Comparative Adherence of Granulocytes to EndothelialMonolayers and Nylon Fiber

ROBRoY MACGREGOR,EDWARDJ. MACARAK,and NICHOLAS A. KEFALIDES,Itnfectious Diseases Sectiotn, Department of Medicine, and the ConniiectiveTissue Research Itnstitute, University of Pennsylvania School of Aledicine,Philadelphia, Pennsylvania 19104

A B S T RA C T Adherence of grainulocytes to tissueculture monolayers of endothelium averaged 26.2±1.3% SEM, which was similar to their adherenceon 50-mg nylon fiber columns (27.7+3.6%). In con-trast, adherence to epithelial cells, fibroblasts, kidneycells, and plastic Petri dishes without monolayerswas only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclicnucleotides and adherence-modifying plasma factorsinduced chainges of adherence to endothelium similarto those in nylon fiber columns. Adherence of granulo-cytes in whole blood was the same as for purifiedgranulocytes in Hanks' balanced salt solution.

Exposure of endothelial monolayers to 0.18%trypsin for 10 min reduced subsequent granulocyteadherence to 25.2% of control values. Incubation oftrypsin-treated monolayers with nutrient medium for4 h did not improve adherence, but values returnedto normal or above by 24 h, with or without serumproteins present in the nutrient medium.

The similarity of granulocyte adherence to nylonfiber and to endothelial monolayers in vitro suggeststhat results with the nylon fiber assay reflect in vivogranulocyte-endothelium interaction. Furthermore,the endothelial monolayer offers a new model forstudying this cell-cell relationship in vitro.

INTRODUCTION

The adherence of granulocytes to vascular endo-thelium is necessary before their diapedesis into thetissues. In 1961, Garvin used columns packed withglass beads to measure granulocyte adherence (GA)'

This work was presented in part at the American Federa-tion for Clinical Research eastern meeting, Boston, Mass.,January 1977.

Received for publication 1 August 1977 and in revisedform 28 October 1977.

' Abbreiation used in this paper: GA, granulocyte ad-herence.

in vitro, and defined some characteristics of the ad-herence phenomenon (1). Recently, we confirmed hisfindings, uising a simpler in vitro adherence assaywhich utilizes sptiun nylon fiber columns and smallvolumes of heparinized whole blood (2). Clinicalstudies with the nylon fiber assay have shown ad-herence to be increased in conditions of granulocytemargination and rapid egress from the intravascutlarcompartment (3, 4), and to be decreased in conditionsof poor granulocyte delivery such as during treatmentwith steroids or other anti-inflammatory agents (2, 5).Thus, GA values obtained from the nylon fiber assayappear to be directly related to the ease of cell move-ment out of the vascular compartment.

Despite these cliinical correlations, questions remainregarding the appropriateness of applying data de-rived from granulocyte adherence to an inert nyloinfiber surface in vitro to the phenomenon of granulo-cyte-endothelial cell interaction in vivo. The develop-ment of effective techniques for culturing endothelialcells (6-9) now offers an opportunity to study the ad-herence of granulocytes to endothelial monolayersin vitro, and to compare adherence characteristics onmonolayers with those in nylon fiber columns.

METHODSThe nylon fiber assay for GAhas been described in detail inan earlier publication (2). Carefully weighed amounts ofspun nylon fiber are packed into Pasteur pipettes to a columnlength of exactly 15 mm. 1-ml volumes of heparinizedwhole blood are applied to the top of triplicate columnsand allowed to flow through by gravity at 37°C. Comparisonof the pre- and postcolumn granulocyte counts permitscalculation of the percent of granulocytes adhering to thecolumn. Results of the triplicate columns are averaged foreach blood specimen and expressed as the percent granulo-cyte adherence. Values for the triplicate columns fall within15%±+ the mean value. Because the percentage of adherencefound on the endothelial monolayers was low, columnspacked with 50 mgof fiber, which result in a mean adherenceof 27.7+3.6% in normals, were used in all experimentscomparing adherence on endothelium to that on nylon.

The Journal of Clinical Investigation Volume 61 March 1978 - 697-702 697

All experiments were repeated a minimum of three timesand the meani values were compared by the Student's t test.All cell counits were performed with an electroniic particlecounter.

Cultures of human endothelial cells were establishedfrom humani umbilical cord veins. Umbilical cords wereobtained soon after delivery and placed in Medium 199(Kansas City Biological, Lenexa, Kans.). Umbilical veinendothelial cells were isolated using the method of Gim-brone et al. (8) and grown in Medium 199 as modified by Lewiset al. (6). The Medium 199 was supplenmented with 20% fetalbovine serum (Rehatuin, Reheis Chemical Co., BerkeleyHeights, N. J.) and antibiotics (gentamicin, 50 ,g/ml andamphotericin B, 2.5 ug/ml). All endothelial cells used inthis study were primary moniolayers, 6-9 days of age, andthree to five generations after isolation. All monolayers wereconfluent. In addition, Petri dishes containing monolayers ofhuman lung fibroblasts (WI-38), human epithelium (HEP-2),and rabbit kidney cells (RK-13), grown until confluent inEagle's minimal essential medium supplemented with 7%fetal calf serum, were supplied by Dr. Harvey Friedman, ViralDiagnostic Laboratory, Children's Hospital of Philadelphia.Total cell protein content was measured for each cell mono-layer by the Lowry technique, to determine whether cell con-centration was roughly analogous for each cell type.

In most experiments, heparinized (5 U/nl) venous wholeblood (leukocyte counts 5-1(,(0((/mm3) from normal donorswas used to measure GA. Medium 199 was decanted fromthe tissue cultures and 1 ml of blood was added to triplicateplates, just covering the 35-mm diameter surfaces. Thetissue culture-blood overlay wats incubated at 37°C and 100%humidity for 15 min without agitation, and then the bloodwas aspirated. Comparison of the granulocyte counts be-fore and after incubation allowed calculation of the percentageof granulocytes adhering to the monolayer. In some experi-ments, a pure granulocyte suspension was prepared byFicoll-Hypa(lue (Ficoll, Pharmacia Fine Chemicals Inc.,Hypaque, Winthrop Laboratories) density gradient sedi-mentation using the technique of Boyum (10). After separa-tion, the granulocytes were washed three times in modifiedHanks' solution and suspended in Hanks' balanced salt solu-tion at a concentration of 5-10 x 106 cells/nl. The suspensionwas layered over the monolayer, incubated, and the percentGAcalculated as above. In experiments evaluating the effectof adherence-modifying plasma factors, heparinized wholeblood was centrifuged at 80 g for 10 min at 40C, the cellbutton was washed three times with modified Hanks' solu-tion, and then resuspended volume for volumiie in eithernormal ABO-compatible plasma or in plasma containing thefactor being studied. Previous studies have shown thatnormal cells can be separated and resuspended in thismanner without affecting their adherence (3, 5). Plasma con-taining the factor which augments granulocyte adherence (3)was obtained from patients with acute inflammatory diseases.Plasma containing the factor which inhibits adherence (5)was obtained from normal volunteers 4 h after the ingestionof 40 mg prednisone. All plasma was stored at -70°C untilused.

Dibutryl cyclic AMP and 8-bromo cyclic GMP(SigmaChemical Co., St. Louis, Mo.) were prepared in 0.001 mMstock solutions in normal saline and used within 1 h ofpreparation.

In experiments evaluating the effect of trypsin treat-ment of the endothelial monolayers on GA, a 2.5% stocksolution of trypsin (Grand Island Biological Co., GrandIsland, N. Y.) was prepared in Earle's balanced salt solutionand kept at -20°C until used. At the time of experiment, thenutrient medium was decanted from the endothelial mono-

layers and trypsin1 sol1ution1 was added in concentrationsfrom 0.06 to 0.18%. Then the monolayers were incubatedfor 10 min at 37°C, and the trypsin inhibitor (AldrichChemical Co. Iic., Milwaukee, Wis.), in Earle's balancedsalt solution, was added to the monolayer and inciubatedfor 30 min. After removal of the trypsin inhibitor, themonolayers were covered with modified medium 199 plus20% fetal calf serumil anid iincubiated at 37°C until time for thedeterminatioin of granutilocyte adhereniee (immediately aftertrypsinization, 4, or 24 h later). In somile experimenits, thefetal calf serum was omitted to assess its importance to themonolayer's regenerationi of normiial granutilocyte adherenceproperties.

RESULTS

The mean adherenice of granulocytes in hepariniizedwhole blood to humian endothelial monolayers was26.2+1.3% SEMin 10 experimenits, compared to ad-herence of 4.3±0.6% in Petri dishes not containing amonolayer (Fig. 1). Adherence oni triplicate mono-layers varied <15%+ the mean value. In contrast togranulocytes, few lymphocytes adhered to the monio-layer.

To determine if the adherenice results simply repre-sented settling of the cells, other monolayers wereoverlayered with blood and either incubated withoutmotion, or gently agitated every 30 s for 15 min. Thestationary monolayers had 20.1±0.8% granulocyteadherence, and the agitated monolayers had 25.7±3.0%.

Granulocyte adherence to other cell types in tissueculture was greater than to the empty Petri dishesthough less than to the endothelial monolayer: 12.4

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FIGURE 1 Granulocyte adherence to different types of cellmonolayers and to nylon fiber columns. Isobars representmean values for a minimum of three experiments, brackets±+SEM. Adherence to epithelium, fibroblasts, and kidneycells was greater than to empty Petri dishes, but less thanto endothelium or nylon fiber (P < 0.05, Student's T test).

698 R. R. MacGregor, E. J. Macarak, and N. A. Kefalides

TT I

I

± 1.3% on human epithelial cells, 9.9±1.0% on humanlung fibroblasts, and 11.1±+1.1% on rabbit kidneycells. Adherence to the other cell types was signifi-cantly less than adherence to endothelium in eachcase (P < 0.05, Student's t test). Total protein contentfor each monolayer was 127±28.3 ,tg/ml SEM forendothelium, 304±10.6 for epithelium, 336±69.7 forfibroblasts, and 551+79.8 for rabbit kidnev. Thus, thegreater adherence to endothelial monolayers does notseem to result from more cell surface area present withthis cell type than with the other monolayers. Thefact that endothelium had the lowest protein content ofthe four cell types probably results from the very flatprofile and thus less cytoplasm for these cells com-pared to fibroblasts or kidney cells. When a purepreparation of granulocytes was suspended in Hanks'balanced salt solution, a similar differential adherencewas noted between different cell types, whereas meanadherence to endothelium was 46.4+7.8% SEM,adherence to epithelium was 17.6+1.4%, to fibroblasts21.6±3.2% and to rabbit kidney cells 17.1±3.8%.Adherence to nylon columns was 42.7±3.1%. Ad-herence of the pure granulocyte suspension wassignificantly greater to endothelium than to anyof the other cell types (P < 0.05, Student's t test).Because adherence values on the endothelial mono-layers were very close to those reported previously(2) for normal blood on 50-mg ("low adherence")nylon columns (Fig. 1), we used such columns in thesubsequent experiments comparing granulocyte ad-herence on endothelial monolayers with that on nylonfiber columns.

To determine whether granulocytes which fail toadhere the first time are capable of adherence if re-incubated with a fresh monolayer, experiments wereperformed in which the same aliquot of whole bloodwas incubated sequentially onto two different endo-thelial monolayers. Mean adherence to the firstmonolayers was 20.1±0.8% SEM, and to the secondmonolayers was 17.5±3.4%. Thus, adherence appearsto be a random phenomenon rather than represent-ing a subgroup capable of adherence within the totalpopulation of granulocytes.

Fig. 2 illustrates comparative granulocyte adherenceto the two surfaces under conditions previously shownto modify adherence to nylon fiber. Plasma frompatients with acute inflammation, which contains anadherence-augmenting factor, caused a significant in-crease in granulocyte adherence measured with eithersurface: from 26.2±1.3 to 47.4±4.1% on endothelium,and from 27.7±3.6 to 54.2±4.7% on nylon (P < 0.05for each, Student's t test). Plasma from patients treatedwith prednisone, containing the factor which inhibitsadherence, decreased the granulocyte adherence toboth surfaces: 20.5±1.9 and 19.6±1.4%, respectively.Addition of 0.01 mMcyclic GMPor cyclic AMP to

50- NYLONFIBERw COLUMNSw

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NORMAL AGA IGA 0.01 mM 0.01 mMPLASMA PLASMA PLASMA cGMP cAMP

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FIGURE 2 Adherence of granulocytes suspended in variousmedia to endothelium and nylon fiber. Isol)ars representmean values for a minimum of three experimiienits, brackets±SEM. AGA factor plasma is from patients with auig-mented grantulocyte adherence associated with inflammiation,and IGA factor plasma is from patients wvith inhibitedgranulocyte adherence after prednisonie admiinlistrationi.Cyclic nucleotides were added to normal plasma in the lasttwo experiments. On both surfaces, adherence of graniulo-cytes suspended in normal plasma diflered significantly fromadherence in the other media (P < 0.05, Studenit's t test).

heparinized whole blood, respectively known toincrease and decrease adhereinee to nylon (11), inducedsimilar changes on both surfaces (significantly differentfrom normal, P < 0.05, Student's t test, but not sig-nificantly different between the two suirfaces). Addi-tion of 0.01 ,utM propranolol to whole blood raised GAto 38.9±1.7% on endothelium, and 34.9±1.0% onnylon (data not shown); 0.01 ,uM atropine reducedadherence to 9.8+3.2% or endothelium, and 9.1± 1.6% on nylon. All changes were significantlydifferent from normal, P< 0.05, Student's t test.

To determine whether GA to endothelial mono-layers requires other 1)lood cell types, platelets, orplasma proteins, a pure suspension of 5-10 x 1(6granulocytes/ml in Hanks' balanced salt solutioni wasprepared by Hypaque-Ficoll density sedimiientation.No erythrocytes remained. The adherence of thissuspension was measured on the endothelial mono-layer with and without the addition of 0.01 mnMcAMPor cGCIP. Adherence of the pure granulocytes inHanks' balanced salt soluitioni averaged 47.9+2.7%SEMI for three experimenits. Addition of 0.01 mMcAMPreduced adherence to 23.9±5.6%, whereas cGCIPincreased it to 69.6+5.5%; )oth changes were signiifi-cantly different from normnal (P < 0.05, Student's ttest). In contrast, suspenidinig the purified granulocvtesin normal plasma led to the same level of adhereniceseen when the cells were in Hanks' balaniced salt solu-tion, 46.5±4.7%. Thus, plasma proteiins are inot re-

Graniulocyte Adherence to Entdothelial Monolayer.s and Nyloin Fiber 699

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FIGURE 3 Effect of trypsin treatment of the endothelial monolayers on subsequent granulo-cyte adherence. (A) Isobars represent mean values for a minimum of three experiments, brackets+SEM. Significant inhibition of adherence occurred only after exposure of the monolayers to0.18% trypsin (P < 0.001, Student's t test). (B) 0.02% increments were used between 0.12 and0.18%; significanit inhibition occurred only with 0.16% (P < 0.02) an'1 with 0.18% trypsin(P < 0.001, Student's t test).

quired for granulocyte adherence to endothelial cells,nor are other cell types necessary either for normaladherence or for the modification of adherence bycyclic nucleotides.

The intensity of granulocyte adherence to trypsi-nized endothelial monolayers is shown in Fig. 3.No significant change in adherence was noted untilthe trypsin concentration was raised to 0.18%. After a10-min exposure to this concentration, the mono-layer could only support a mean granulocyte adherenceof 6.2+1.3%, as compared to 24.6+1.4% with the un-trypsinized cells (P < 0.001, Student's t test). The via-bility of monolayer cells after exposure to 0.18% trypsinwas 96.1% by trypan blue exclusion, and microscopicexamination demonstrated that the monolayer re-mained confluent and appeared normal. To define thecritical concentration of trypsin necessary to inhibitGA to the monolayer, the studies were repeated, with0.02% increments in trypsin concenitration between0.12 and 0.18% (Fig. 3). Only the adherence after0.16 and 0.18% exposures was significantly less thanon uintreated monolayers (P < 0.02 and 0.001 Student'st test); changes induced by lower concentrationswere not significant.

The reversibility of the trypsin effect on GAis shownin Fig. 4. Fig. 4A first shows that incubating theendothelial monolayer with soybean trypsin inhibitoralone has no effect on subse(luent GA. Second, treat-ing the monolayers for 10 min with 0.18% trypsin,followed by a 30-min incubation with trypsin inhibitor,led to a significant decrease in subseqluent GA (P

< 0.05, Student's t test). Finally, overlaying thetrypsinized monolayers with the standard nutrientmedium for 4 h did not improve subsequent GA, butincubation for 24 h after trypsinization completely

A Bwz0 30-

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CONTROL/+TRYPSIN 0 4 24- CONTROL 0 4 24INHIBITOR Hours After Hours After

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SERUM IN NUTRIENT MEDIUM SERUM-FREENUTRIENT MEDIUM

FIGURE 4 Reversibility of trypsin-induced inhibition of'granulocyte adherence to endothelial monolayers. Isobarsrepresent the mean of at least three experiments, brackets+SEM. In the experiments displayed in A, 20% fetal calfserum was present in the nutrient medium covering themonolayers after their exposure to 0.18% trypsin; in B experi-ments, serum was omitted. Adherence was still low 4 h aftertrypsinization but returned to normal or above normal underboth conditions.

700 R. R. MacGregor, E. J. Macarak, and N. A. Kefalides

corrected the inhibited adherence. Although the ad-herence value after 24 h was well above control, thedifference was not significant (P < 0.1, >0.05, Student'st test). The experiments were repeated with 10% fetalcalf serum excluded from the post-trypsinizationnutient medium Fig. 4B. Correction of the impaired GAwas complete after the 24-h incubation; thus serumproteins are not req(uired in the nutrient medium fortrypsinized endothelial monolayers to regenerate theproperties necessary for normal GA.

DISCUSSION

The developmiient of techniques for establishing invitro cultures of huminan endothelial cells (6-8) hasallowed the study of many aspects of endothelialfuncetion inaccessible up to now. Several charac-teristics documilenit that the monolavers are composedof endotheliumiii: first, the tissue culture morphologyshows plate-like cells arranged in a tight nmonolayer,without any overgrowth. In contrast, fibroblasts appearas spindle-shaped cells, and smiooth muscle cells over-grow one aniother, giving a clumped appearanice tothe cultur-e (12). Second, the presence of rod-shapedorganelles called WA'eibel-Palade bodies is uni(que toendotheliumii (12). Third, immunofluorescence micros-copy (lemonostrates that antihemnophilic factor is presentin the cells of the endothelial monolayer, in contrast,to fibroblasts and smooth muscle cells (13). These threecharacteristics were demonstrated by the monolayersuised in the present studies.

The greater adherence of granulocytes to monolayersof all cell types tested than to the empty plastic Petridishes is consistent with the well-known attribute ofmetazoan cells to adhere (14). However, adherence tothe endothelium was more than twice as great as to theother cell linies, suggesting a specific adaptation be-tweeni granulocvtes anid endothelial cells for attach-mllent. Sulch ain adaptation or receptor would facilitategranulocyte delivery, because they mulst attaclh to theeiclothelial sturf'ace before diapedesis into the tis-sties (15).

The pLarallelismi of adherence values fouind between50-nmg coltiimniis aind the enidothelial monolavers wvasstrikiing. In everv situsation where adhereniee to the iv-lon fiber coltumnis hal(l been previously reported to bealterecl, similar chaniges were noted with the endo-thelial nonolavers. Although conicerni has been ex-pressed regardinig the extrapolationi of data derivedfromi atcllherenice to nylonl fiber to the more physiologicalevenit of granulocvte adherenice to endotheliumil,presenit stuidies indicate that the adherenice charac-teristics of the two sturf'aces are similar. Thus, adher-enice miieastured with the in v itro nyvlon fiber assayappears to rieflect il vivo graniutlocyte-endotlheliuminiteraictionis. All of the previouis sttudies u-tilizinig the

nylon fiber assay (2-5) have supported the conceptthat the intensity of adherence, measured with thenylon fiber columns, affects the ease of movement ofgranulocytes out of the vascular compartment; thepresent study directly confirms the concept thatchanges in adherence, observed with the nylon fiberassay, reflect adherence to endothelium.

When separated granulocytes are suspended in amedium devoid of plasma proteins, the adherence toendothelium and nylon remains similar; moreover,changes in adherence induced by cyclic nucleotidesand pharmacologic agents are similar for both surfaces,and are equal when whole blood is used. Conse-quentlv, adherence to endothelium does not requireplasma proteinis or the preseince of any other celltype, and modifications of adherence do not occurthrough the mediation of other cells. The higher degreeof adherence that is noted when purified granulocytesare used in place of whole blood, may result from theexposure of cells to HNpaque-Ficoll, or the time re-quired to process the cells.

Try psinization has been used to remove protein-containing components from cell membranes ofmany differenit cell types without killing them (16-18).Such treatment has been shown to remove as much as15% of the dry weight of the cell (16), and 39% of itssialic acid content (17), while leaving 100% of the cellsimpermeable to trypan blue. In the present studies95% of cells remained viable by trypan blue exclusion,and phase-contrast examination of the monolayershoved no visible abnormality after a 10-min exposureto various concentrations of trypsin. However, con-centrations of 0.16% and greater caused major reduc-tion of granulocvte adherence to the monolayers, sug-gesting that endothelium has a protein or glycoproteinsul)stance on its membrane which promotes the attach-ment of grantulocv tes. This substance could be aspecific receptor or perhaps a nonspecialized mem-l)rane componienit containiing a charged moiety, suchas sialic acid. The return of adherenice to normalor increased levels bv 24 h after trypsinization sug-gests that the substance is replaceable by the cell,even in the absence of exogenious serumii proteins.

An alternative explaniationi for the actioin of trypsinoIn adherence is found in a receint report by Revel et al.(19) showing that trvpsinization causes the Imlonlo-layer cells to become round and lose their flattenedappearance. A similar effect oni endothelial mono-layers would present ac more difficult surfatce coinfigura-tion for adherence of graniulocv-tes, even though themoniolaver remiiainied intact and of normal appearanceas seeni using phase-cointrast microscopy. Thereafter,the returni to norimial adherence chalracteristies couldreflect a returni of the cells to a flattened configurationirather thlan the regenerationi of a surface componienit.Scanninig electroni microscopic study of the monolayer

Gran uilocilte Adhierenice to Endothelial Mlonolaiyers andcl Nilloon Fiber 701

before and after trypsinization is necessary to evaluatethese possibilities.

The availability of endothelial cells grown in mono-layers now permits in vitro studies of the characteristicsof granulocyte adherence to endothelium, and appearsto represent an in vitro analog of granulocyte margina-tion before diapedesis. The close correlation of adher-ence results on endothelial monolayers to those withnylon fiber columns, suggests that experiments usingthe nylon fiber assay system for granulocyte adher-ence are indicative of granulocyte-endothelium inter-actions in vitro; this cell-cell interaction in vitromay reflect events in the intravascular compartment.

ACKNOWLEDGMENTS

The authors thank Dr. Jaffe for his suggestions, the excellenttechnical assistance of Mrs. Elvira Ventura, Ms. PamelaHoward, Missy Cooper, and Terry Kirk, as well as Mrs.Kathleen Ludden's secretarial aid. Dr. Elias Abrutyn madehelpful editorial suggestions.

This work was supported by U. S. Public Health ServiceGrants AA-00332, AM-14526, HL-15061, and HL-18827.

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16. Codington, J. F., B. H. Sanford, and R. W. Jeanloz.1970. Glycoprotein coat of the TA3 cell. I. Removal ofcarbohydrate and protein material from viable cells.

J. Natl. Cancer Itnst. 45: 637-647.17. Snow, C., and A. Allen. 1970. The release of radioactive

nucleic acids and mucoproteins by trypsinized andEDTA-treatnment of baby hamiister cells in tissue eulture.Biochem J. 119: 707-714.

18. Hughes, R. C. 1976. Membrane glycoproteins. In AReview of Structure and function. Butterworth & Co.(Publishers) Ltd., London. 43-44.

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702 R. R. MacGregor, E. J. Macarak, and N. A. Kefalides