Cloning and Expression of Legionella pneumophila Antigens inEscherichia coli
N. CARY ENGLEBERG,' DAVID J. DRUTZ,1'2'3 AND BARRY I. EISENSTEIN1l2*Departments of Medicine' and Microbiology,2 The University of Texas Health Science Center at San Antonio, and the
Veterans Administration Hospital,3 San Antonio, Texas 78284
Received 3 October 1983/Accepted 23 January 1984
To isolate and characterize Legionella pneumophila antigens, we constructed a genomic library of L.pneumophila serogroup 1 (strain 130b). L. pneumophila DNA fragments (2.5 to 7.5 megadaltons) obtainedby partial digestion with Sau 3A endonuclease and size fractionation on a sucrose density gradient wereinserted into the dephosphorylated BamHI site of vector pBR322; CaCl2-treated Escherichia coli cells ofstrain HB101 were transformed with hybrid plasmids. To detect expression of antigens, 2,559 ampicillin-resistant transformants were transferred to nitrocellulose paper, lysed in situ, and screened by enzymeimmunoassay (EIA) with E. co/i-absorbed rabbit anti-L. pneumophila sera. A total of 77 (3%) of the colonieswere reactive by EIA; 31 (1.2%) were strongly reactive, and 6 were strongly reactive by EIA without colonylysis. Analysis of 29 stable, strongly reactive clones by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and electroblotting showed antigenic bands in 18 clones by EIA with E. coli-absorbedantisera. Absorption of antisera with heat- and Formalin-killed L. pneumophila antigen eliminated ordimninished the reactivity of the antigenic bands in representative clones. These studies confirm that severalL. pneumophila antigens can be cloned and expressed in E. coli.
Since the recognition of Legionella pneumophila as thecausative agent of Legionnaires disease in 1977, vigorousresearch has produced an impressive body of informationabout the disease, principally in the areas of clinical medi-cine and public health (13). During the same period, howev-er, insight into the immunology and pathogenesis of legionel-losis has developed slowly, and today our understanding ofthese aspects of infection remains incomplete. In general,laboratory-based research has been complicated by difficul-ties in cultivating and handling the organisms and by theproblem of maintaining their virulence on artificial media.
In early studies by Wong and co-workers, complex L.pneumophila antigens, prepared by ion-exchange and liquidchromatography, were isolated, and fractions having seroty-pic specificity and cross-reactivity were identified and ana-lyzed (24-26). The serotypic antigen fraction is composed ofa pronase-sensitive lipid-protein-carbohydrate complex thatinduces active immunity to infectious challenge in mice.Cross-reacting antigens, which are almost entirely composedof protein, were found to elicit dermal hypersensitivity.Other workers have studied L. pneumophila antigens by thetechniques of crossed immunoelectrophoresis (3, 4, 10),counterimmunoelectrophoresis (19), and most recently bypolyacrylamide gel electrophoresis and protein blotting(M. S. Hindahl and B. H. Iglewski, Abstr. Annu. Meet. Am.Soc. Microbiol. 1983, C104, p. 329; W. Ehret, G. Anding,and G. Ruckdeschel, 2nd International Symposium on Le-gionella, Atlanta, Ga., M-8, 1983). Although these studieshave confirmed the existence of discrete serotypic andcrossreacting antigens, it will be necessary to purify thesemolecules to establish their immunogenicity and to fullycharacterize their role in the host response to infection.To circumvent the problems inherent in working with L.
pneumophila and the difficulties of biochemical purificationof antigens of interest, we chose to isolate antigens at thegenetic level by using recombinant DNA technology. Since
* Corresponding author.
others have shown that genes from a variety of bacterialpathogens can be transcribed and translated by Escherichiacoli (14, 16-18, 20, 23), we chose a simple cloning strategythat did not include specific engineering for gene expression.We constructed a library of the L. pneumophila genome byusing vector pBR322. We then screened the library forexpression of L. pneumophila antigens by using a modifiedfilter binding assay (8, 14) in which colonies bound tonitrocellulose paper are tested for antigens by in situ enzymeimmunoassay. In this article, we report the isolation ofseveral E. coli clones that express a variety of L. pneumo-phila antigens.
MATERIALS AND METHODSBacterial strains. L. pneumophila serogroup 1 (130b) was
used for all cloning procedures and antigen preparations. Forboth purposes' L. pneumophila was isolated from infectedguinea pigs and passed only once on buffered charcoal-yeastextract agar (7). E. coli K-12 strain HB101 (mk-, rk-, recA)was the recipient for hybrid plasmid transformations.Enzymes and chemicals. Restriction endonucleases and T4
DNA ligase were obtained from Bethesda Research Labora-tories, Bethesda, Md. Calf intestinal alkaline phosphatase,lysozyme (grade I), protein A-Sepharose, 5-aminosalicylicacid, ampicillin, and tetracycline were purchased from Sig-ma Chemical Co., St. Louis, Mo. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin was purchasedfrom Cappell Laboratories, Cochranville, Pa. The color-forming reagent 4-chloro-1-naphthol and nitrocellulose paperfor electroblotting were purchased from Bio-Rad Labora-tories, Richmond, Calif. Nitrocellulose disks for filter bind-ing assays (type HA) were purchased from Millipore Corp.,Bedford, Mass.; Whatman 3MM chromotography paper wasobtained from American Scientific Products, McGaw Park,Il.
Construction of the clone bank. DNA was extracted fromL. pneumophila cells harvested from buffered charcoal-yeastextract agar plates in phosphate-buffered saline (PBS), pH7.2. To assure against contaminating bacteria, samples of
this suspension were plated on buffered charcoal-yeast ex-
tract and sheep blood agar and inoculated into brain heartinfusion broth. There was no growth on blood agar or brainheart infusion broth. Pure growth of L. pneumophila was
detected on buffered charcoal-yeast extract agar and was
confirmed by direct immunofluorescence with fluorescein-labeled, polyvalent rabbit antisera (Centers for DiseaseControl, Atlanta, Ga.). L. pneumophila DNA was extractedand purified by the method of Nakamura et al. (15). PurifiedDNA was partially restricted with Sau 3A restriction endo-nuclease, and the digestion fraginents were applied to a 10-ml 5 to 40% sucrose gradient in 1 M NaCl-20 mM Tris-hydrochloride-5 mM EDTA (pH 8.0) and centrifuged at100,000 x g for 21 h. Gradient fractions (0.5 ml) were
analyzed by agarose gel electrophoresis, and fractions con-
taining restriction fragments of 2.5 to 7.5 megadaltons were
pooled.Vector pBR322 was prepared for cloning by complete
digestion with BamHI followed by 5' dephosphorylationwith alkaline phosphatase (12). The latter procedure resultedin a 2- to 3-log reduction in recircularization and ligation ofthe vector as compared with untreated linear pBR322.
Size-fractionated L. pneumophila Sau 3A restriction frag-ments were ligated to dephosphorylated pBR322 with T4DNA ligase and used to transform E. coli strain HB101rendered competent by treatment with 0.5 M CaCI2 (5).Transformants were selected on Louria-Bertani agar con-
taining ampicillin (40 ,ug/mI) (12). Forty ampicillin-resistant(Apr) transformants were screened for tetracycline sensitiv-ity (Tcs) on Louria-Bertani medium containing tetracycline(15,ug/ml).
Preparation of L. pneumophila cells for immunization. L.pneumophila cells from six buffered charcoal-yeast extractagar plates were harvested, pooled, and suspended in 6 ml ofPBS (pH 7.3).F,ormalin-killed (FK) cells were prepared from3 ml of this suspension by adding Formalin to a finalconcentration of 2% and holding overnight at 4°C. Heat-killed (HK) cells were prepared by heating the rerhaining 3ml of suspended cells to 100°C for 30 min. Both preparationswere checked for nonviability at 24 h.
Preparation of antisera. New Zealand rabbits were inject-ed subcutaneously with 2 ml ofHK (1:5 dilution) or FK (1:10dilution) cells at 0, 2, 4, and 6 weeks. Four rabbits were
immunized; two received HK cells and two received FKcells. Sera were collected at 7 weeks or later and were storedwith preimmune sera at -70°C.Immune sera (0.5-ml samples) were absorbed 4 times with
E. coli HB101 (pBR322). For each absorption, cells from 175ml of a stationary-phase culture were washed, mixed withsera, and rotated at 4°C for 1 h; sera were recovered bycentrifugation at 5,000 x g for 5 min after each absorption.Sera used for the filter-binding assay were also absorbedwith cells harvested from Louria-Bertani agar plates andwith sonicated E. coli. For the sonicate absorption, cellsfrom 500 ml of stationary-phase growth suspended in 10 mlof PBS were disrupted with a Branson Sonifer probe sonica-tor and then mixed with sera for 1 h at4°C. The serum-
sonicate mixture was then cleared by centrifugation at100,000 x g for 1 h in a Beckman SW 40 rotor. Fiftymicroliters of E. coli-absorbed serum was also absorbedtwice with mixtures of 0.1 ml of FK and 0.1 ml ofk-K L.pneumophila cells for 8 h at4°C. All sera were heated to56°C for 30 min before use; the coagulum that formed in theserum-sonicate mixtures was pelleted and removed by cen-
trifugation at 10,000 x g for 5 min.ELISA. Sera were tested for anti-L. pneumophila activity
by an enzyme-linked immunosorbent assay (ELISA) byusing whole L. pneumophila cells fixed to 96-well microtiterplates (Dynatech Laboratories, Inc., Alexandria, Va.). FKcells corrected to optical density at 550 nm of 1.5 with PBS(pH 7.2) were further diluted 1:25 with PBS. A 0.1-ml sampleof dilute FK cells was added to each well, and the microtiterplates were allowed to dry overnight at 42°C. Plates werewashed three times with PBS containing 0.5% Tween 20,serial dilutions of test sera (0.1-ml samples) were added, andplates were incubated for 2 h at room temperature on aMinimix agitator (Fisher Scientific Co., Silver Spring, Md.).After three washes with PBS-0.5% Tween 20, 0.1 ml ofperoxidase-conjugated goat anti-rabbit immunoglobulin(1:1,500 dilution) was added to each well, and the plateswere again agitated for 2 h. After three final washes withPBS-0.5% Tween 20, 0.1 ml of a color-forming substratesolution (0.08% 5-aminosalicylic acid and 0.006% hydrogenperoxide, pH 6.0) was added to each well. After agitation for30 min, absorbance at 450 nm was measured in a MR580MicroElisa Auto Reader (Dynatech). An absorbance of 0.5or more was considered positive.To assess the effectiveness of serum absorption, a similar
ELISA test was used in which live E. coli whole cells werefixed to the microtiter plate wells by the methods describedabove.FB-EIA. Apr transformants were spotted onto agar plates
with sterile toothpicks (ca. 325 colonies per plate), grownovernight, and then blotted onto dry nitrocellulose filterdisks; 1 to 2 RI of FK cells was also spotted onto each filteras a positive control. Colonies were lysed in situ by themethod of Meyer et al. (14). Briefly, filter disks were placedsequentially, colony side up, onto 3MM paper in a series offour petri dishes saturated respectively with (i) 0.1 N NaOH,(ii) 1.5 M Tris-hydrochloride (pH 7.4), (iii) 300 mM NaCI-30mM sodium citrate, and (iv) 70% ethanol for 5 min each.Each filter was then dried in a vacuum at 60°C for 2 h.Antigen-bearing colonies wete detected by an enzyme im-munoassay (EIA). Dry filters were placed in Tris-bufferedsaline (TBS; 50 mM Tris-hydrochloride, 150 mM NaCl, pH7.5) containing 3% gelatin to block nonspecific protein-binding sites. After gentle rotation at room temperature for 2h, the filters were transferred to a solution of absorbed,pooled antisera (1:800 dilution in 1.5% gelatin-TBS) andincubated overnight at room temperature. The followingmorning, the filters were rinsed with distilled water, washedfour times in TBS, and incubated in a solution of peroxidase-conjugated goat anti-rabbit immunoglobulin (1:3,000) for 2 h.After a final rinse and series of four washes in TBS, thefilters were immersed in a color development solution con-sisting of 0.05% 4-chloro-1-naphthol and 0.015% hydrogenperoxide in a 5:1 solution of TBS-methanol. All coloniesshowing color development were reanalyzed by a modifiedfilter binding (FB)-EIA in which the alkaline lysis andneutralization steps (i.e., on saturated 3MM paper) wereeliminated, and freshly blotted colonies were placed directlyinto the vacuum oven.
Protein electroblotting of positive clones. Clones that werestrongly reactive by FB-EIA were analyzed by sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresisand protein blotting. Each clone was grown to the stationaryphase in 3.0 ml of Louria-Bertani broth containing ampicillin(50 jig/ml). Cultures were washed once with PBS, trans-ferred to Eppendorf tubes, suspended in 0.375 ml of samplebuffer (75 mM Tris-hydrochloride, 5% 2-mercaptoethanol,2% SDS, 10% glycerol, 0.002% bromophenol blue, pH 6.8),and heated to 100°C for 2.5 min. SDS-polyacrylamide gel
electrophoresis was performed in a vertical slab gel electro-phoresis tank (Hoefer Scientific Instruments, San Francisco,Calif.) by the method of Laemmli (11). Samples of 0.04 mlwere run at 25 mA (constant current) through the 5%polyacrylamide stacking gel (pH 6.8) and at 50 mA throughthe 15% polyacrylamide separating gel (pH 8.3). Electro-transfer of proteins to nitrocellulose was performed by theprocedure of Towbin et al. (22) in a Trans-Blot Cell (Bio-Rad) at 30 mV for 12 h with a buffer containing 0.025 M Trisbase, 0.192 M glycine, and 20% methanol (pH 8.3). Rena-tured proteins on nitrocellulose were visualized with an EIAsimilar to that described above. Nitrocellulose sheets werepreincubated in a blocking solution of TBS with 0.05%Tween 20 and then transferred overnight to a similar solutioncontaining 1:750 rabbit antisera. After four washes with0.05% Tween 20-TBS, sheets were incubated in a 1:3,000dilution of peroxidase-conjugated goat anti-rabbit immuno-globulin in 0.05% Tween 20-TBS for 1 h. After a rinse indistilled water and four additional 0.5% Tween 20-TBSwashes, the sheets were exposed to the color developmentreagent used in the FB-EIA. The molecular weight of eachindividual band was estimated by comparing its coefficient ofmigration to a logarithmic plot of the migrations of proteinstandards run on the same gel and visualized with Coomassieblue stain.
Selected clones were also analyzed by protein blottingunder less denaturing conditions by a method of samplepreparation that did not require boiling. Cultures (1 ml) ofeach clone were centrifuged, washed once with PBS, andsuspended by vigorous vortexing in 0.04 ml of 25 mM Tris-hydrochloride-10 mM EDTA (pH 8.0) with lysozyme (2 mg/ml). After 10 min of incubation at room temperature, 0.04 mlof 2 x sample buffer was added, and the samples wereclectrophoresed as described above.
RIP. Radioimmunoprecipitation (RIP) was performed withabsorbed rabbit antisera as previously described (6), exceptthat protein A-Sepharose (40 ILI) was used instead of proteinA-containing Staphylococcus aureus.
Agarose gel electrophoresis of recombinant plasmids. Plas-mids were extracted from selected clones by the modifiedrapid alkaline lysis method of Birnboim and Doly (2, 9).Samples were mixed (1:5) with concentrated gel loadingbuffer (0.25% bromophenol blue, 30% glycerol) and added tothe wells of a 0.8% agarose gel, prepared in Tris-acetatebuffer (40 mM Tris-acetate, 2 mM EDTA, pH 7.85). Electro-phoresis was performed in a horizontal gel electrophoresissystem (Bethesda Research Laboratories), with Tris-acetatebuffer at 1 V/cm for 18 h. Gels were stained in ethidiumbromide (0.5 p.g/ml) for 45 min at room temperature, illumi-nated on a Chromato-Vue Transilluminator (Ultra-VioletProducts, Inc., San Gabriel, Calif.), and photographed witha Polaroid MP-3 Land camera. Restriction endonucleasedigestions were performed under conditions specified by themanufacturer to produce complete digestion.
RESULTS
ELISA of rabbit antisera. Rabbits imtnunized with eitherFK or HK cells developed antibody titers of >1:1280 asmeasured by the FK (whole cell) ELISA. Anti-L. pneumo-
phila activity was not detected in any preimmune serum attiters of 1:10 or greater. Absorption of antisera with E. colidid not reduce the anti-L. pneumophila activity by more thanone dilution, but did reduce the anti-E. coli titer from 1:320to 1:40 in the E. coli whole cell ELISA.
Characterization of E. coli transformants. E. coli HB101
TABLE 1. Characterization of cloned L. pneumophila antigensMolecular
E. coli transformants masses of(isolate no.) cloned
a RIP resolved individual bands of 19K, 20K, 23.5K, and 28K.
was transformed by hybrid plasmids at an efficiency of 103per ,ug of vector DNA. A sample of 40 Apr transformantswas transferred to media containing tetracycline; all 40 wereTc'.We screened 2,559 Apr transformants by FB-EIA; 77
(3.0%) of these clones produced detectable blue color. Ofthese, 31 (1.2%) were considered to be strongly reactive, and46 (1.8%) were considered weakly reactive. All 77 reactiveclones were transferred to a single nitrocellulose disk andanalyzed by a modified FB-EIA, which excluded the chemi-cal lysis steps. In this assay, 11 clones produced detectablecolor; 6 were strongly positive (clones 11, 13, 40, 41, 44, and70), and 5 were weakly positive (clones 33, 47, 61, 65, and73). Of these 11 clones, 10 were also strongly reactive in theinitial FB-EIA; clone 47 was weakly reactive in both assays.
Protein blotting of strongly reactive clones. We analyzed 29of the strongly reactive clones by SDS-polyacrylamide gelelectrophoresis and protein blotting (two clones were unsta-ble and could not be maintained in antibiotic-containingmedia). Of the 29 clonles, 18 had protein bands detected byprotein blotting with E. coli-absorbed rabbit antiserum, butnot with preimmune serum (Table 1). Clones 11, 13, 40, 41,44, and 70, which gave strong signals by FB-EIA with andwithout chemical lysis, all expressed the same antigenicproteins. Trhese antigens appear as a confluence of bands inthe 19K to 23K range (Fig. 1, lane c). Because a faintlyreactive 19K to 20K band was seen in immunoblots of all E.coli strains, we analyzed a representative clone (no. 11) byadditional techniques to confirm the separate identity of thecloned antigens from the background band. Protein immun-oblots probed with unabsorbed antisera confirmed that 19Kantigens are present in both the recipient E. coli strain and L.pneumophila; E. coli clone no. 11 expresses both reactivities(Fig. 2). In addition, analysis of clone no. 11 by RIP withabsorbed antisera resolved four bands of 19K, 20K, 23.5K,and 28K that are distinct from E. coli background antigens(Fig. 3). Also in this analysis, an E. coli antigen between 19Kand 20K was precipitated. None of the other cloned antigenbands that we identified by immunoblotting coincided withbackground antigenic bands.To confirm that the antisera recognized L. pneumophiia
antigens cloned in E. coli, we absorbed antisera with L.pneumophila FK and HK cells. This additional absorptionmarkedly diminished the reactivity against the antigenicbands in clone 11 and completely removed reactivity againstone of the six clones that expressed a 24K antigen (Fig. 1). Ina similar experiment, reactivity against the higher-molecu-lar-weight antigens in clone 12 was also reduced or removed(data not shown); quantitatively poor expression of the 17K
FIG. 1. Protein blot analysis of recombinant plasmid-bearing E.coli; samples shown here were prepared from the recipient straincontaining pBR322 (lane a), isolate 11 (lanes b through d), andisolate 21 (lanes e through g). Blots were assayed with preimmunesera (lanes b and e), immune sera absorbed with E. coli (lanes a, c,and f), or immune sera absorbed with killed L. pneumophila (lanes dand g).
antigen has not yet permitted any firm conclusions about thepostabsorptive reactivity of this band.Repeat blotting with less denaturing conditions failed to
uncover antigenic bands in any of the 12 clones that werepositive by FB-EIA and negative by protein blotting.
Agarose gel electrophoresis of recombinant plasmids. Plas-mids from clones 11, 13, 40, 41, 44, and 70 were isolated andanalyzed by gel electrophoresis. Electrophoresis of unre-stricted plasmid extracts confirmed that each clone con-tained only one recombinant plasmid (data not shown). Afterdigestion with HindIII, only clones 41 and 44 had plasmidswith identical restriction fragments; clones 11 and 40 con-tained similar but not identical plasmids (Fig. 4). Thesefindings were confirmed by restriction analysis with Sau 3A,BamHI, AvaI, and AccI.
DISCUSSIONWe have demonstrated that L. pneumophila antigens can
be cloned and expressed in E. coli. The screening FB-EIA
a b c
FIG. 2. Section of a protein immunoblot of solubilized E. coliclone 11 (lane a), E. coli (pBR322) (lane b), and L. pneumophila(lane c) with unabsorbed rabbit antisera. Note the presence of boththe E. coli and L. pneumophila 19K bands in E. coli clone 11.
25.7K -
18.4K -
-28 K
-23.5 K
-004,0 0 -20K_-1- 9 K
!4t_,,...__:.
FIG. 3. RIP and SDS-polyacrylamide gel electrophoresis of solu-bilized E. coli (pBR322) (lane a) and E. coli clone 11 (lane b) withabsorbed rabbit antisera. Tritiated molecular mass standards areindicated in left margin.
with antisera absorbed with the recipient strain preventedthe selection of cloned, contaminating E. coli genes. The L.pneumophila origin of the antigen bands from representativeclones was confirmed by their failure to react with eitherpreimmune sera or immune sera absorbed with killed L.pneumophila. Because reactivity against a native E. coliantigen that comigrated with a cloned antigen at approxi-mately 19K could not be completely deleted from ourantisera after extensive absorption with E. coli, it wasnecessary to confirm the separate identity of the clonedantigen. SDS-polyacrylamide gel electrophoresis and im-munoblotting with unabsorbed antisera demonstrated thepresence of this antigen in solubilized L. pneumophila andshowed that the L. pneumophila and E. coli antigen bands inclone 11 were not identical. Moreover, RIP of clone 11antigens with (whole cell) E. coli-absorbed antisera resolvedfour distinct bands of 19K, 20K, 23.5K, and 28K. In thesetwo studies, the native E. coli antigens migrated slightlydifferently relative to the smallest of the series of clonedantigens. Because of differences in antigen preparation forthe two techniques and because of the difficulty in accuratelyassigning molecular weights within such a narrow range, weare reluctant to attempt any matching of the bands detectedby RIP and immunoblotting. Although not apparent in Fig. 1,activity against a 28K component has been observed onother immunoblots of clone 11 antigens conducted underdifferent experimental conditions (data not shown).The expression of the same series of four antigens in five
distinct clones suggests that some or all of the individualbands may result from stepwise degradation of a largerantigen. A similar series of antigens (61K, 66K, 68K) isrepresented in clone 12 (although it is not yet clear why two
FIG. 4. Agarose (0.8%) gel electrophoresis of Hindlll digests ofrecombinant plasmids from six strongly reactive clones that expressthe same series of L. pneumophila protein antigens. Completedigests of pBR322 and plasmids from clones 11, 13, 40, 41, 44, and70 were run in lanes a through g, respectively. pBR322 multimerswere included as molecular mass references (lane h). Note that onlythe plasmids of clones 41 and 44 have identical restriction patterns.
other clones, 50 and 63, selectively expressed certain ofthese bands, but not others). The observation that certainclones were positive by FB-EIA without colony lysis sug-gests that their encoded antigens may be surface expressed;peptide cleavage associated with membrane translocationmay partially explain the multiple antigenic bands.The cloning of individual antigens will permit us to study
the immune response to infection in an animal model. In thenude mouse model, both cellular and humoral mechanismsappear to be important for protection (D. J. Drutz, P.DeMarsh, J. Richard, W. Owens, R. Rolfe, P. Edelstein, andS. Finegold. Exp. Cell Biol. 50:325, 1982; unpublishedobservations). Cloned antigens can be used to quantitate andcharacterize the humoral immune response to infection andto investigate cross-reactivity with other L. pneumophilastrains. In an animal model in which immune serum providespassive protection, cloned antigens can be used to absorbantisera selectively and to identify potential protective anti-gens. In models in which protective immunity is predomi-nantly cell mediated, they can be used in a similar manner todetect T-lymphocyte responsiveness selectively. In studyingthe immunogenic components of L. pneumophila, separationof antigens by cloning is particularly useful since manyimportant polypeptide surface components cannot be ade-quately purified by biochemical means (1).
ACKNOWLEDGMENTSWe thank John Abraham, Janice Clements, Peter DeMarsh,
Douglas C. Dodd, Suzy Engleberg, Cynthia Freitag-Hall, EricPearlman, Robert Thornburg, and Janine Trempy for their assist-
ance during the conduct of these studies. We also wish to acknowl-edge the valuable advice of Philip Bassford, William Haldenwang,Itzhak Kahane, and Lola V. Stamm.
B.I.E. is supported in part by a Public Health Service ResearchCareer Development Award from the National Institutes of Health.
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