular
juvenile rheumatoid arthritis (25, 26, 27), and juvenile
ankylosing spondylitis (28). In addition, an HLA-DP
association has recently been demonstrated in primary biliary cirrhosis
(29), in childhood common acute lymphoblastic leukemia
(30), and also in Thai individuals with enhanced
vaccine-induced Ab response to a malaria sporozoite Ag
(31). However, some of these associations have not been
confirmed (32, 33), and in none of these examples has the
association with HLA-DP been related to the restriction element used by
T cells involved in the disease. It has been shown in other studies on
the association of MHC class II with chronic autoimmune disorders that
single amino acid substitutions at positions 57 and 71 of HLA-DQB1 and
HLA-DRB1 chains influence the susceptibility to insulin-dependent
diabetes mellitus and rheumatoid arthritis, respectively (34, 35). In addition, amino acid changes in DP are associated
with juvenile rheumatoid arthritis and CD (36, 37, 38).
Despite the evidence of DP restriction for all of the T cell
clones examined, significant, although less impressive, inhibition was
also seen with the anti-DR mAb, L243. Based on the observation
that cross-linking of HLA-DR on B cell lymphoma leads to apoptosis
(18), we were able to demonstrate that the inhibition seen
in our system was due, at least in part, to cell death of the B-LCL
presenting peptide (Fig. 4). The reasons for the discrepant effect of
anti-DR and anti-DP isotype-specific Abs is unclear, but may be
best explained by the substantially low level of expression of the DP
molecules. These observations highlight an important potential artifact
in the interpretation of Ab-blocking data.
To further analyze the restriction element used by the Be-specific T cell clones, we used murine DAP.3-DP2 transfectants to present Be. Only 3 of 12 clones tested responded. One possible explanation for the failure of the murine transfectants to stimulate most of the Be-specific clones is the lack of human accessory molecules. We have shown previously that coexpression of human LFA-3 by HLA-expressing DAP.3 cells can lead to substantial augmentation of human T cell responses (39). An alternative possibility is that the T cells recognize Be bound to a particular peptide displayed by human, but not mouse cells. Species-specific differences in DR-bound peptides have been demonstrated before, in the context of T cell allorecognition using DR-expressing DAP.3 transfectants and human B-LCL (40).
In light of these considerations it remains unclear as to how Be is recognized by T cells. One possibility is that Be binds directly to the HLA-DP molecule, leading to modification of peptide binding or of DP conformation. Alternatively, Be may interact with self-peptides bound in the groove of the HLA-DP molecule, leading to the display of "altered self". These two possibilities are under investigation.
The immunogenetic analysis referred to above highlighted the importance
of the Glu at position 69 in the DP-chain; although most commonly
represented in the DPB1*0201 allele, susceptibility was also
conferred by less common alleles with this residue in the DP-chain
(13, 41). However, this study demonstrates the functional
importance of this residue by showing that presentation in the context
of a Glu69 expressing molecule was a mandatory
requirement for T cell activation as none of the clones responded to Be
presented by B cells expressing DPB1*0402, an allele that
differs only at position 69 in the DP-chain. The three-dimensional
structure of HLA-DP has yet to be solved; however, in the predicted
structure, based on the known structures of several other class II
molecules, the side chain of residue 69 in the DP-chain points into
the peptide-binding groove (42, 43). As a consequence it
is likely to interact with bound peptide and be inaccessible to TCR
contact.
One of the striking features of the DP association with CBD is that a
single amino acid appears to determine susceptibility or resistance.
Based on these observations, it is easier to envisage that the
influence of Glu69 results from direct binding of
the positively charged Be to this negatively charged site on the
DP-chain. However, a role for HLA-DP in Be presentation cannot be
excluded, based also on the recent observation by Wang et al. that
HLA-DPA1 alleles may also be related to CBD (41). Sequence
homologies of the TCR and CDR3 regions and the extensive
homology between one of the four clones and a dominant sequence
detected in BAL-derived T cells from CBD patients (20)
provide strong evidence of Ag-driven TCR selection. Whether this
selection is driven by Be itself or by a conserved peptide to which Be
is bound is unknown.
Finally, the pattern of cytokine production by the Be-specific clones provides further evidence that these in vitro observed responses to Be reflect in vivo events in that the T cell clones appeared mostly to be of the Th1 subset that would be predicted to be pathogenic in a chronic inflammatory lung disease.
These data give a functional basis to the genetic association between CBD and HLA-DP Glu69 previously reported by us (13) and represent a step forward to the understanding of the pathogenesis of CBD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Giovanna Lombardi, Department of Immunology, ICSM, Hammersmith Hospital, Du Cane Road, London W12ONN, U.K.
3 Abbreviations used in this paper: CBD, chronic beryllium disease; BAL, bronchoalveolar lavage; B-LCL, B lymphoblastoid cell line; Xth IHW, Xth International Histocompatibility Workshop; PI, propidium iodide; CD, celiac disease.
Received for publication August 28, 2000. Accepted for publication November 28, 2000.
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 G. Diaz, M. Amicosante, D. Jaraquemada, R. H. Butler, M. V. Guillen, M. Sanchez, C. Nombela, and J. Arroyo Functional analysis of HLA-DP polymorphism: a crucial role for DP{beta} residues 9, 11, 35, 55, 56, 69 and 84-87 in T cell allorecognition and peptide binding Int. Immunol., May 1, 2003; 15(5): 565 - 576. [Abstract] [Full Text] [PDF]
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 K. Irifune, A. Yokoyama, N. Kohno, K. Sakai, and K. Hiwada T-helper 1 cells induce alveolitis but do not lead to pulmonary fibrosis in mice Eur. Respir. J., January 1, 2003; 21(1): 11 - 18. [Abstract] [Full Text] [PDF]
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M. Amicosante, F. Berretta, A. Franchi, P. Rogliani, C. Dotti, M. Losi, R. Dweik, and C. Saltini HLA-DP-unrestricted TNF-{alpha} release in beryllium-stimulated peripheral blood mononuclear cells Eur. Respir. J., November 1, 2002; 20(5): 1174 - 1178. [Abstract] [Full Text] [PDF]
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A. P. Fontenot, L. A. Maier, S. J. Canavera, T. B. Hendry-Hofer, M. Boguniewicz, E. A. Barker, L. S. Newman, and B. L. Kotzin Beryllium Skin Patch Testing to Analyze T Cell Stimulation and Granulomatous Inflammation in the Lung J. Immunol., April 1, 2002; 168(7): 3627 - 3634. [Abstract] [Full Text] [PDF]
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 M. D. ROSSMAN, J. STUBBS, C. W. LEE, E. ARGYRIS, E. MAGIRA, and D. MONOS Human Leukocyte Antigen Class II Amino Acid Epitopes . Susceptibility and Progression Markers for Beryllium Hypersensitivity Am. J. Respir. Crit. Care Med., March 15, 2002; 165(6): 788 - 794. [Abstract] [Full Text] [PDF]
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C. Saltini, L. Richeldi, M. Losi, M. Amicosante, C. Voorter, E. van den Berg-Loonen, R.A. Dweik, H.P. Wiedemann, D.C. Deubner, and C. Tinelli Major histocompatibility locus genetic markers of beryllium sensitization and disease Eur. Respir. J., October 1, 2001; 18(4): 677 - 684. [Abstract] [Full Text] [PDF]
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B. Nemery, A. Bast, J. Behr, P.J.A. Borm, S.J. Bourke, Ph. Camus, P. De Vuyst, H.M. Jansen, V.L. Kinnula, D. Lison, et al. Interstitial lung disease induced by exogenous agents: factors governing susceptibility Eur. Respir. J., July 1, 2001; 18(32_suppl): 30S - 42s. [Abstract] [Full Text] [PDF]
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), Co (
), and Ni
(•). After 48 h [3H]TdR was added and the plates
were harvested 18 h later. Proliferation is expressed as
cpm.
) or 8305, DPB1*0402 (