In Vitro Idiotypic Suppression of Chronic LymphocyticLeukemia Lymphocytes Secreting Monoclonal

Immunoglobulin MAnti-Sheep Erythrocyte Antibody

CONSTANTINA. BONAand ANTHONYS. FAUCI, Laboratory of Itmmtiuniology anid theLaboratory of Clinical Iinvestigation, Nationial Inistitute of Allergy antd InfectiousDiseases, Nationial Inistitutes of Health, Bethesda, Mariylatd 20205

A B S T RA C T A patient with chronic lymvlphocyticleukemia was found to have B cells with surface imn-munoglobulin (Ig)M manifesting anti-sheep erythrocyte(SRBC) specificity together with a high titer serummon-oclonal anti-SRBC IgM antibody. By imimilluiniziiiga sheep with the monoclonal IgM antibody, followedby multiple absorptions against normal human IgM, anlanti-idiotype (Id) antibody was obtained. The serumnIgM anti-SRBC antibody was then demonstrated toshare the same idiotypic determinants with the surfaceIgM with anti-SRBC specificity on the patient's B cells.

The anti-Id antibody suppressed the spontaneous se-cretion of anti-SRBC antibody as well as the pokeweedmiitogen-induced anti-SRBC antibody production asmeasured by a hemnolysis-in-gel plaque-formning cellassay. In contrast, pokeweed mitogen-induced anti-SRBCplaque-formning cell responses of normiial incdi-viduals were not suppressed by the anti-Id antibody.Thus, this study demonstrates in vitro suppressioni ofhuman B-cell function by anti-Id antibody.

INTRODUCTION

The association of anti-idiotypic (Id)' anitibo(lies withcertain immunoregulatory phenomenia has been clearlydemonstrated in animal models. For example, idliotype-specific suppression of the growth of mouse myelomiiacells can be demonstrated in mice with anti-Id anti-bodies (1-4). Furthermore, anti-Id antibodies are ableto suppress the synthesis by normal B cells of antibodliescarrying the relevant idiotypes. This suppression canbe the result of either a direct effect of anti-Id anti-bodies on the precursors of antibody-forming cells, as

Receivedfor publication 6June 1979 and in revised form 30October 1979.

' Abbreviations used in this paper: CLL, chronic lympho-cytic leukemia; E, erythrocyte; Id, idiotype; Ig, immnitiio-globulin; PFC, plaque-fonning cell; PWM,pokeweed mitogen;SRBC; sheep erythrocyte; TCE, T cell-enriched.

shown in the inulin and phosphorylcholine systems(5, 6), or by generation of suppressor T cells that caninhibit helper functions in the arsonate and streptococ-cal systems (7, 8).

In the present paper, we report studies of a patientwith chronic lymphocytic leukemia (CLL) who hadhigh titer serumi monoclonial immilunoglobulin (Ig)M anti-sheep erythrocyte (SRBC) antibody, and whose B cellsexpressed surface IgM with anti-SRBC specificity. Ananti-Id antibody was produced by immunizing sheepwith the monoclonal serum anti-SRBC antibody,followed by multiple absorptions against poolednormal serum IgM. The anti-SRBC anitibody in theserum and on the surface of the patient's B cells wasshown to be idiotypically identical. Furthermiore, theaniti-Id antibody suppressed the spontanieous secretionof anti-SRBC antibody by the leukemic B cells as wellas the pokeweed mitogen (PWM)-induced ainti-SRBCantibody production as measured by a hemolysis-ini-gel plaque-formiinig cell (PFC) assay.

METHODSPatienit. The patient is a 64-yr-old miale who was liagniosed

as having CLL. The diagniosis of CLL is based on a totalleukocyte counit ranginig from 7,000 to 20,000 cells/mm11113 with arange of 40-90% lymphocytes. Up to 80% of the lymphocytesbear surface Ig (IgM, K-light chaini). The patienit has neverreceived therapy for his CLL.

Cell suspensioms. Heparinize(l venous blood was obtainie(dfromn the patienit and from normiial voltunteers; monioniuclearcell suspenisions were obtainie(d by stanidardl Hypa(lue-Ficolldensity centrifuigationi.

Fractioniationi of inionionuclear cell suspensionis. T cell-enriched (TCE) suspenlsionls were obtainie(d by two separatemethods. In the first method, TCE suspenlsionls were oh-taimiedl by rosettinig lviynphocytes vith sheep erythrocvtes (E),followed by cenitrifugationi of rosetted cells over Hypa(que-Ficoll gradients as previously dlescribe(d (9). The alterniativemethod of obtainiing TCE suspenisions was to pass m11on1o-nuclear cells over a nvlon wool columnli.

Cuilture coni(litiot s. Culture cond(litionis for the genieration

The Journal of Clitnical Itnvestigationi Volumole 65 March 1980 761 -767 761

of anti-SRBC PFCafter polyclonal stimulation of human bloodlymphocytes with PWMwere carried out according to meth-odologies that have been previously described in detail(10, 11).

Assay for PFC. Lymphocytes were assayed for directPFCagainst SRBCby an ultrathin layer complemenit dependenthemolysis-in-gel technique as previously described in detail(10, 11). This was performed on cells obtained immediatelyafter separation from blood (spontaneous PFC) and after 7d in culture with or without PWM.

Preparation of anti-Id antisera. Three strain 13 guineapigs were immunized with 100 ,tg of the patient's mono-clonal IgM anti-SRBC antibody emulsified in Freund'scomplete adjuvant. Starting 4 wk after immunization, theguinea pigs were bled weekly and sera were screenedin an immunodiffusion assay for antibodies to IgM. Sheep3501 (National Institutes of Health Animal Farm, Poolesville,Md.) was immunized with 3 ml of packed autologous SRBC(3501-SRBC) coated with 100 ,ug of the patient's monoclonalIgM anti-SRBC antibody emulsified in Freund's completeadjuvant. Adsorption of the antibody was performed byincubation of 3501-SRBC with the monoclonal IgM for 1 hat room temperature. After immunization, the sheep werebled weekly and precipitating antibodies were dletected byimmunodiffusion. Guinea pig and sheep anti-Id sera weremade specific for the patient's monoclonal IgM by five adsorp-tions, on an immunoadsorbent prepared according to Avremeaset al. (12), from a pool of normal IgM as well as from aWaldenstrom's macroglobulin (kindly provided by Dr. HenryMetzger, National Institutes of Health, Bethesda, Md.).

Determination of anti-Id activity of guinea pig and sheepantisera. After five adsorptions of an immunoadsorbentprepared from normal IgM or a macroglobulin, determina-tion of anti-Id activity was performed by two methods: (a)A hemagglutination assay in microtiter plates using horseerythrocytes coated by the chromic chloride method (13) withthe patient's monoclonal IgM, the patient's IgG, and IgM fromnormal healthy subjects. The titers were expressed as 1/log2of the highest dilution of antisera giving agglutinationi. (b)A radioimmunoassay in microtiter plates using sheep or guineapig anti-Id antibodies and l25I-labeled patient's monoclonal IgM.

In addition, a hemagglutination inhibition assay was usedto test the ability of anti-Id antibodies to react with thecombining site of monoclonal IgM as described previously (13).

Effect of anti-Id antisera on anti-SRBC PFC. To deter-mine the effect of anti-Id antibody on spontaneous anti-SRBCPFC, freshly drawn lymphocytes from the patient and fromthree normal individuals were immediately placed in thePFC assay with agar into which various dilutions of thesheep and guinea pig pre- and postinmuinization antiserawere added. After 2 h of incubation guinea pig complementwas added and incubation was continued for an additionalhour. PFC were then counted using a dissecting microscope.

To determine the effect of anti-Id antibody on PWM-induced PFC, various dilutions of the above antisera wereadded on day 0 directly to lymphocyte cultures from the pa-tient and three normal individuals in the presence andabsence of PWM.PFC were counted after 6 d of culture. Inseparate experiments, to control for the carry-over of anti-Idantibody from the cultures to the assay system, cultures weredone for 6 d in the presence of PWM, but without anti-Idantibody. At the end of the 6-d culture period, a 1:30 dilutionof anti-Id was added to the cultures immediately beforeharvesting. The cultures were then washed three times andcells were placed in the PFC assay.

Determination of the presence of Id or anti-Id on cells.To determine the presence of Id on the B cells of the patientand normals, mononuclear cells were first incubated for 45 min

at 370C with either control media or a 1:100 dilution ofsheep preimmunization IgG or sheep IgG anti-Id. Afterincubation, cells were washed and stained with fluoresceinatedgoat anti-human Ig (14) to determine whether the anti-Ilblocked the expression of surface Ig.

To determine whether the idiotypic determiniants werepresent on T cells, TCE suspensions were obtained from twonormal individuals by the E-rosette enrichment method (9)and by the nylon wool column method, and from the patientby the nylon wool method. The E-rosette enrichment methodwas inadequate in obtaining purified TCE suspensions fromil thepatient, because the patient's B cells expressed surface Igwith anti-SRBC activity and consequently bound to theSRBCto form E rosettes (15).

The sheep control and anti-Id antisera were conjugated withfluorescein isothiocyanate, and the TCE suspensions of thepatient and normals were assayed for the presence of i(liotypicdeterminants.

To determine whether anti-Id determinants were present oniT cells, nylon-purified TCE suspensions fronm the patientand two normal individuals were preincubated for 30 min at37°C with a 1:10 dilution of the patient's IgM, IgG, and anormal IgM. Cells were washed and binding of the differentIg was determinedl by reacting the cells with fluoresceinatedgoat anti-human Ig.

RESULTS

Fractiotnation of seortuml oni Sephadex G-200 colinimns.Normal serum pooled from four donors, as well asserum from the patient, were fractionated separatelyon Sephadex G-200 columns (Pharmacia Diagniostics,Div. of Pharmacia Inc., Piscataway, N. J.). SRBChemagglutinins were tested on fractions that includedboth IgM and IgG. Anti-SRBC hemagglutination ac-tivity was found predominaintly in the IgM fractionsof the patient. These fractions were pooled and used toraise anti-Id sera.

Determination of an ti-Id activity of guinlea pig an(1sheep antisera. Data in Table I demonstrate that bothguinea pig and sheep antisera contain anti-IgM anti-bodies but that after five adsorptions on pooled normalhuman IgM only anti-Id antibodies were detected.These five-times-adsorbed sera were subsequentlyused in the radioimmunoassay. A dose-effect relation-ship between the dilution of these antisera and thebinding of 1251-labeled IgM (patient) was observedindicating that both guinea pig and sheep antiseracontain anti-Id antibodies (Fig. 1).

Specificity of anti-Id antisera. The ability of guineapig and sheep anti-Id antisera to react with thecombining site of the patient's IgM anti-SRBC anti-bodies was studied in a hemagglutination inhibitionassay. The data presen-ted in Fig. 2 and Table II showthat only sheep anti-Id antibody was able to inhibit thehemagglutinationi activity of the patient's IgM aniti-SRBC antibodies. These data suggest that anti-Idantibodies produced in sheep were comnbiniing sitespecific, whereas guinea pig antibodies were directedagainst some other antigenic determinant, probablylocated on the framework of the variable region of the

762 C. A. Bona and A. S. Fauci

TABLE IHemnaggluitinatiotn Titer (log2) of Guinea Pig

and Sheep Anti-idiotypic Sera

Horse erythrocytes coated with:*

Numiiber of Waldeni-a(lsorptionis str6m's

Origin of on human Patient Patient macro- Normiialanti-Id serumii Ig.\M I 1g\ IgG globulini 1g\1

Guinea pig 0 >24§ 0 20 194 6 0 3 15 4 0 0 0

Sheep 0 20 0 14 154 5 0 2 15 5 0 0 0

* Chromic chloride method was used to coat horse erythro-cytes with various proteins (see Methods).t 1 ml of ammonium sulfate-precipitated Ig fraction, pre-pared from Sephadex G-200-separated IgM, was adsorbed on100 mg of immunoadsorbent from four normal pooled serum.§ Hemagglutination titer (log2).

IgM anti-SRBC antibody, but apparently not related tothe combining site.

Effect of antti-Id antisera otn sponitanieous anlti-SRBCPFC. The effect of various dilutions of the anti-Idantisera on the spontaneous anti-SRBC PFC from thepatient is shown in Fig. 3. Neither sheep nor guineapig preimmunization sera inhibited spontaneous anti-SRBC PFC at any dilution tested. However, thesheep anti-Id that is most likely specific for the anti-SRBC combining site completely suppressed PFCresponses at dilutions of 1:100 and 1:300, and sup-pressed to almost 50%of control at a dilution of 1:1,000.On the other hand, the guinea pig anti-Id, which, asshown in Fig. 2 and Table II, is likely specific fordeterminants other than the combining site of theidiotypic antibody, failed to suppress PFC responses.

Because normal individuals do not express sponta-neous anti-SRBC PFCbefore culture with PWM,it wasimpossible to determine the effect of the various anti-Idantisera on unstimulated normal lymphocytes.

Effect of anti-Id antisera on1 PWVM-inidtuced anti-SRBCPFC. The effects of various dilutions of anti-Idantisera on the PWM-induced anti-SRBC PFC of anormal individual and the patient are shown in Figs.4 and 5, respectively. Neither preimmunization noranti-Id antisera from sheep or guinea pig suppressedthe PWM-induced PFC of the normal individual (Fig.4). This experiment was done three times using threedifferent normnal subjects and the results were similarin each, i.e., no suppression. With the patient therewas no suppression with either sheep or guinea pigpreimmnunization sera. However, sheep anti-Id anti-serumn, which blocks the combining site of the patient'smonoclonal IgM anti-SRBC antibody, markedly sup-

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FIGURE 1 Binding of various antisera to '25I-labeled patientIgM. Neither sheep nor guinea pig preimmunization antiserabound to the patient's IgM. However, both sheep and guineapig postimmunization antisera, after multiple adsorptions withpooled normal IgM and a macroglobulin from a patient withWaldenstrom's macroglobulinemia, did not bind to normalIgM (Table I). Hence, both sheep and guinea pig post-immnunization antisera were directed against the patient's IgMidiotype. *, sheep preimmunization; 0, sheep anti-Id; A,guinea pig preimmllunization; A, guinea pig anti-Id.

pressed the PWM-induced anti-SRBC PFC at a dilu-tion of 1:30. In addition, there was a lesser degree of,but nonetheless obvious suppression at higher dilutions(Fig. 5). On the other hand, the guinea pig anti-Id anti-serum, which reacts with noncombining site deter-minants, did not suppress.

It was highly unlikely that the suppression seen inthe PWM-stimulated cultures, which contained sheep

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FIGuRE 2 Inhibition by various antisera of the hemagglutina-tion of SRBCby the patient's monoclonal IgM. Both the sheepantibody directed against normal IgM and the patient's IgGand guinea pig antibody directed against the patient's IgMfailed to inhibit hemagglutination of SRBCby the patient'sIgM. However, sheep antibody directed against the patient'sIgM clearly inhibited hemagglutination in a concentration-dependent fashion. *, sheep anti-IgM (patient); 0, sheep anti-IgM (normal); A, sheep anti-IgG (patient); A, guinea piganti-IgM (patient).

Idiotypic Suppression of Chronic Lymphocytic Leukemia Lymphocytes

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TABLE IIInhibitiotn by Anti-idiot ypic Sera of Hemagglutintation

of SRBCby Patient's Monioclotnal IgM

Heinagglutination inhibition titerOrigin of serum (log2)

Guinea pig preimnmunization 0Guinea pig anti-Id 0Sheep preimmunization 0Sheep anti-Id 6

anti-Id antibody, was the result of the carry-over of theanti-Id antibody from the culture to the assay system,which then blocked not the secretion of antibody butthe binding of anti-SRBC antibody to the target erythro-cytes. In control experiments in which anti-Id antibodyin a dilution of 1:30 was added to the cultures not at thebeginning of the 6-d culture but at the termination,immediately before harvesting, no suppression of PFCresponses was observed.

Determination of Id on B cells. The patient's mono-nuclear cells and those of two normals were incubatedfor 45 min at 37°C with either control media or a 1:100dilution of sheep preimmunization IgG or sheep IgGanti-Id. After incubation, cells were washed and stained

140 k

DILUTION OFANTISERUM

FIGURE 4 Effect of various antisera on the PWM-inducedanti-SRBC PFC response of normal lymphocytes. The anti-sera were added to cultures on day 0 with PWM,and anti-SRBC PFC responses were measured after 6 d of culture.Neither the pre- nor postimmunization antisera from sheep orguinea pig suppressed the anti-SRBC PFC responses. 0,sheep preimmunization; 0, sheep anti-Id; A, guinea pigpreimmunization; A, guinea pig anti-Id.

0 1:100 1:300 1:1

DILUTION OF ANT

FIGURE 3 Effect of various dilutions oftaneous (day 0) anti-SRBC PFC resp(lymphocytes. Only the sheep anti-Id suranti-SRBC PFC responses. 0, sheep Isheep anti-Id; A, guinea pig preimmunianti-Id.

with fluoresceinated goat anti-human Ig. Preincubationwith neither sheep preimmunization nor sheep IgGanti-Id blocked the subsequent staining of the normal Blymphocytes with fluoresceinated goat anti-human Ig.With the patient, preincubation with sheep preim-munization IgG did not block, but sheep anti-Id com-pletely blocked the detection of surface Ig by thefluoreseeinated goat anti-human Ig reagent.

Presence of Id on T cells. Purified T cells from the000 1:3000 1:10,000 patient and two normals were assayed for the presence

of Id by staining with the fluoresceinated sheep IgGrISERUM anti-Id. Id determinants were not detected on T cellsFanitisera on the spoIn- of either the patient or the normal.mnse of the patient's Presence of anti-Id on T cells. Purified T cells fron.)pressed spontanleouis

reimunization; . .the patient and two normals were preincubated for 30pzraetiOnm;AngZione pg min at 37°C with a 1:10 dilution of the patient's IgM,IgG, and normal IgM. Cells were then washed and

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FIGURE 5 Effect of various anitisera on the PWM-inducedatiuti-SRBC PFC response of the patient's lymphocytes. The

antisera Nwere added to cutltures on daty 0 together with P\V\I,aind anti-SRBC PFC respoonses were meastired after 6 d ofculture. ()nly the sheep anti-I(l antiserulm suippressed the anlti-SRBCPFC responses. *, shleep preiiiimntunizationi; 0, sheep

aniti-Id; A, guiniiea pig preimnmunization; A, guinea pig anti-I(l.

staine(l with fluoreseeiiaited sheel) anti-huimian Ig. Nostainiing was observed with patient or norimial T cells.Hencee, anlti-Id was not detectable on the patient'sT cells.

DISCUSSION

.Most pLatients with C(LL milaniifest a monoclonal piro-liferation of B cells (16, 17). Although surface Ig cani bedeiionstrated on the surface of a major portion of theleukeinic lymllplhocytes in patients with CLL, there isfrequently a defect in the ability of these cells to

terimiinally differentiate into Ig-secretinig cells (16-18).However, the miaturational .defect is variable amilongpatienits in that leuikemic B cells froii certain (CLLpatients lhave been demonstrated to synthesize and

secrete Ig (19). In fact, in line with the muonoclonalniatuire of the B-cell proliferation, a recenit stuidy haselegantly described a patieint wvith CLL in whiomil theIgMI onl the surface inemnbrane of the lyvmphocytes, theintracellutlar Ig.M in the differeintiate(l plasmna cells,aind the aloinocloinal IgM in the serumiii were slhown toI)e idiots pically ideintical (20).

The ('Ll, patienlt reported in the presenit stuidy pro-vid(lc a uini(quie opportunity to examiniiie the effect ofanti-Id antisera on the reguilattion of' huian B-cellfiunctioni. The tw o feattiures that allowed this approachwvere that the CLL lymiphocytes were capable ofsecreting Ig\I, aind( the Ig.M in the serumil was idio-typically identical to the suirfaice IgM on the leuikemilicB cells. More ilnportantly, the B cell surface IgNM1 andthe seruimii IgM had specificity for SRBC, milaking thespontaneous and miiitogei-indtee(d IgM secretioni easilydetectable at the single cell level by an hemicolysis-in-gel anti-SRBC( PFC assaty (10, 11). A CLL pittient whoseletikemilic B cells expressed surface Ig that reacted withSIIBC to formii rosettes has been previously reported(21). Ilowever, Ig secretioni was inot determiniie(l. In thep)resent study, not only did the patient's leukeimic Bcells spontanMeolIsly secrete anti-SRBC antibody, butthey -ouild also be iniuitced ly PW.M to differentiatefulrthier in culture to secrete the SRBC-specific antibody.Because the PXVM-induced ainti-SRBC response byhiiuman peripheral blood B lyinphocytes is a T-cell-dependenit phenoinenorl (9), one would expect that thepattienit's T cells could provide lhelper activity. As a

imatter of faet, this was the case as fuirther demoinstratedby the fact thtat this pLatient's irradiated T cells pro-vided ade(Ituate help toward PWNI-stinmulated anti-SRBCPFC responises of norimial allogeneic co-cultuires.2In this regard, we have previously reported the abilityof T cells fromil CLL patients to provide helper effectsin this systemn (22). This is somilewhat at variance withpreviouis reports of al helper T-cell defect in CLL (23).However, the latter stuidies were not employiing thePWMI-induiced anti-SRBC PFC systemi.

In the present stuidy, using ptirified serum mono-clonal IgMI with anti-SRBC specificity, we succeededin raisinig aniti-Id antibody. The anti-Id not only directlyblocked the spointaneous secretion of anti-SRBC anti-body by the leukeinic B cells, but also blocked theP%WM-induced anti-SRBC PFC responses of these cellsafter 6 d in culture. The suppression was less dramaticthani that of the spontanieotisly secreting B cells, but itw.vas nonetheless profoutnd.

The miechlianisns whereby this ainti-Id antibody sUp-pressed the secretion of aniti-SRBC antibody by thepatient's leitkemiiic B cells is unclear at present. It ispossible that the anti-Id antibody directly suppressedthe futnctioln of genes that vere differentiated, i.e., the

2 Fauci, A. S. Unipublished observations.

Idiotypic Suppression of Chrotnic Lymtphocytic Leukemia Lymphocytes

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VHand VL genes of the CLL B cells. However, there areother alternatives that have not been completely ruledout. For example, the anti-Id antibody may have beendirectly cytotoxic to the CLL cells. This is unlikely,however, because the viability of the cells was notaltered by the anti-Id antibody. Alternatively, the anti-Id antibody could have been bound to and blocked theanti-SRBC antibody in the agar after it had beensecreted. This might explain the effect of the anti-Idantibody in the experiments in which the antibody wasadded directly to the assay system in the detectionof spontaneously secreting anti-SRBC antibody on day0. However, it is highly unlikely in cultures stimulatedwith PWMand subsequently washed three times thatenough anti-Id antibody could have been carried overinto the assay system to interfere with the binding ofantibody and thus explain the suppression by thatmechanism. To directly control for this possibility,experiments were carried out in which the patient'slymphocytes were cultured for 6 d with PWMin theabsence of the sheep anti-Id antibody. At the termina-tion of culture, a 1:30 dilution of the anti-Id was addedto culture immediately before harvesting and plaquing.The cultures were then washed three times and thecells plated for plaquing. Under these circumstances,there was no inhibition of the PFC response. Thus,although it is still conceivable that there could havebeen carry-over anti-Id from the original PWM-stimulatedcultures that contained anti-Id into the assay system,the latter control experiment makes this highly unlikely.It is also unlikely that the anti-Id blocked the PWMreceptor on the CLL lymphocyte because even thespontaneous secretion of anti-SRBC antibody wasblocked by the anti-Id.

The suppression was not overcome by a powerfulstimulus such as a polyclonal B-cell activator, in thiscase, PWM.This is of interest in that other data haveindicated that tolerance induced by antigen or antibody(allotypic suppression) could be broken by polyclonalB-cell activators (24,25). It should be pointed out, giventhe report of idiotypic determinants on T cells (26), thatneither idiotypic determinants nor anti-Id activitycould be demonstrated on the surface of the patient'sT cells.

Thus, the present study has clearly demonstratedidiotypic suppression of antibody secretion by humnanleukemic B cells in vitro. Although the B cells studiedwere clearly abnormal, these findings emphasize thereal and potential role of anti-Id antibodies in theimmunoregulation of human B-cell function.

ACKNOWEDGMENTS

The authors wish to thank Ms. Gail Whalen and Ms. CynthiaBurch for expert technical assistance, and Ms. Gina Shankeland Mrs. Betty Sylvester for expert secretarial and editorialassistance.

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