tions and sterile inflammatory lesions suffer from majordrawbacks. The low binding affinity of 99mTc..ciprofloxacin(4) to bacteria and the risk of emerging antibiotic-resistantmicroorganisms make this radiopharmaceutical unattractive

for imaging bacterial infections (5). The specificity ofradiolabeled monoclonal antibodies or their F(ab')2 fragments can be expected to be useful for the detection ofinfections (6). However, Rubin et al. have reported thatinfection detection using F(ab')2 fragments and nonspecificpolyclonal immunoglobulin G (IgG) is similar. Clearly, aradiopharmaceutical that binds to a variety of microorganisms with little or no binding to host cells would be better.

Because antimicrobial peptides preferentially bind tobacterial membranes (7), radiolabeling of these peptideswould offer the medical community novel candidates for thedevelopment ofbactena-seeking radiopharmaceuticals (8,9).One of the best-studied antimicrobial peptides is humanneutrophil peptide (HNP)-l, which is a member of thefamily of defensins (10,11). This antimicrobial peptide,

which is stored in the granules of human neutrophils,contributes to bacterial killing during phagocytosis. HNP-1displays antimicrobial activity against gram-positive andgram-negative bacteria, many fungi and some envelopedviruses (12—14).Defensins kill bacteria by a mechanisminvolving electrostatic interactions between the positivelycharged antimicrobial peptide and the negatively chargedbacterial surface molecules. The bacterial phospholipidmembranes then become permeable, resulting in metabolicinjury (11). Therefore, one can imagine that small amountsof radiolabeled defensins may be seen at sites of bacterialinfections. Initially, the potential use of HNP-l for antibacterial therapy of experimental infections in mice was studied(8). In that study, a single injection of HNP-l reduced thebacterial numbers significantly. This antibacterial effect wasfound to be associated with an increased influx of neutrophils into the infected area, a process that appeared to be arequirement for HNP-l to have antibacterial activity in vivo.Radiolabeled HNP-l was found to be trapped in the infectedarea, whereas unbound HNP- 1 was rapidly removed fromthe circulation through renal excretion. Furthermore, it isbelieved that defensin-resistant bacteria may not emergeeasily, because during evolution these peptides have beensuccessful in protecting plants, animals and humans againstthe continuous threat of pathogens (7).

Thisstudywasundertakento evaluatewhether @Tc-labeIedhuman neutrophil peptide (HNP)-1 can be used as a tracer forrapidvisualizationof bacterialinfections.Methods:Micewereinjected intramuscularly with 1 million Staphylococcus aureus orKiebsiellapneumoniaeorganismsand 5 mm later were injectedintravenouslywith0.4 pg (0.8 MBq) @Tc-HNP-1. At variousintervals, detailed information about clearance and accumulationof this tracer at sites of infection and in various organs wasobtainedby scintigraphy. @Tc-IabeIedimmunoglobulinG (IgG),an established marker of infection and inflammation,was usedfor comparison. Results: After injection into S. aureus—or K.pneumoniae—injected mice, @Tc-HNP-1was rapidly removedfromthecirculation,mainlythroughthekidneysandbladder,withhalf-lives of 170 and 55 mm, respectively. Similar half-lives wereobservedfor @‘Tc-IgGin theseanimals.Visualizationoffoci withS. aureus or K. pneumoniae, as indicated by a ratio of 1.3 orhigher between the targeted thigh muscle (containing bacteria)and the nontargeted(contralateral)thigh muscle(T/NT),wasalreadyachieved5 mmafterinjectionof 9@Tc-HNP-1.SimilarT/NTs for @mTc-IgGwere obtained 4 h after injection ofthe tracer,indicating that imaging of foci of bacteria with @Tc-HNP-1ismuchfaster thanwith @Tc-lgG.Toobtain insightintofactors thatcontribute to accumulationof @“Tc-HNP-1at sites of infection,the bindingof this tracerto bacteriaand leukocyteswas assessedusinga peritonealinfectionmodel.Bindingof @mTcHNP-1 to bacteria was approximately 1000 times higher thanbinding to leukocytes. Although the number of bacteria in thepentoneumwas1000-foldlowerthanthenumberofleukocytes,a significant correlation between binding of @Tc-HNP-1tobactena on the one hand and accumulation oftracer on the otherwas still found, in contrastto @Tc-lgG.Conclusion: @TcHNP-1 allows rapid visualizationof bacterial infections. Bindingofthistracertobacteriamostlikelycontributessignificantlytotheaccumulationof 99mTc..HNp1at sites of infection.

Key Words: infectionimaging;bacteria; @Tclabeling;humanneutrophilpeptide-1J NucI Med 1999;40:2073—2080

uclear medicine techniques enable visualization ofinfection and inflammation (1—3).However, the presentradiopharmaceuticals that can distinguish between infec

ReceivedOct.25,1998;revisionacceptedApr.19,1999.Forcorrespondencecontact:ErnestK.J.Pauwels,DepartmentofRadiology,

DivisionofNuclearMedicine,C4-Q,LeldenUniversityMedicalCenter(LUMC),P.O. Box 9600, 2300 RC Leiden, The Netherlands.

ANTIBACTERIAL PEPTIDE FOR DETECTING INFECTIONS Welling et al. 2073

Imaging of Bacterial Infections with99mTcLabeled Human Neutrophil Peptide- 1

Mick M. Welling, Peter H. Nibbering, Akke Paulusma-Annema, Pieter S. Hiemstra, Ernest K.J. Pauwels and Wim Calame

Department ofRadiology, Division ofNuclear Medicine; Departments oflnftctious Diseases and Pulmonology,Leiden University Medical Center, Leiden; and Hercules European Research Center Barneveld, The Netherlands

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On the basis of these considerations, the present scintigraphic study was undertaken to investigate whether 99mTc@labeled HNP-l can be used for rapid imaging of bacterialinfections. Moreover, factors contributing to the accumulation of @Tc-HNP-lat sites of infection were addressed. Weinvestigated two models. The first was a bolus bacterialinjection 5 mm after which the contribution of infiltratingleukocytes was negligible; the second was a full-blownperitoneal infection characterized by proliferating bacteriaand recently immigrated leukocytes.

MATERIALS AND METHODS

ProteinsHNP-l was purified from human neutrophils as described (15).

Briefly, neutrophils were isolated from enriched leukocyte preparations obtained from a blood bank (Leiden University MedicalCenter, Leiden, The Netherlands). Next, purified neutrophils weredisrupted by nitrogen cavitation, cellular debris was removed bycentrifugation at 540g for 10 mm at 4°C and the resultingsupernatant was subsequently centrifuged at 27,000g for 20 mm at4°Cto sedimentgranules.Granuleproteinswereextractedfromthis fraction using 5% (vol/vol) acetic acid and fractionated by gelfiltration on a 2.5 x 100 cm Sephacryl 5-200 H column (Pharmacia, Woerden, The Netherlands) in 5% (vol/vol) acetic acid. Thefractions containing HNP-l were further purified by reverse-phasehigh-performance liquid chromatography on a 4.6 X 250 mm C18column (Vydac; The Separations Group, Hespena, CA) using alinear water-acetonitrile gradient that contained 0. 1% trifluoroacetic acid as an ion-pairing agent. The purity of the HNP-1preparation was assessed by sodium dodecylsulfate—polyacrylamide gel electrophoresis (PAGE), acid urea—PAGEand laserdesorption mass spectrometry. The lipopolysaccharide content of apreparation of 100 @igHNP-l was less than 100 pg, as assessed bythe Limulus assay (Chromogenix, Mölndal, Sweden). Purified,pyrogen-free polyclonal human IgG was obtained from the centrallaboratory of the Red Cross Blood Transfusion Service (Amsterdam, The Netherlands).

Labeling Procedure and Quality ControlHNP-l and IgG were labeled with @Tcas described (16).

Briefly, 10 IlL of a solution of 1 mg HNP-1 per milliliter ofl0-mmol/L sodium phosphate buffer (Na-PB), pH 7.4, was addedto 2 jiL of an aseptic solution containing 0.5 mg stannouspyrophosphate per milliliter. Immediately, 4 pL of a solutioncontaining 10 mg crystalline KBH@(Sigma Chemical Co., St.Louis, MO) per milliliter of 0.l-mol/L NaOH was added. After theaddition of 0.1 mL 200-MBq/mL @‘@Tc—sodiumpertechnetatesolution (Mallinckrodt Medical BV, Petten, The Netherlands), themixture was gently stirred for 30 mm at room temperature.

To remove excess reactants (including free @“Tc),the mixturewas applied to a Sep-Pak C18 cartridge (Waters, Milford, MA)previously flushed with 20 mL 0.0l-mol/L acetic acid (pH 4.0).After being rinsed with 10 mL acetic acid, @°‘Tc-HNP-lwas elutedwith 2 mL methanol (Sigma), and then the methanol was evaporated using hot air. IgG, used as the control tracer, was radiolabeledin an identical fashion without further purification. @“Tc-HNP-land @Tc-IgGwere resuspended in 100 pL Na-PB, and the peptidecontent was determined by a bicinchoninic acid kit (Pierce,Oud-Beijerland, The Netherlands). The recovery of HNP-1 afterSep-Pak C18 chromatography was 96%. Labeling yield was as

sessed by instant thin-layerchromatographyand was 95% for HNP-land 96% for IgG. The stability of @Tc-HNP-land @mTc@IgGwastested by placing them in 20% human serum (vol/vol) for 4 h at37°C,followedby20%trichloroaceticacidprecipitation.

BacteriaStaphylococcus aureus 25923 and Klebsiella pneumoniae 43816

(American Type Culture Collection, Rockville, MD) were preparedin a brain-heart infusion broth (Oxoid Ltd., Basingstoke, UK) keptovernight in a shaking water bath at 37°C.The resulting cultures,containing approximately 1 billion viable bacteria per milliliter,were stored at —70°C.Immediately before the experiment, analiquot of this suspension was rapidly thawed in a water bath at37°Cand diluted in pyrogen-free saline.

Assay for Antibacterial ActivityAn in vitro assay was used to determine whether the labeling

procedure affected the antibacterial activity of HNP-l. Briefly,0.1 mL Na-PB containing various concentrations of HNP-l or

@“@Tc-HNP-1was added to 0.1 mL Na-PB supplemented with 2%(vol/vol) tryptic soybean broth (TSB) (Difco Laboratories, Detroit,MI)and200,000 K.pneumoniaeorganisms.At theonset andafter2and 3 h of incubation at 37°Cin a shaking water bath, the numberof viable bacteria in the samples was determined microbiologicallyusing diagnostic-sensitivity-test agar (DST) plates (Oxoid). As acontrol, Na-PB supplemented with 2% TSB instead of HNP-l wasused.

MiceSpecific, pathogen-free male Swiss mice weighing 20—25g

(Broekman Institute, Someren, The Netherlands) were used throughout the study. The mice were housed in the central animal housingfacilities of the Leiden University Medical Center for at least 1week before the experiments began. Food and water were given adlibitum.

Experimental Infections and Injectionof Radlopharmaceuticals

The animal studies complied with the ethical committee of theLeiden University Medical Center and with national laws relatingto the conduct of animal experiments.

Thigh Muscle Model. Mice were anesthetized with a singleintraperitoneal injection of 0.1 mL saline containing 1 mg fluanisone and 0.03 mg fentanyl citrate (Hypnorm; Janssen Pharmaceutics, Tilburg, The Netherlands). Immediately afterward, a bolus ofapproximately 1 million colony-forming units (CFUs) S. aureus orK. pneumoniae in 0.1 mL saline was injected into the right thighmuscle (17,18). Five minutes later, 0.2 mL saline containing either0.4 jig @‘@Tc-HNP-1(0.58 jimol/L, amounting to 0.8 MBq) or 10jig

@Tc-IgG(0.33 pmol/L, amounting to 0.8 MBq) was injectedintravenously. This technique was termed “injection.―In additional studies, scintigraphy was performed with a nonmicrobicidalsynthetic peptide (KVAKQEKKKKKT), i.e., a residue of ubiquicidin, a new antimicrobial peptide (19). The mice were then killed atintervals by intraperitoneal injection of 0.25 mL saline containing12 mg sodium pentobarbital, and the number of viable bacteria pergram of thigh muscle was determined microbiologically. Someexperiments were performed on mice that were injected with thetracer 18 h after pentoneal infection with S. aureus. This techniquewas termed “infection.“The infected thigh muscles were removedand, after weighing, homogenized in 4 mL phosphate-bufferedsaline (PBS), pH 7.4. Appropriate dilutions of the homogenatewere plated onto DST plates (Oxoid), and the number of colonies

2074 THE JOURNALOF NUCLEARMEDICINE•Vol. 40 •No. 12 •December 1999

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was counted after overnight incubation at 37°C.The results wereexpressed as the number of CFUs per gram of infected tissue. Allcultures with negative findings were assigned the value 100CFU/mL, the lower limit of detection.

Peritoneum Model. A peritoneal K. pneumoniae infection modelwas used to quantify the binding of tracers to both bacteria andleukocytes from the site of infection. In short, 0.1 mL salinecontaining approximately 1 million CFUs K. pneumoniae wasinjected into the peritoneal cavity of anesthetized mice (8,20), and5 mmlater,0.2 mLsalinecontaining0.4 jig @“Fc-HNP-lor 10pg

@9'c-IgGwas injected intravenously as a control. The mice werethen killed at intervals by an intraperitoneal injection of sodiumpentobarbital, and the peritoneal cavity was lavaged by intraperitoneal administration of 4 mL ice-cold PBS supplemented withheparin (50 IUImL; Leo Pharmaceutical Products BV, Weesp, TheNetherlands). The abdomen was gently shaken for 60 s, after whichthe peritoneal leukocytes and bacteria were harvested and centrifuged for 5 mm at llOg at 4°C.The radioactivity and the number ofcells in the pellet, i.e., the leukocytes, were counted. Subsequently,the supernatant, containing the bacteria and the unbound @mTc@peptide, was centrifuged for 5 mm at 2000g at 4°C.The radioactivity in the pellet, i.e., of the bacteria, was counted, and the number ofCFUs was determined as described. The remaining supernatant,containing the unbound peptide, was also counted for radioactivity.

For analysis of the relationship between the binding of tracers tothe various populations of leukocytes and the accumulation oftracers in the peritoneal cavity, monocytes, lymphocytes andgranulocytes were purified from the leukocyte fraction. In short,leukocytes in the pellet were resuspended and then adjusted aboveisotonic Percoll (Sigma) with three layers having density grades of1.070, 1.087 and 1.100 (55%, 70% and 81 %, respectively) andcentrifuged at 1500g for 5 mm at 4°C.Next, the various fractionswere collected, and the radioactivity associated with the cells wasdetermined (21,22).

ScintigraphyThe pharmacokinetics of @‘Tc-HNP-l,or of @‘@Tc-IgGas a

control, in S. aureus— or K. pneumoniae—injected mice wereassessed using planar scintigraphy. Before the scintigraphy, asubcutaneous injection of 0.1 mL saline containing 0.2 mgdiazepam (Valium; Hoffmann-La Roche, Mijdrecht, The Netherlands) was administered. Then the mice were placed supine on asingle-head, planar gamma camera (GCA 7100/UI; Toshiba, Tokyo, Japan) equipped with a low-energy general-purpose parallelhole collimator. Both hind legs of each mouse were spread out andfixed with surgical tape, and whole-body images were acquiredevery 60 5 during the first hour after tracer injection. Four hoursafter tracer injection, a 5-mm image was acquired. The camera wasconnected to a computer (GMS 5500/UI; Toshiba), and highresolution images were obtained in a 256 X 256 matrix. The energypeak was set at 140 keV, with a 20% window.

On the scintigraphic images, anatomically rendered regions ofinterest (ROIs) were drawn over the heart, liver, kidneys, bladder,stomach, lungs and both thighs to provide data about the uptake of

@‘@Tc-HNP-l.An ROt of 5 X 5 pixels was drawn over the focus ofinfection in the right thigh muscle (targeted). The region wascopied to the same location of the contralateral thigh muscle(nontargeted), and after the radioactivity in both regions wascounted, the targeted-to-nontargeted ratio (TINT) was calculated(21). Accumulation of@―Tc-labeledtracers at sites of infection wasexpressed as the ratio of the counts in the ROl drawn over the

targeted and nontargeted thighs ofeach mouse. Clearance of tracersfrom the circulation was assessed by determining their half-life inROIs drawn over the heart at various intervals. The radioactivity inthe heart was corrected for decay and subsequently expressed as thepercentage injected dose (%ID).

StatisticalAnalysesDifferences between the values for @mTc@HNP@I and @mTc@IgG

in the various infection models were analyzed using the MannWhitney test. The relationship between accumulation of tracers inthe pentoneal cavity on the one hand and radioactivity associatedwith bacteria or leukocytes on the other was calculated usingmultiple-regression analysis. The level of significance was set atP<0.05.

RESULTS

Quality ControlAfter incubation of 200,000 CFUs K. pneumoniae with

unlabeled HNP-l or @Tc-HNP-l at a concentration of50 jig/mL for 2 and 3 h in vitro, no viable bacteria could berecovered, indicating that the labeling procedure did notaffect the antimicrobial activity of HNP-l. Neither unlabeledIgG nor 99mTc4gGaffected the number of viable bacteria. Inaddition, more than 95% of the radioactivity associated with

@Tc-HNP-lor @Tc-IgGremained after incubation inNa-PB supplemented with 20% serum for 4 h at 37°C.

Pharmacokinetics

In mice injected with a bolus of S. aureus or K.pneumoniae, @@nTc@HNP@lwas rapidly removed from thecirculation, with half-lives of 175 ±28 mm (mean ±SEM)and 55 ± 9 mm, respectively (n = 4), indicating thatelimination of this tracer in K. pneumoniae—injected mice issignificantly (P < 0.05) faster than in S. aureus—injectedmice. Moreover, @°@Tc-HNP-1 rapidly accumulated in thekidneys, bladder and liver (Figs. lA and B, Table 1) and, to alesser extent, in the spleen (5—7%ID) and lungs (3—5%ID),whereas no radioactivity was observed in the thyroid andstomach of these mice. Furthermore, only minor differenceswere seen in half-life and biodistribution of 99mTc@HNP@lbetween mice that received an S. aureus bolus injection andmice with S. aureus infection (Tables 1 and 2). The half-livesfor @Tc-IgGin the circulation ofS. aureus—or K pneumoniae—injected mice were 250 ±35 and 70 ± 11 mm, respectively (n = 4), showing that the clearance rate of this

tracer from the circulation did not differ from that of@Tc-HNP-l. @°@Tc-IgGwas rapidly taken up by the liver

and kidneys and was excreted through the bladder (Table 1).

Accumulation at Sites of InfectionWithin 5 mm after injection of 99mTc@HNP@linto mice

that had received a bolus injection of S. aureus or K.pneumoniae, the site of infection had already been localized,as indicated by a significant T/NT in comparison with thatafter @Tc-IgGinjection. Maximum values were seen 15—60

mm after injection of 99mTc@HNP@l(Fig. 2). Furthermore,the TINTs 5 and 15 mm after injection of the tracers weresignificantly (P < 0.05) higher for @Tc-HNP-1 than for

ANTIBACTERIAL PEPTIDE FOR DETECTING INFECTIONS Welling et al. 2075

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Intervalafter K.pneumoniaeinjection @Tc- injeCtedmiceS.

aureusinjected miceS.

aureusinfectedmice(mm)

protein Kidney Bladder LiverKidneyBladderLiverKidneyBladderLiver5

HNP-1 15±4tt 17±7*t 18±5t11±3*11±3*7+4*17±68±411±4*@lgG23±4t 5±lt 20±430±83±117±417±76±319±415

HNP-1 16±5* 16±9 11±6*13±5*11±710±518±4j10±48±4lgG27±7t 7±5 19±539±94±316±516±4@8±212±560

HNP-1 16±7* 20±4* 6±6*15±3*16±106±3*2O±8@@+9*@8±2*lgG24±8 7±2 17±828±85±219±621±5f7±216±6240

HNP-1 11±4*t 10±7* 8±6*20±313±97±6*23±821±7*@10±5*lgG16±5t 8±5 19±819±58±718±522±414±313±51440

HNP-1 10±4*t 7±2 11±4*29±911±55@5*24±610±78±5lgG16±2 9±2t 14±317±49±217±517±513±510±5*P

< 0.05,HNP-1comparedwithlgG.tP< 0.05,K.pneumoniaeinjectioncomparedwithS.aureusinfection.1:P< 0.05, S. aureus injectioncompared with S. aureusinfection.Data

are mean (±SD)activity per organ, expressed as percentage injected dose, for 8—14animalsfromthreeexperiments.

p'@.A

Liver—..‘@ Kidney

4—Bladder

In@

Infection Infection

FIGURE 1. Representativescintigraphicimagesbetween1 and 60 mmafterinjectionof @‘Tc-HNP-1inmicewithK.pneumoniaeinfected thigh muscle. (A)Low-contrast images show organ uptake (arrows) of@'Tc-HNP-1 . (B) High-contrast images show uptake of

@Tc-HNP-1in infectedthighmuscle (arrows).

@°‘Tc-IgG(Fig. 2), whereas the TINTs observed 4 h afterinjection were similar for @Tc-HNP-1and 99mTc@IgG.Incontrast to the TINTs observed for @‘@Tc-HNP-1, those for

@Tc-IgGincreased significantly (P < 0.05) during the 4-hperiod. Interestingly, the TINTs for @Tc-HNP@but not for99mTcIgG, were significantly (P < 0.05) higher in mice withan S. aureus infection than in mice with a bolus injection of

S. aureus (Fig. 3). In additional studies with the cationicsynthetic peptide (KVAKQEKKKKKT) derived from ubiquicidin, TINTs never exceeded 1.30.

Effect on Number of Viable BacteriaThe possibility was considered that the TINTs for @‘°Tc

HNP-l do not increase over time because the peptide

TABLE 1Biodistributionof @Tc-LabeledHumanNeutrophilPeptide(HNP)-1and @mTc-LabeledImmunoglobulinG (IgG)

in Mice Injected with Staphylococcus aureus or Kiebsiella pneumonia or Infected with S. aureus

2076 THE JOURNALOF NUCLEARMEDICINE•Vol. 40 •No. 12 •December 1999

Liver@ Kidney Infection

@,—Bladder

Liver______:@h Kidney

E—Bladder

R15

Liver—.--.@@ Kidney

I@ Bladder

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4

0

I-

a)0)

0

0

a)0)

I-

1

## **

*

TABLE 2Effectof99mTcLabeledImmunoglobulinG (lgG)andHumanNeutrophilPeptide(HNP)-1onBacterialNumbersperGram

of Thigh Muscle in Mice Intramuscularly Infected with Staphylococcus aureus or Kiebsiella pneumoniae

Experimental @Tc-HNP-1 @Tc-lgG

Microorganism condition 0 h 4 h 24 h 4 h 24 h

S.aureus Injection 1.7 ±0.5 x 10@ 6.9 ±5.2 x 10@ 7.1 ±2.7 x 10@@ 2.2 ±0.9 x 10@ 1.0 ±1.6 x 10@Infection 4.7±1.5x 10@ 3.1±0.3x 10@ 6.7±4.Ox10@ 3.5±0.5x 10@ 1.5±0.4x 10@

K.pneumoniae Injection 9.2 ±0.5 x 10@ 8.2 ±2.7 x 10―@ 1.4±2.1 x 10―* 7@9@ 8.9 x 10@ 2.8 ±5.8 x 10@

*P < 0.05,comparedwithnumberofviablebacteriarecoveredfrommiceinjectedwithlgG.Datamean(±SD)ofat least6 animals.

reduces the number of viable bacteria (8). Therefore, the injection (Table 2). The antibacterial effect of HNP-l proved

number of viable bacteria in the thigh muscle was assessed to be considerably less in S. aureus—infected mice than in K.at intervals after injection of 99m@@4.fl44p1 or 99mTc@IgG.In pneumoniae—infected mice. Furthermore, the number ofmice that received a bolus injection of S. aureus or K. viable organisms in mice with an S. aureus infection was

pneumoniae, @Tc-HNP-l, in contrast to @°@Tc-IgG,greatly significantly (P < 0.05) higher than in mice that had

reduced the number of viable bacteria 4 and 24 h after received an S. aureus bolus injection.

5 15 60 240

Time(mm)after injectionof radiopharmaceuticalFIGURE2. Target-to-nontargetratiosforeither @Tc-HNP-1(whitebars)or @Tc-lgG(verticalllyhatchedbars)inmicethatreceivedbolus injection of K. pneumoniae and for @mTc-HNP-1(black bars) and @Tc-IgG(horizontally hatched bars) after administration oftracerintomiceinjectedwithbolusofS.aureus.Resultsaremean(±SD)ofatleast9animalsfromthreeseriesofexperiments.@ <0.05, compared with @mTc-lgG;#P < 0.05 for HNP-1 in S. aureus infections compared with HNP-1 in K. pneumoniae infectionsaccording to Mann-Whitney test.

AmAcri@iw@ PEPTIDE FOR DETECTINGINFECTIONS•Welling et al. 2077

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r@-i

6

#*

*

#*

5 15 60 240

FIGURE 3. Target-to-nontargetratiosof @Tc-HNP-1(whitebars)and @Tc-lgG(verticallyhatchedbars)in micethat receivedbolus injectionof K.pneumoniaeand for @Tc-HNP-1(blackbars) and @Tc-lgG(horizontallyhatchedbars)after administrationintomice injected with bolus of S. aureus. Results are mean (±SD)of at least 9 animals from three series of experiments.@ < 0.05,comparedwith @Tc-IgG;#P < 0.05 for HNP-1 in bolus-injectedmice comparedwith HNP-1 in infectedmice accordingtoMann-Whitney test.

Binding to K. pneumonlae and LeukocytesIn Peritoneal Cavity

The pentoneum model was used to obtain insight intobinding of@Tc-HNP-l to bacteria and leukocytes at sites ofinfection. The results for @“@‘Fc-HNP-1revealed that approximately 37% ofradioactivity in the pentoneal washings couldbe recovered in the fraction containing leukocytes, approximately 23%, in the fraction containing free bacteria and theremaining 40% in the washing fluid. The respective valuesfor @Tc-IgGwere approximately 10%, 10% and 80%.After corrections based on these findings were applied (Fig.4), @Tc-HNP-1 binding to bacteria proved to be approximately 1000 times higher than to leukocytes, whereasbinding of @°‘Tc-IgGwas roughly the same for leukocytesand bacteria.

In agreement with the results in the thigh muscle model,the number of viable bacteria found in pentoneal washingswas much lower for K. pneunwniae—infected mice injectedwith @Tc-FINP-l than for those injected with @‘@Tc-IgG(8). Furthermore, the number of leukocytes in peritoneal

washings was higher (P < 0.05) for mice injected with@Tc-FINP-lthan for mice injected with @Tc-IgG.In addition, 4 and 24 h after injection, multiple-regression

analysis revealed that binding of @‘@Tc-HNP-1to bacteria(r 0.81, P < 0.05), macrophages (r = 0.97, P < 0.04),granulocytes (r = 0.76, P < 0.05) and lymphocytes (r =

0.64, P < 0.05) contributed to accumulation of this tracer atthe site of infection. Interestingly, for @°@Tc-IgG,only thebinding to macrophages (r = 0.93, P < 0.05) and granulocytes (r = 0.64, P < 0.05), not to bacteria, correlated withaccumulation of this tracer in the infected peritoneal cavity.

DISCUSSION

The main conclusion from this study is that @Tc-HNP-lcan be used for rapid visualization of bacterial infections.This conclusion is based on the observation that TINTs

higher than 1.3 for @Tc-HNP-1 had already occurredwithin 5 mm after administration of the tracer to mice that

had been injected intramuscularly with either gram-positive

2078 THEJoui@i OFNUCLEARMEDICINE•Vol. 40 •No. 12 •December 1999

Time(mm)after injectionof radiopharmaceutical

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4

BA

lime(h)

lime(h)

24

8 8•!

4

2

io2 10_i io@ io1 io2

FIGURE4. Bindingof @Tc-labeIodhumanneutrophilpeptide(HNP-1)and @Tc-immunoglobuIinG (lgG)to 1 millionbacteriaorperitonealleukocytesafterinjectionintomiceinfectedwithK.pneumoniae.At intervalsafterthisinjection,peritonealcavitywaslavaged and, after selective centrifugation, fractions containing free bacteria (black bars) and leukocytes (white bars) were counted fornumbersandforradioactivity.

or gram-negative bacteria. This rate is considerably fasterthan that for 99mTc4gG, an established marker of infectionand inflammation (1, 18), for which similar TINTs wereobserved 4 h after administration of the tracer. The observation that the TINT for @°@Tc-HNP-lremained roughlyconstant over the 4-h postinjection period contrasts with therises in TINT for @Tc-IgG.An explanation for the finding

that the TINT for 99m@@pJ@p@ does not increase after 30mm could be elimination of bacteria in the thigh muscle onintroduction of the peptide. Agreeing with this view is anearlier report that the number of viable bacteria in the thighmuscle of mice decreased after injection of @Tc-HNP-1 butincreased after injection of @Tc-IgG(8).

The suggestion that binding of @mTc@HNP@lto bacteria isthe major factor contributing to accumulation of this tracerin bacterial infections is based on the following findings.First, in mice with an infected peritoneal cavity, binding of

@°@Tc-HNP-l,but not 99mTc..IgG, to bacteria was found tocorrelate positively with accumulation of tracer at the site ofinfection. Moreover, binding of @Tc-HNP-l to bacteriawas at least 1000 times higher than to leukocytes in vivo.

However, 2 h after injection of @mTc@HNP@1, the number ofleukocytes in the infected peritoneal cavity of mice wasalready more than 100 times higher than the number ofbacteria. Poor binding of @Tc-HNP-l to relatively highnumbers of leukocytes could explain why binding of @‘@TcHNP- 1 to leukocytes was also found to correlate well withaccumulation of this tracer in the peritoneal cavity. Part ofthe @Tc-FINP-lactivity related to leukocytes can likely beattributed to binding of @Tc-HNP-l to phagocytosed

bacteria. Unfortunately, binding of tracers to leukocytescould not be distinguished from binding of tracers tobacteria associated with these leukocytes. In addition, not allactivity in the peritoneal washings could be attributed tocells. Other possible modes of action, such as aspecificleakage of protein into the site of infection, binding of thetracer to proteins (22) or other noncellular components andreduction of blood flow, may also contribute to accumulationin the peritoneal cavity.

Second, the observation that the TINTs for @Tc-HNP-lin mice infected with S. aureus are much higher than in miceinjected with S. aureus may be attributed to the higher

ANTIBACTERIAL PEPTIDE FOR DETECTING INFECTIONS Welling et al. 2079

99mTc•IgG 99mTc•@NP•1

24—

@@1notdetermined

2 —@notdetermined

100 iOi i02@@@ io6Binding(nmol/mOI'mncells) Binding(nmo@millioncells)

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numbers of bacteria in mice with an infection. Alternatively,binding of @Tc-HNP-1to cytokine-activated leukocytes orother cell types present at the site of infection, e.g.,endothelial cells, may explain the difference in TINT. Thispossibility is less likely because in vitro binding studies haveshown that cytokine-activated human leukocytes and endothelial cells from the umbilical cord vein bind negligibleamounts of HNP-l (MM Welling et al., unpublished data,1999). In addition, 4 h after injection of FINP-1 in mice thatreceived a bolus injection of S. aureus, similar TINTs wereobserved for HNP-l, despite an influx of leukocytes duringthis period.

Another important finding pertains to differences in@Tc-HNP-lpharmacokineticsin mice infected with S.

aureus or K pneumoniae. Elimination of @Tc-HNP-1 fromthe circulation and uptake of this tracer by various organsand the infected thigh muscle are faster in mice infected withK. pneumoniae than in mice infected with S. aureus.Although a definitiveexplanationcannot be offered, theseverity of the K. pneumoniae infection, which is indicatedby the high number of circulating leukocytes in these mice,may well be responsible for these results. Other possibleexplanations, such as differences in the binding capacity ofHNP-l to S. aureus or K. pneumoniae, cannot be ruled out.

@°@Tc-HNP-lhas severaladvantagesover currentlyusedtracers for detection of bacterial infections. The smallmolecular size of HNP-l enables rapid entrance of the tracerat the site of infection, as illustrated in this study. Adverseeffects in patients, e.g., triggering of antibodies or allergicreactions against this peptide, are not likely because HNP-lis of human origin. Furthermore, the highest concentrationof HNP-1 used in this study (400 ng per mouse; i.e., 260ng/mL blood) is not toxic to human cells. During infection in

humans, the level of HNP-1 in blood has been reported toincrease from 40 ng/mL to 1 @.tg/mL(23,24). Rapid uptake of

@“@Tc-HNP-1 by the kidneys and bladder, followed byexcretion through the urine, reduces the radiation burden inorgans. When considering the possibility that bacteria become resistant to this antimicrobial peptide, one shouldremember that HNP-l eliminates bacteria by causing bacterial plasma membranes to become permeable and, subsequently, subject to metabolic injury (7). This fact makesunlikely the development of bacteria that are resistantagainst HNP-1 . Besides, during evolution, defensins havebeen been successful in protecting plants, animals and

humans against the continuous threat ofpathogenic microorganisms (25).

CONCLUSION

99m@@@}fl@p4 allows rapid visualization of bacterial infec

tions, in part because of binding of this tracer to themicroorganisms at these sites.

ACKNOWLEDGMENTS

The authors appreciate the technical assistance of PetraDibbets-Schneider and Bram Sinon of the Department of

Radiology, Division of Nuclear Medicine; Maria van denBarselaar of the Department of Infectious Diseases; andSandra van Wetering of the Department of Pulmonology(Leiden University Medical Center).

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2080 T@ Joui@i OF NUCLEARMEDICINE•Vol. 40 •No. 12 •December 1999

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1999;40:2073-2080.J Nucl Med.   Mick M. Welling, Peter H. Nibbering, Akke Paulusma-Annema, Pieter S. Hiemstra, Ernest K.J. Pauwels and Wim Calame 

Tc-Labeled Human Neutrophil Peptide-199mImaging of Bacterial Infections with

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