Measurement of air-borne pyrogens by the in vitro pyrogen test (IPT) based on human whole blood cytokine response

Eun>flt'(1Illournal of PurenteraI&Phurmaceuticai Sciences2003:8(3): 65-69 0200 3PareRteral$oc iety

Measurement of air-borne pyrogens by the in vitro pyrogen test (IPT) based on human whole blood cytokine response

Ilona Kindinge r',Stetan Fennrich' ,BertZuckert,GunterLinselt.Sonjavon Aulock'and ThomasHartung'

·UniversityofKonstanz,Germany

trree

Theimpa ctofenviro nment almicr o-o r ga nismsaswellastheirfra gmen ts and componen ts, especially endoto xfns,on humanhea lth is lncr easla glyrecogn ised .Differ en t syndromes havebeendescribedin connec tio nwithinhaledair -bo r nemicr ob iologicalcont ami nation, e.g,sick buildi ngsynd ro me, hum id ifierlung,organic dust toxic synd ro me (O DTS) and"Mondayillness" .Air-conditio ning syste msinte nsifythis proh lem, asdoes thecollecti on ofbiologicalwastein households,which represents a substa nt ialsou rce ofal r -b orn epollulion .

In1995 we descr ibeda newmet hodforthe detection orpyroge niccontam lnanon'-',This sensitive test(invitropyrogen test:IPT) usesthenat ura lreaction of theimmune system to detecta broad spect r umof pyro gensin humanblond. Safety testsininj ectabledr ugs representthemainappl ica tion and thetest hasbee n successfullyvalidated forinclusion intothe Euro pea n Pha rmacopoeia.

Her e,the testwas adapted to thedetectionofenvir o nme ntalair-bo r ne pyro gens.Air wasdrawn th ro ughafilterwhichwasIhenincu bat eddirectly wit hdiluted humanwhole blood.The releaseof thetnttarnmato rycyto ktne,interteukfn-If(II..~IP),wasmeasuredby ELlSA.lnanimalstab les,upto 3x llY'endo tox inequivalent units (EEU)we refound per cub icmeter of air.

Theproblem

Both ourwell-being andour healthdep end substa ntia lly on thequalityoftheair inourenvironment.Thequalityofthe room climate ismainlydete rmined bythecompo nents ' humidit y.airtemperatureand pollutant content,includi ng dust , environmental micro-organisms and their decay products

Inparticula r,fungalsporesand bacteria,aswellastheir toxins,arethemainmicrobiologicalcompo nentsofthe air.

The pollutionof inte riorairwithbacteriaand moul ds representsthe causalfactorfor"sickbuildi ngsyndrome'".

This syndromeis charac terisedtypicallybylackofwell- being,faintness,lackofconcentration, headache.affectionof mucousmembranes,and increasedincidenceof allergyand infect ion. Accord ing to a study bythe World Health Organi sat ion(WHO).30%ofthe 3millionpeople employed in air-conditioned roo ms suffer these symptoms'.

Investigation sat the University of Kiel demons trated a connectio n betweenpulmonarydisease.Le.thefreq uencyof

CnlTl'SpnRding8 ul hor: Thom asHan ung.MDPhD.Biochemical Phar macology.University ofKonstanz..POBM655. 78457 Kon stan z.

TeJ:+49 753111ll 4 116:fax:+49 7531 1l84 J17:

email:Thomas.Han uRg @uRi-koRstanz.de

infection s andallergies.and thepresenceofenvironmental micro -organi sm s (bacteria and moulds)in hou seholds' .

Themaincauseofmicrobiologicalcontaminationofliving areaswith bact eriaandfungi iscontinuousdampnessinoron buildi ngmaterials.Thisproblem is exacerbatedby the continuous trendtowardsmore efficie ntsealing ofbuildings tosaveenergy.Acontinuo usexchange ofairisobstruc tedby these sealingmeasures.Theresultismoisturein thebuilding materials andsubseque ntincreasingloadsofmicrobiological pollutantsin the air.Duetothis connection,itcanbeassumed that themicrobio logical burdenin living and workareaswill increasingly cause health prob lemsinthenext fe w years.

Therefore,researchmethodsandevaluationcriteriamust be improvedfor the characteri sation ofenvironme ntal micro- orga nismloadsinlivingandworkarea s.

So far,microbio logicalconta minationof househ old s is no rmally,if atall, evaluatedonlybydetermination ofthe concentrationof fungalspores in room air. whic h is insufficientforaglobalevaluation ofthemicrobialload as:

Moul dsreprese ntonlyone formofmicro biological pollutant:animpo rtantcausal relevanceisattri b utedto Gram-negativebacteriaandtheircndotoxins in"sick building syndrome".

First publ. in: European journal of parenteral and pharmaceutical sciences 8 (2003), 3, pp. 65-69 Konstanzer Online-Publikations-System (KOPS)

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-79498

URL: http://kops.ub.uni-konstanz.de/volltexte/2009/7949/

Themeasurementof theloadofair-borne mouldsis based onclassical microbiological methods,Le.only micro-organisms which can be cultured are represented.Deadfungiand bacteria,whichare not detected incultures, pose anadditional health riskto humans.

Thedeterminationofthe concentration ofmicro- organisms,Le.colony-formingunitsper airvolume.

doesnotreflect thepathogenicityor inflammatory potentialofdifferent fungiorbacteriaforhumans.

A comprehensive estimate of environmental micro- organismloadshouldinclude a varietyof relevantmicro- organismsandtheir toxicmetabolicproductsandreflect theirpathogenicityfor humans.

Besidethe micro-organismsthemselves.theircellwall constituents, i.e.whatremainsafter themicro-organisms aredead.representasubstantial riskfactor forthe well- being and health of humans.These structures (eg.

endotoxins andglucans)arerecognisedbyhumanimmune cells,e.g,after inhalation.leadingtoaninflammatory reaction,the causeof the abovementionedhealthproblems.

The loadwithsuchenvironmentalmicro-organisms is approximately threetimeshigherin householdsthatcollect biologicalwastewithinthehousehold;thefungalload is abouteight-fold higher. If carpetsareused as floor covering,these valuesrise dramaticallyup to 8OD-foldt-.

The workingenvironment represents anotherrelevant area whereworkers are increasinglyexposedto air- conditioningsystems; forexample,in 1993threemillion peopleintheFederalRepublic ofGermanyworkedin offices withair-conditioning' .Today,thefollowinghealth problems are described in connection with air- conditioningsystems:sick building syndrome, building- relatedillness,infectionsandallergies

The detection of endotoxin as a marker for contamination with pathoge nic micro-organisms is considered avery important factor;six-foldincreased endotoxin quantities, Le. 36-800nglm³airareconsidered a significantload ",Gram-positivemicro-organisms are also found to asubstantialexte nt;howevertheydonot seem to correlate with the pathogenic importance suggested bymicro-biologicalinvestigations,Le. based onthecountof living organismsthat can becultured.

However.itshouldbenotedthatthere isno specificin vitrotestsystem todetectdead materialfromGram- positivebacteriaasthereisforGram-negativeendotoxin, Le. the Limulus Amoebocyte Lysate assay (LAL).

Therefore.amoredetailed evaluation is stillawaited;

similarconsiderationsapplytofungalconstituents.

TheWHOestimated morethan10yearsagothatabout onethirdofall neworreconstructedbuildingsare so- called potentially"sick"buildi ngs".Since the health problemsapplylesstonaturally ventilated thantofully air-conditioned buildings,thenumbers areincreasingin parallelwiththe increasing useofairconditioning.

Systemswhichoperatewithair humidifiers appearto representa specialrisk factorsincehighhumidityfavours contamination andmicro-organism growth.Dueto energy saving measures,theregulationof thehumidity is often omittedandhumidityincreases.BothGram-negativeand

Gram-positivebacteria.aswel lasfungiandamoebae, are causally relatedto "humidifier lung",which isparticularly common in the printing industry'. The so-called

"humidifier fever"isdirectly correlatedwithendotoxlns", Itis wellknownina variety of working environments that illnessesfrequently occur onMondays,when the bodyisabruptlyconfrontedwithamultiplicityof air- borne contaminationsafterarecuperativeperiodover the weekend. This"Monday syndrome",characterised by general malaise combined with shortageof breath.

coughingand thoracictightness.improvesduring the course oftheworkingweek.Inhalation of organicdust, endotoxinsandother factorsarethoughttobe causesof thisillness,knownin the cottonindustrybythename

"byssinosis'".

People employedin petshops,animalstables,zoos.

poultryfarms orrefuse sortingfacilitiesarealso affected Inaddition,workersinthe steel industryare particularly exposed,becausethe reclrculating coolingemulsions(oill water mixtures) spread pyrogenic pollution after microbiologicalovergrowthespeciallywith Pseud omon as species.Thisis exacerbatedbythehightemperatures and thedampenvironment.

Detection possibilities

Essentially,thefollowing procedures are available:

1. Microbiological testing methods, where sample material is incubated together with a nutrient substrate.Depending on the substrare.specific micro- organismscangrow, whichcanthenbeidentified.

Incubationtakesupto14days.

2. 'Rapid tests'forlivemicrobiological contamination are bioluminescence and impedance monitori ng, whichalso requireprevious growthof the organism.

The opticalsystemof directepifluorescenceis very time-consum ing, insensitive and unsuitable for routine analysisof large sample numbers.With methods of direct fluoresce nce marking (e.g ChemScansystem,based on measurement withan ultrasensitivelaserscanner), livingmicro-organisms aredetected veryrapidly.Itisintendedforraw materialsor finishedproducts, e.g.in pharmaceutical manufacture,andunsuitablefor air control.because the naturally occurring micro-organism spectrum woulddisturbthistest substantially.

3. Testofendotoxincontent.Afterseparationofair- borne endotoxins from filters.these structurescanbe detectedspecificallyand verysensitivelyinthe LimulusAmoeboc yteLysatetest (LAL)(seealso procedures forthe determination of the endotoxin concentrationinthe workingenvironment,BIA working folder "Measurement of dangerous materials",Index 9450).

Thesemicrobiological differentiation methods arc useful for analysingthelivingmicro-organismspectrumwhile deadonesescape detection.Notably,thesemethodsare very labourand materialintensive.Thisishardlyuseful forroutinepurposes,andthedirecteffect onhumansisnot reflected.Inaddition, putativelyinfectiousmaterialis

nofilter LPSdriedontilter LPSpluslil1er

withoutthefilter ortothe eo-inc ubationofLPS with untreatedfilters.

Air samplesfromanimalstableswerethen examined.

The contaminationof airin animalstablesisparticularly problematic for humansworkingthere"andwasused here to checkthe applicabilityofthe test.

Air was drawn onto Sum nitrocellul ose filters (Sartorius,Germany)withaPGPdust samplingsystem (Str6hleinGmbH,German y) ata rate of 3.5Ilmin for 60 min.The exposedfilters wereincub ated with8mlsaline and2mlbloodovernightinaPetridish.IL·lprelease was measured in the supemataru. Control endotoxins employe d inincreasi ngconcentratio nsoncleanfilter materialinduced an increasinginterleukin-l Brelease,i.e.

therewas a concentration/response- relationship(Figure 2,left part).Contaminate dsamples from a shee pstable showed clear signalresponses(Figure 2,right part) depende ntontheirload.

Airsamplesfrom two pigstables(K andV) and samplesfromoutsideairweregainedbyimpingemer u'".

The air was bubbledthrough5Dmlpyrogen-freewater (Aq uaadiniectabi le,Braun Mclsunge nAG, Melsungen, Germany) inorderto dissolvethepollution by AGI-3D impingers (AGI=AllGlass Impinger) operatedfor 20 Figu re1:Release of IL-ljJby humanwhole blood in response to nitrocellulosefilterscoatedwithdifferentconcentrationsofLPS in comparison toLPSwithoutfilterSand incubationofLPS with untreatedfillers.

Pyrogens are intla mmation-inducingsubstanceswh ich, dependinguponthe route ofuptake , canlead to genera l malaise,breathingproblems,fever, circulatoryandorgan failureup tofatal shock inhumans.These disease symptomscan alsomanifest after uptake ofendotox invia thelung andarenowadays regarded as aunique syndro me in environ menta l medicine10.

Thehumanimmunesystemreacts verysensitively to environmental micro-organisms or their products and initiatesappropriatedefencemechanisms.Itis unimporta nt for the hostdefence whether microbiologicalmaterial (livingmicro-organisms,endotoxlnsor otherpyrogens) threatensthe organis mdirectlyviathe blood, skinorthe respiratory system. After contact with relevant contaminations, white blood cells (monocytes/

macrophages) release pro-inflammatory signalmolecules such as intcrleukin- IB (lL·l P).The pro-inflammatory cytokineschangetheset-pointfor thermo-reg ulationinthe brain andcause a feverreaction intheorganism. We used thisreactionto developatest for pyrogenic contaminations indrugsbased onwholeblood incubatio n'A'!'!' .Blood from a healthy donor isdiluted in clinical salineand incubated with theliquidsampleat 37"Cfor 6--24hours.IL-lPrelease is quantifiedby ELlSAmeasurement.Recovery ofan endotoxinspikeinthe sample rules out interference.

Foradaptationof the testto thedetection of air-borne pyrogens,airwasdrawn onto8mmnitrocellulosefilter s (Sartorius,Germany)using a PGP dust samplingsystem (StrohleinGmbH,Germany)orbubbledthrough pyrogen- free water usingAGI-3Dimpingers.The bloodcanbe

~:::I:t~~t~i;:~:ie;;~hl i~;i:lter

r - - - ,

prod uced and has to be processedand disposed ofunder appropriatesafety conditions.

Endotoxinscanbe usedasmarkers forcontaminatio n with Gram-negative bacteria. TheLAL test isawell establishedand sens itive method.Ho we ver,onlyarinsing solutionof thecontaminatedfilter canbe examined.

Tigh tly boundandnon-extrac tableendo toxinsescape detection .

8~i?~r§,g~i8(i)m~;:;

LPS spikeper filter samples !rom sheepstable

= 3000

!

Figure2:Release ofIL·1~in human whole blood initiated by nitrocellulosefilters contaminatedwithdefinedLPS-concenlrations or tillerscontaminated in a sheepstableby collecting3.5IImin ofair for1 hour(S1toS4)

Resultsofthe new whole blood met hod (IPT)

First,wetestedwheth erthenitrocellulosefiltersinterfe re withthewholebloodtest.Different concentrationsof lipopolysacc haride (LPS)fromSalmonella abortus eoui were appliedtothefilters ina volumeofID!!1and leftto dry.TheIL-IPresponse tothesefiltersplacedin 24-well tellcultureplates and coveredwithImlwholeblood diluted1:5 insalinewas comparedwiththeresponse of theblood toLPS witho utfiltersortountreatedfilters plus LPS adde ddirectlytothe incubation.As canbeseen in FigureI,thefilter alone doesnot induce significa nt

cytokinere lease.However.itappea rs toimpro vethe L- ----'

pre sent ationofLPS to theblood,resultinginamore sens itiveIL-I ~respo nse(l2.5pgwere detected ) and in hig her levels of cytokinerelease compared tothesamples

Table1: Overviewofthemicrobialair contaminationintheinvestigaledstables

air hygen icparameters sheepstab le pigstableK pigstable V Qutsideair

inhalabledust(mg!m3] 0.5 3.1 2.e 0.1

inhalableendotoxin(EUIfn3) 512 3994 3841 15

Gram-negative

bacteria[CFU/m3) 10 115 100 3

totatbacteria[CFU/m3j 40 000 175000 132 000 2055

moulds[CFU/m3j 94007 247 230 n.c

IPT[mg IL-1IYm³⁾ 0.4' 25.8t 12.3t O.1t

Arithmetic average01atleast 5measurements n.c.'"not done

CFU'" colonylormingunits EU'"endotoxinunits 'measured ontitters tmeasured by impingement

We showedthatintheproblem at ic fieldofanima lstables air contaminatio n with living enviro nme ntal mic ro- organisms and deadmateri alcanbeassessed withthein vitropyrogentest.The testmethodwasadaptedfor these specific condi tions(filtration andimpingement for the collectionofair-bo rnecontamination).Air-borne pyrogcns arenot onlyof importanceinanimalstables. butfor the entire human livingand working area. The specia l advantag esofthetestlieinitsspecies-spcciflclty. the med icalrelevance.thebroaddetectionspectru mandthe possibil ityofcheckingthebloodofthe affectedperson directly.Thereisno comparabletestsystem that cancover both deadandlivingmaterial in anintegralmanner.

Weintendtoevaluatethis inno- vative method furtherwithpartners from environme ntaland occupa- tional medicine wad. safety and industry toopeimisethemethodo- logical development s described here andto adapt themto the differentenvironme ntalconditions relevant tohumans.The crucial advantageof thenewtestisthat the reactionmimicsthereactionthatair contamination would cause in humans.therebyreflecting potential dangersforhumans.Firstresults promisethata simple.standardise d metho d willbeavailableshortlyfor the measurem ent of air-borne pyrc gens. especiallysince asta n- dardisedkitversio nof theIPTis no w availabl e (Charles River

I ,

I

Anderse nsam plers were operated forI minatanair nowrate of 28.3Vmintoobtain thetotalnum berof airborne aerobicbacteria (on5%shee pbloodagar)andfor 20 min to obtainthe number of airbo rneGram -negative aerobic bact eria (on MacConk ey 3agar.Oxoid.wesel.

Genn any).Thesheep blood agarplateswereincubatedat 37°Cfor 24h;the MacC onkey agarplateswereincubated at 37"C or 22<>C for 24h. All bacteria grownon MacConkey agarweretestedfor the irGra m-reactionby stainingto givethe realnumber ofGram -negative bacteri a inthesamples.Fortheairbornemoul dsthe samplerswere

~LJ!ILJIIILJIII---LJ!ILJlllLII'---,,-=-=....IIII0LIequippedwithDG-18 agarplates and operatedforI minat

i

lIJ Thedata gainedwiththewholeblood method withfilters

L .::"".::mp.::'"=-- --..Jorbyimpingementwere relatedtothecontaminationin 1m³

Figure 3: Measurement01contaminated airvia impingement: Air-forcomparisonwiththeothermeasured parameters.

borne pyrogens couldbedetectedinpig stables(K1-K4.V1-V4)while Thesignalsmeasur ed inthewho leblood testcorre late

::;~~~~t:~~a~: ;~~i~lel:~r~~~:~UCed

~~U7~~y ~~~:r~hn~ I:;:~~e~f ~n~~r~~~1 :~~ti~~i~~~O~~~~:~

minutesatanairflo wrateof 12.5Vmin . Sam ples ofthis experi ments willbenecessarytoexaminein moredet ail washingliquidconta minated withair-bornemateri als how wellthismethodreflect stheriskof expo sure offann were employ edinthewhole blood test asliquidsamples workers orhighly sensitiveindividuals andto optimisethe (IOOJ!Isam ple.900tI1salineandlOO~1blood ) (F igu re3). method ology.Le.optimisationoffiltermaterial. poresize.

Themicrobiological conta minat ioninthe exam ined furthersim plifica tio nof incu bation procedure.etc.

animalsta bleswas alsocharacterised incomparisontothe

outs ideairwithstandardcultureproced uresandthe LAL Conclus ion testin parallel withthe wholebloodtest(T able1).The

PGP dust-sampling syste m(Strohlein GmbH.Genn any ) wasusedto co llecttheinhalabledust fraction.ThePGP systemwas operated at anairflowrate of3.5Vmin for60 min. For measure ment of endotoxin. the expose d nitroc ellulo sefilters wererocked (22rpm;WS 5shaker.

Edmund Btihler GmbH.Tubingcn.Germ any ) for2hin50 ml pyrog en-free water. After centrifugationof the washingfluid with20(Xlg.anallquorwasmeasuredinthe LAL assayQCL1000(Bio whtuaker.Walkersville,MD. USA) asrecommen ded bythemanu factur er.Theamount ofendotox in in thesupernata ntofthesamplerfluidswa s conve nedfromEUlml to EUlm^lusin gtheairflowrateof the sampler and the sam plingtime.

Endosarc).This is animportant prerequisite forthe development of internationalguidelines and thresholdsand respectivepreventivemeasuresin thelong term.The test mayeven be adaptedtogauge the inflammatory reaction of animals living in the stables and to compare their sensitivitytothatof humans.

Ackno wledgements

The authors wouldliketothank AstridLeja and Gregor Pinski fortechnical assistance.

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