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43TCNICAS DE ESTUDIO DE FAGOCITOSIS IN VIVO Y SU APLICACIN EN LA INVESTIGACIN EN LA INVESTIGACIN...PROCEDURES TO STUDY THE IN VIVO PHAGOCYTOSIS AND APPLICATION FOR THE INVESTIGATIN OF IMMUNOMODULATORY...

Tcnicas de estudio de fagocitosis in vivo y suaplicacin en la investigacin de la actividadinmunomuduladora de antibiticos

Procedures to study the in vivo phagocytosis and application for theinvestigation of immunomodulatory activity of antibiotics

ALKOUWATLI K1, LEIVA M2, RUIZ-BRAVO A2, JIMNEZ-VALERA M2*

11111 Department of Biochemistry and Microbiology, Faculty of Pharmacy, University of Damascus, Syria.Department of Biochemistry and Microbiology, Faculty of Pharmacy, University of Damascus, Syria.Department of Biochemistry and Microbiology, Faculty of Pharmacy, University of Damascus, Syria.Department of Biochemistry and Microbiology, Faculty of Pharmacy, University of Damascus, Syria.Department of Biochemistry and Microbiology, Faculty of Pharmacy, University of Damascus, Syria.22222Department of Microbiology, Faculty of Pharmacy, University of Granada, Spain.Department of Microbiology, Faculty of Pharmacy, University of Granada, Spain.Department of Microbiology, Faculty of Pharmacy, University of Granada, Spain.Department of Microbiology, Faculty of Pharmacy, University of Granada, Spain.Department of Microbiology, Faculty of Pharmacy, University of Granada, Spain.

*****Author for correspondence. E-mail: [email protected] for correspondence. E-mail: [email protected] for correspondence. E-mail: [email protected] for correspondence. E-mail: [email protected] for correspondence. E-mail: [email protected]

R ESUM ENR ESUM ENR ESUM ENR ESUM ENR ESUM EN

La depuracin de partculas de la sangre es una medida de la capacidad funcional del sistema fagocticoLa depuracin de partculas de la sangre es una medida de la capacidad funcional del sistema fagocticoLa depuracin de partculas de la sangre es una medida de la capacidad funcional del sistema fagocticoLa depuracin de partculas de la sangre es una medida de la capacidad funcional del sistema fagocticoLa depuracin de partculas de la sangre es una medida de la capacidad funcional del sistema fagocticomononuclear, responsable de la eliminacin sistmica de microorganismo patgenos, inmunocomplejosmononuclear, responsable de la eliminacin sistmica de microorganismo patgenos, inmunocomplejosmononuclear, responsable de la eliminacin sistmica de microorganismo patgenos, inmunocomplejosmononuclear, responsable de la eliminacin sistmica de microorganismo patgenos, inmunocomplejosmononuclear, responsable de la eliminacin sistmica de microorganismo patgenos, inmunocomplejosy clulas apoptsicas. Esta capacidad puede ser alterada por agentes modificadores de la respuestay clulas apoptsicas. Esta capacidad puede ser alterada por agentes modificadores de la respuestay clulas apoptsicas. Esta capacidad puede ser alterada por agentes modificadores de la respuestay clulas apoptsicas. Esta capacidad puede ser alterada por agentes modificadores de la respuestay clulas apoptsicas. Esta capacidad puede ser alterada por agentes modificadores de la respuestabiolgica, entre los que figuran numerosos agentes antimicrobianos. En este trabajo se compar labiolgica, entre los que figuran numerosos agentes antimicrobianos. En este trabajo se compar labiolgica, entre los que figuran numerosos agentes antimicrobianos. En este trabajo se compar labiolgica, entre los que figuran numerosos agentes antimicrobianos. En este trabajo se compar labiolgica, entre los que figuran numerosos agentes antimicrobianos. En este trabajo se compar laefectividad de la medida de la capacidad de depuracin de ratones BALB/c inoculados con distintosefectividad de la medida de la capacidad de depuracin de ratones BALB/c inoculados con distintosefectividad de la medida de la capacidad de depuracin de ratones BALB/c inoculados con distintosefectividad de la medida de la capacidad de depuracin de ratones BALB/c inoculados con distintosefectividad de la medida de la capacidad de depuracin de ratones BALB/c inoculados con distintos

microorganismos (una levadura, dos bacterias Gram-positivas, extra- e intracelular, y dos bacteriasmicroorganismos (una levadura, dos bacterias Gram-positivas, extra- e intracelular, y dos bacteriasmicroorganismos (una levadura, dos bacterias Gram-positivas, extra- e intracelular, y dos bacteriasmicroorganismos (una levadura, dos bacterias Gram-positivas, extra- e intracelular, y dos bacteriasmicroorganismos (una levadura, dos bacterias Gram-positivas, extra- e intracelular, y dos bacteriasGram-negativas, asimismo extra- e intracelular). La levaduraGram-negativas, asimismo extra- e intracelular). La levaduraGram-negativas, asimismo extra- e intracelular). La levaduraGram-negativas, asimismo extra- e intracelular). La levaduraGram-negativas, asimismo extra- e intracelular). La levadura Candida albicansCandida albicansCandida albicansCandida albicansCandida albicans fue seleccionada, por sufue seleccionada, por sufue seleccionada, por sufue seleccionada, por sufue seleccionada, por suapropiada cintica de depuracin y su resistencia natural a agentes antibacterianos, para estudiar laapropiada cintica de depuracin y su resistencia natural a agentes antibacterianos, para estudiar laapropiada cintica de depuracin y su resistencia natural a agentes antibacterianos, para estudiar laapropiada cintica de depuracin y su resistencia natural a agentes antibacterianos, para estudiar laapropiada cintica de depuracin y su resistencia natural a agentes antibacterianos, para estudiar lamodificacin de la fagocitosis in vivo por el antibitico macrlido azitromicina. El tratamiento conmodificacin de la fagocitosis in vivo por el antibitico macrlido azitromicina. El tratamiento conmodificacin de la fagocitosis in vivo por el antibitico macrlido azitromicina. El tratamiento conmodificacin de la fagocitosis in vivo por el antibitico macrlido azitromicina. El tratamiento conmodificacin de la fagocitosis in vivo por el antibitico macrlido azitromicina. El tratamiento conazitromicina durante 10 y 20 das disminuy la capacidad de depuracin del sistema fagoctico-mononuclear.azitromicina durante 10 y 20 das disminuy la capacidad de depuracin del sistema fagoctico-mononuclear.azitromicina durante 10 y 20 das disminuy la capacidad de depuracin del sistema fagoctico-mononuclear.azitromicina durante 10 y 20 das disminuy la capacidad de depuracin del sistema fagoctico-mononuclear.azitromicina durante 10 y 20 das disminuy la capacidad de depuracin del sistema fagoctico-mononuclear.PALABRAS CLAVE: Fagocitosis. Depuracin. Modelo murino. Microorganismos extracelulares. Microorganismos intracelulares.Azitromicina. Tratamiento de larga duracin.

A B S T R A C TA B S T R A C TA B S T R A C TA B S T R A C TA B S T R A C T

The blood stream clearance of particles is a measure of the functional capacity of the mononuclearThe blood stream clearance of particles is a measure of the functional capacity of the mononuclearThe blood stream clearance of particles is a measure of the functional capacity of the mononuclearThe blood stream clearance of particles is a measure of the functional capacity of the mononuclearThe blood stream clearance of particles is a measure of the functional capacity of the mononuclearph ag oc yt ic sy st em , wh ic h is re sp on sib le fo r th e sy ste mi c el im in at io n of pa th og en ic mi cr oo rg an is ms ,ph ag oc yt ic sy st em , wh ic h is re spo ns ib le fo r th e sy ste mi c el im in at io n of pa th og en ic mi cr oo rg an is ms ,ph ag oc yt ic sy st em , wh ic h is re sp on sib le fo r th e sy ste mi c el im in at io n of pa th og en ic mi cr oo rg an is ms ,ph ag oc yt ic sy st em , wh ic h is re spo ns ib le fo r th e sy ste mi c el im in at io n of pa th og en ic mi cr oo rg an is ms ,ph ag oc yt ic sy st em , wh ic h is re sp on sib le fo r th e sy ste mi c el im in at io n of pa th og en ic mi cr oo rg an is ms ,immunocomplexes and apoptotic cells. This capacity may be altered by biological reponse modifiersresponse,immunocomplexes and apoptotic cells. This capacity may be altered by biological reponse modifiersresponse,immunocomplexes and apoptotic cells. This capacity may be altered by biological reponse modifiersresponse,immunocomplexes and apoptotic cells. This capacity may be altered by biological reponse modifiersresponse,immunocomplexes and apoptotic cells. This capacity may be altered by biological reponse modifiersresponse,in which numerous antimicrobial agents are present. In this work, the effectiveness of the measurementin which numerous antimicrobial agents are present. In this work, the effectiveness of the measurementin which numerous antimicrobial agents are present. In this work, the effectiveness of the measurementin which numerous antimicrobial agents are present. In this work, the effectiveness of the measurementin which numerous antimicrobial agents are present. In this work, the effectiveness of the measurementof clearance capacity was compared in BALB/c mice that were inoculated with different microorganismsof clearance capacity was compared in BALB/c mice that were inoculated with different microorganismsof clearance capacity was compared in BALB/c mice that were inoculated with different microorganismsof clearance capacity was compared in BALB/c mice that were inoculated with different microorganismsof clearance capacity was compared in BALB/c mice that were inoculated with different microorganisms(a yeast, two extra and intracellular gram-positive bacteria, and two extra and intracellular gram-negative(a yeast, two extra and intracellular gram-positive bacteria, and two extra and intracellular gram-negative(a yeast, two extra and intracellular gram-positive bacteria, and two extra and intracellular gram-negative(a yeast, two extra and intracellular gram-positive bacteria, and two extra and intracellular gram-negative(a yeast, two extra and intracellular gram-positive bacteria, and two extra and intracellular gram-negativebacteria). As a means to studying the in vivo modification of phagocytosis by the macrolid antibiotic,bacteria). As a means to studying the in vivo modification of phagocytosis by the macrolid antibiotic,bacteria). As a means to studying the in vivo modification of phagocytosis by the macrolid antibiotic,bacteria). As a means to studying the in vivo modification of phagocytosis by the macrolid antibiotic,bacteria). As a means to studying the in vivo modification of phagocytosis by the macrolid antibiotic,azithromycin, the yeast Cazithromycin, the yeast Cazithromycin, the yeast Cazithromycin, the yeast Cazithromycin, the yeast Candida albicansandida albicansandida albicansandida albicansandida albicans was chosen for its appropriate clearance kinetics and its naturalwas chosen for its appropriate clearance kinetics and its naturalwas chosen for its appropriate clearance kinetics and its naturalwas chosen for its appropriate clearance kinetics and its naturalwas chosen for its appropriate clearance kinetics and its naturalresistance to antibacterial agents. Treatment with azithromycin for 10 and 20 days reduced clearanceresistance to antibacterial agents. Treatment with azithromycin for 10 and 20 days reduced clearanceresistance to antibacterial agents. Treatment with azithromycin for 10 and 20 days reduced clearanceresistance to antibacterial agents. Treatment with azithromycin for 10 and 20 days reduced clearanceresistance to antibacterial agents. Treatment with azithromycin for 10 and 20 days reduced clearancecapacity of the mononuclear phagocytic system.capacity of the mononuclear phagocytic system.capacity of the mononuclear phagocytic system.capacity of the mononuclear phagocytic system.capacity of the mononuclear phagocytic system.KEYWORDS: Phagocytosis. Clearance. Mouse model. Extracellular microorganisms. Intracellular microorganisms. Azithromycin.Long-term therapy.

1. INTRODUCCIN

La fagocitosis es un mecanismo crucial enla inmunidad innata1. Las clulas fagocticas

1. INTRODUCTION

Phagocytosis is a crucial mechanism in innateimmunity1. Phagocytic cells are chemotacti-


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ALKOUWALTLI K, LEIVA M, RUIZ-BRAVO A, JIMNEZ-VALERA M

son atradas quimiotcticamente hacia los fo-cos inflamatorios inducidos por la presenciade agentes patgenos en los tejidos. Alterna-tivamente, el sistema fagoctico mononuclearconsiste en un nmero de fagocitos fijos en

ciertos rganos, como bazo e hgado, queretiran las partculas extraas presentes en lasangre2. Esta actividad de depuracin de lasangre es un parmetro importante de la ca-pacidad de defensa del organismo frente amicroorganismos, inmunocomplejos y clulasmuertas por apoptosis. Por tanto, una defi-ciencia en la capacidad de depuracin tienecomo consecuencia un mayor riesgo de infec-ciones y de trastornos inmunopatolgicos3,4 .Se han propuesto diversas tcnicas para el

estudio experimental de la depuracin de lasangre por fagocitos. Algunas se basan en laretencin de partculas inertes, como carbncoloidal, por los fagocitos tisulares. Otras uti-lizan microorganismos vivos, que se inoculanpor va intravenosa para cuantificar la tasa dedepuracin del torrente circulatorio en fun-cin del tiempo. Los llamados agentes modi-ficadores de la respuesta biolgica o BRMs(de Biological Response Modifiers) se defi-nen por los efectos moduladores que ejercenen el sistema inmune5. Uno de los mecanis-mos ms comunes de accin de los BRMs esa travs de sus efectos sobre las clulas fago-cticas. Los BRMs pueden modificar la capa-cidad de fagocitosis, la eficiencia de los me-canismos microbicidas intracelulares y lasecrecin de citokinas por fagocitos. Esto tie-ne consecuencias directas sobre la resistenciaa la infeccin, e indirectas por la accin in-munorreguladora de las citokinas que modu-lan tanto la respuesta de anticuerpos como lainmunidad celular5.Actualmente es bien cono-

cido que numerosos agentes antimicrobianosposeen actividad BRM6. Muchos de ellos ejer-cen efectos sobre fagocitos7. Los antibiticosmacrlidos poseen interesantes propiedadesinmunomoduladoras, posiblemente relaciona-das con su acumulacin intracelular a con-centraciones varias veces superiores a lasextracelulares, especialmente en fagocitos8. Eltratamiento con macrlidos es beneficioso paraenfermos con patologa de inflamacin pul-monar crnica, como fibrosis qustica, pan-

bronquiolitis difusa, bronquiectasia y asma9,10.Las propiedades anti-inflamatorias de los

cally attracted towards inflammatory sourcesinduced by the presence of pathogenic agentsin tissues. Alternatively, the mononuclearphagocytic system consists of a number offixed phagocytes in certain organs, such as

the spleen and liver, which remove foreignparticles from blood2. This blood clearanceactivity represents an important parameter inthe defence capacity of the organism againstmicroorganisms, immunocomplexes and celldeath due to apoptosis. Accordingly, a defi-ciency in clearance capacity may bring abouta greater risk of susceptibility to infection andimmunopathological disorders3,4. Several tech-niques in the experimental study of bloodclearance by phagocytes have been proposed.

Some of these have been based on the reten-tion of inert particles, such as colloidal car-bon, by tissue phagocytes. Others use livemicroorganisms, which are inoculated intra-venously, in order to quantify blood streamclearance rates in terms of time. The so ca-lled modifying agents of biological respon-se, (Biological Response Modifiers(BRMs)are defined by the modulating effects on theimmune system5. One of the most commonaction mechanisms of BRMs is the effects thatthese have on phagocyte cells. BRMs canmodify phagocyte capacity, the effectivenessof intracellular microbicidal mechanisms andthe secretion of mechanisms cytokines byphagocytes. These have direct effects on in-fection resistance, and indirect effects due tothe immunoregulatory effect of the cytokines,which modulate both antibody response andcellular immunity5. It is currently well knownthat numerous antimicrobial agents present BRMactivity6. Many of these affect the phagocytes7.The macrolide antibiotics possess interesting

immunomodulating properties, which are pos-sibly related to an intracellular accumulationat concentrations that are several times higherthan extracellular concentrations, especially inphagocytes8. Treatment with macrolides isbeneficial for patients with chronic pulmona-ry inflammation, such as cystic fibrosis, diffu-se panbronchiolitis, bronchiectasis and asth-ma9,10. The anti-inflammatory properties ofmacrolides are attributed to the interferencewith the production of proinflammatory cyto-

kin production by phagocytes8,10.The objecti-ve of the present work is to comparatively


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macrlidos se atribuyen a interferencia con laproduccin de citokinas pro-inflamatorias porfagocitos8,10. El objetivo del presente trabajoes determinar comparativamente la cintica dedepuracin de distintos microorganismos, ex-

tra- e intracelulares, y aplicar esta metodolo-ga al estudio de la influencia de la adminis-tracin durante periodos prolongados delmacrlido azitromicina sobre la actividad fa-goctica in vivo.

2. MATERIALES Y MTODOS

Animales

Se utilizaron ratones BALB/c, hembras, de5 a 6 semanas de edad y de alrededor de 20g de peso corporal, procedentes de la Unidadde Animales de Experimentacin, pertenecienteal Centro de Instrumentacin de la Universi-dad de Granada. Los animales se mantuvie-ron, libres de patgenos, con acceso ad libi-tum a agua y pienso estndar estriles.

Microorganismos

Se usaron cepas de la coleccin de culti-vos del Departamento de Microbiologa, per-tenecientes a las especies Escherichia coli,Salmonella enterica serovar Typhimurium,Micrococcus luteus, Listeria monocytogenesy Candida albicans. Las cepas se cultivaronen Agar tripticasa soja (TSA; Difco) inclinadoen tubos. Tras 24 de incubacin a 37C, seprepararon suspensiones en tampn fosfatosalino isotnico, a pH 7.2 (PBS), estril. Laconcentracin microbiana se ajust por turbi-

dez a aproximadamente 107 microorganismospor ml.

Ensayo de depuracin

Los animales fueron sometidos a una lige-ra anestesia etrea, para proceder con como-didad a la inoculacin de la suspensin mi-crobiana, por va intravenosa. Cada ratn recibiuna nica inyeccin de 100 l en el plexo

venoso retrorbital del ojo izquierdo, registran-do el tiempo en un cronmetro. Transcurridos

determine kinetic clearance of different extraand intracellular microorganisms, and to applythis methodology to the study of the influen-ce of the macrolides azithromycin over pro-longed periods of time on in vivo phagocyte

activity.

2. MATERIAL AND METHODS

Animals

Female BALB/c mice of 5 to 6 weeks ofage and about 20g in body weight were used.These were obtained from the Animal Expe-rimentation Unit of the University Instrumen-

tation Centre of Granada. The animals weremaintained free of pathogens, with ad libitumaccess to water and sterile Standard chow.

Microorganisms

Strains of the species Escherichia coli, Sal-monella enterica serovar Typhimurium, Micro-coccus luteus, Listeria monocytogenesand Can-dida albicans were obtained from themicrobiology departments culture collection.These were subsequently cultivated in trypticsoy agar (TSA;Difco) in tubes in slanted po-sition. After 24 hrs of incubation at 37C, sterileisotonic phosphate buffered saline suspensio-ns (PBS) at ph 7.2 were prepared. Microbialconcentration was adjusted to approximately107 microorganisms per ml by turbidimetry.

Clearance testing

The animals were given light ether anaes-thesia, in order to carry out intravenous mi-crobial suspension inoculation more easily. Eachmouse was given a single injection of 100lin the retro orbital venous plexus of the lefteye and time was measured with a stop wat-ch. At 1, 5 & 10 min from inoculation, etheranaesthesia was repeated, in order to carryout blood extractions of a volume of approxi-mately 150l, using a sterile Pasteur pipette.So as to avoid coagulation, the blood obtai-

ned was transferred to tubes containing 10 lof heparin. From each blood sample two se-


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1, 5 y 10 min de la inoculacin, se repiti laanestesia etrea para realizar extracciones devolmenes de sangre de alrededor de 150 l,mediante pipeta pasteur estril. La sangre sellev a tubos con 10 l de heparina, con el

objeto de evitar la coagulacin. De cada muestrade sangre se realizaron dos diluciones seria-das (10-1 y 10-2) en PBS estril, y 100 l dela muestra original y de cada dilucin se de-positaron en la superficie de placas de TSA yse extendieron por ella mediante una esptulade vidrio. Las placas se incubaron 24 h a 37C,tras lo cual se procedi al recuento de colo-nias. Se dieron como vlidos los recuentoscomprendidos entre 30 y 300 colonias porplaca. Los resultados se expresaron como

unidades formadoras de colonias (CFU) porml de sangre.

Tratamiento con azitromicina

El macrlido azitromicina se obtuvo co-mercialmente (ZitromaxR 500 mg de Pfizer),en forma de polvo destinado a la preparacinde disoluciones para perfusin. Cada animalrecibi una dosis de 7.1 mg por Kg y da(extrapolada de las dosificaciones usadas enterapia humana), por va intraperitoneal. Seexamin el efecto de tratamientos de 10 y 20das de duracin. En cada caso, el ltimo dadel tratamiento, los animales fueron sacrifica-dos, junto con un lote similar tratado duranteel mismo tiempo con el mismo volumen delvehculo acuoso.

Anlisis estadstico de los resultados

La significacin estadstica de las diferen-cias entre animales tratados y controles notratados se determin mediante la prueba dela t de Student. Los lotes experimentales cons-taron de 4 animales. Los valores de P < 0.05se consideraron significativos.

3. RESULTADOS

La primera parte de este estudio se centr

en la comparacin de diversos microorganis-mos de prueba para el ensayo de fagocitosis

rialized dilutions (10-1 & 10 -2) in sterile PBSwere carried out 100 l of original sampleeach dilution and cach were then depositedover the surface of the TSA dishes and spreadusing a glass spatula. The dishes were incu-

bated for 24 hrs at 37C, after which a countof the number of colonies was carried out.Counts of between 30 and 300 colonies perdish were considered as valid. The resultswere expressed in terms of colony formingunits (CFU) per ml of blood.

Azithromycin treatment

The macrolide antibiotic, azithromycin, was

obtained commercially (ZitromaxR

500 mg,Pfizer), in powdered form for use in the pre-paration of perfusion solution. Each animalreceived a dose of 7.1mg per kg per day (ex-trapolated from doses used in human thera-py), through intraperitoneal injection. The effectof the treatment after 10 and 20 days wasexamined. In each case, the animals weresacrificed on the last day of treatment toge-ther with a similar batch that were treated forthe same period of time with the same volu-me of aqueous vehicle.

Statistical analysis of the results

The statistical significance of the resultsobtained from the differences between treatedand untreated control animals was determinedusing the Student t test. Experimental batchesconsisted of 4 animals. The values of P < 0.05were considered as significant.

3. RESULTS

The first part of this study was focussed onthe comparison of varying test microorganis-ms for the in vivo phagocytosis tests, in whi-ch clearance of previously intravenously ad-ministered particles was measured in terms oftime. This comparison was carried out using ayeast, Candida albicans, and four bacteriaspecies. Two of these were Gram-negative,

one an extracellular type (Escherichia coli) andthe other intracellular (Salmonella enterica


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in vivo, en el que se mide la depuracin, enfuncin del tiempo, de partculas previamenteadministradas por va intravenosa. En esta com-paracin se incluyeron una levadura, Candidaalbicans, y cuatro bacterias, dos Gram-nega-

tivas, de las cuales una extracelular (Escheri-chia coli) y otra intracelular (Salmonella ente-rica serovar Typhimurium), y dos Gram-positivas, una extracelular (Micrococcus lu-teus) y otra intracelular (Listeria monocytoge-nes).

La Figura 1 muestra los resultados de losensayos realizados con C. albicans. Las tasasde depuracin (referidas al recuento inicial deCFU, realizado al minuto de la inoculacin)fueron del 65% a los 5 minutos y del 94% a

los 10 minutos (Figura 1A). Al representar losvalores de CFU en escala logartmica (Figura1B), se obtuvo una buena rectificacin de lagrfica de depuracin, con un valor R2 = 0.9918,y la siguiente ecuacin:

Y = -0.139 X + 4.330

FIGURA 1FIGURA 1FIGURA 1FIGURA 1FIGURA 1. Cintica de depuracin de C. albicans.

FIGURE 1FIGURE 1FIGURE 1FIGURE 1FIGURE 1. Kinetic clearance of C. albicans.

0

5

10

15

0 5 10 15

CFUpormldes

angre(x103)

A

-

1

2

3

4

5

6

0 5 10 15

Tiempo (min) post-inoculacin

CFUpormlde

sangre(log)

B

Los resultados de depuracin de E. coli sepresentan en la Figura 2. Las tasas de depu-racin fueron del 68% a los 5 minutos y del90% a los 10 minutos (Figura 2A). En escalasemilogartmica, se obtuvo un valor R2 = 0.9966

y la siguiente ecuacin:Y = -0.110 X + 5.393

FIGURA 2.FIGURA 2.FIGURA 2.FIGURA 2.FIGURA 2. Cintica de depuracin de E. coli

FIGURE 2.FIGURE 2.FIGURE 2.FIGURE 2.FIGURE 2. Kinetic clearance of E. coli

0

50

100

150

200

250

0 5 10 15

CFUpormldesangre(x103)

A

empo m n) post- nocu ac n

1

2

3

4

5

6

0 5 10 15


CFUpormldesangre(log)

B

serovar Typhimurium). Similarly, an extrace-llular and intracellular Gram-positive bacteriawere used, these being Micrococcus luteus andListeria monocytogenes respectively.

Figure 1 shows the results of the tests ca-

rried out with C. albicans. Clearance rates (withinitial CFU counts carried out at one minuteafter inoculation taken as reference) were foundto be 65% at 5 minutes and 94% at 10 minu-tes (Figure 1A). By representing CFU valueson a logarithmic scale (Figure 1B), a goodrectification of the clearance graph was obtai-ned, with a R2 = 0.9918, value, and the follo-wing equation:

Y = -0.139 X + 4.330

The results for E. coli clearance are repre-sented in Figure 2. Clearance rates from 68%at 5 minutes and 90% at 10 minutes wereobtained (Figure 2A). On a semilogarythmicscale, a R2 = 0.9966 value was obtained with

the following equation:Y = -0.110 X + 5.393


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La Figura 3 muestra los resultados obteni-dos con M. luteus. Se encontraron unas tasasde depuracin 98% (5 minutos) y 99% (10minutos) (Figura 3A). Al pasar a la escalasemilogartmica, la rectificacin no fue bue-

na, debido al gran descenso en el nmero deCFUs registrado ya a los 5 minutos, con R2 =0.7845 y la siguiente ecuacin:

Y = -0.219 X + 5.075

FIGURA 3. Cintica de depuracin de M. luteus

FIGURE 3. Kinetic clearance of M. luteus

0

50

100

150

200

0 5 10 15

CFUpermlofblood(x103)

A


1

2

3

4

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Time (min) post-innoculation

CFUpermlof

blood(log)

B

La Figura 4 corresponde a S. enterica sero-var Typhimurium. Las tasas de depuracinfueron 47% (5 minutos) y 58% (10 minutos).En escala semilogartmica, se obtuvo R 2 =0.8989. La ecuacin fue:

Y = -0.041 X + 4.859

FIGURA 4. Cintica de depuracin de S. enterica serovar Typhimurium.

FIGURE 4. kinetic clearance of S. enterica serovar Typhimurium.

empo m n) post- nocuac n

1

2

3

4

5

6

0 5 10 15


CFU

pormldesangre(log)

B

0

20

40

60

80

100

0 5 10 15

CFU

pormldesangre(x103)

A

En la Figura 5 se presentan los datos obte-nidos con L. monocytogenes. Se registrarontasas de depuracin de 67% (5 minutos) y92% (10 minutos). La escala semilogartmicagener una recta (R 2 = 1.0000), con la s i-guiente ecuacin:

Y = -0.122 X + 5.671

Figure 3 shows the results obtained fromM. luteus, where clearance rates of 98% at 5minutes and 99% at 10 minutes were obtai-ned. (Figure 3A). After converting to the se-milogarythmic scale, rectification values were

not found to be good, due to a sharp decreasein the number of recorded CFUs at 5 minutes,with con R2 = 0.7845 and the following equa-tion:

Y = -0.219 X + 5.075

Figure 4 corresponds to S. enterica serovarTyphimurium. Clearance rates of 47% at 5minutes and 58% at 10 minutes were obtai-ned. R2 = 0.8989 was obtained on the semi-logarythmic scale, giving the equation:

Y = -0.041 X + 4.859

Figure 5 shows the data obtained for L.monocytogenes. Clearance rates of 67% at 5minutes and 92% at 10 minutes were recor-ded. The semilogarythmic scale gave a straig-ht of (R2 = 1.0000), with the following equa-tion:

Y = -0.122 X + 5.671


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49TCNICAS DE ESTUDIO DE FAGOCITOSIS IN VIVO Y SU APLICACIN EN LA INVESTIGACIN EN LA INVESTIGACIN...PROCEDURES TO STUDY THE IN VIVO PHAGOCY TOSIS AND APPLICATION FOR THE INVESTIGATIN OF IMMUNOMODULATORY ...

El estudio com parativo de los diferentesmicroorganismos de prueba presenta unas ta-

sas de depuracin, a 5 y 10 minutos, de ran-gos similares para C. albicans, E. col i y L.monocytogenes. M. luteus fue muy sensible ala fagocitosis in vivo, siendo eliminado ya enun 98% a los 5 m inutos de la inoculacin: poreste motivo, la regresin en la grfica semilo-gartm ica no f ue aceptable. Por el cont rario,S. enterica serovar Typhimurium demostr unanotable resistencia a la depuracin, ya que latasa a 10 minutos f ue inf erior incluso a lastasas a 5 minutos de los otros microorganis-

mos.

FIGURA 5. Cintica de depuracin de L . monocytogenes

FIGURE 5. Kinetic clearance of L. monocytogenes

0

100

200

300

400

0 5 10 15

Tiem o min ost-inoculacin

CFUpormldesangre

(x103)

A

empo m n pos - nocu ac n

1

2

3

4

5

6

0 5 10 15


CFUpormldesangr

e(log)

B

FIGURA 6. Efecto del tratamiento con Azitromicina durante 10 das en la cintica de depuracin de C.

albicans

FIGURE 6 Effect of Azithromycin treatment for 10 days on kinetic clearance of C. albicans.

0

5

10

15

20

25

0 5 10 15

CFUpermlofblood(x103)

Control

Treated

A


1

2

3

4

5

6

0 5 10 15

Time (min) post-innoculation

CFU

permlofblood(log)

B

Entre los tres microorganismos con un

comportamiento aceptable, la levadura C. al-

bicans fue seleccionada para la segunda parte

del trabajo, ya que su condicin de clula

eucariota le hace resistente a la mayora de

los agentes antibacterianos a ensayar como

BRMs. Se realizaron, pues, ensayos de depu-

racin de C. albicans en ratones tratados con

The comparative study of the different test

microorganisms presented clearance rates at 5

and 10 minutes within similar ranges for C.albicans, E. coli and L. monocytogenes. M.

luteus was found to be sensitive to in vivo

phagotosis, eliminated to the degree of 98%

at 5 minutes after inoculation: consequently,

regression on the semilogarithmic graph was

not acceptable. Conversely, S. enterica sero-

var Typhimurium showed noteworthy resis-

tance to clearance, given that the rates obtai-

ned at 10 minutes were even lower than those

obtained at 5 minutes from other microorga-

nisms.

Of the three microorganisms that present

acceptable behaviour, the yeast C. albicans

was chosen for the second part of the study,

given that as a eukaryotic cell, it is resistant

to most antibacterial agents to be tested as

BRMs. Clearance t est with C. albicans were

therefore carried out in mice treated with azi-

thromycin for 10 and 20 days. The re sults


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azitromicina durante 10 y 20 das. Los resul-tados de las determinaciones tras 10 das sepresentan en la Figura 6. El tratamiento con elantimicrobiano no afect a las rectas de re-gresin: los valores promedio de R 2 fueron

0.907 0.1092, para los animales testigos, y0.997 0.0017 para los tratados (diferenciasno significativas). Sin embargo, la tasa dedepuracin en los primeros 5 minutos fue sig-nificativamente mayor en los animales testi-gos (81%) que en los tratados (65%) (P< 0.05).Por tanto, el tratamiento con azitromicinadurante 10 das retard la cintica de depura-cin de C. albicans, aunque a los 10 minutoslas diferencias perdieron la significacin.

obtained after 10 days are presented in Figure6. Antimicrobial treatment did not affect thestraights of regression: the average values forR2 were 0.907 0.1092, for control and 0.997 0.0017 for treated animals (no significant

difference). However, clearance rates withinthe first 5 minutes were significantly higher incontrol (81%) than in treated animals (65%)(P < 0.05). Therefore, azithromycin treatmentover a period of 10 days delayed kinetic clea-rance of C. albicans, even though at 10 minu-tes, significant differences were not apparent.

-

1

2

3

4

5

6

0 5 10 15


CFUpormldesangre(log)

B

0

5

10

15

20

25

30

0 5 10 15

CFUpormldesangre(x103)

Testigos

Tratados

A

FIGURA 7.FIGURA 7.FIGURA 7.FIGURA 7.FIGURA 7. Efecto del tratamiento con Azitromicina durante 20 das en la cintica de depuracin de C.albicans.

FIGURE 7FIGURE 7FIGURE 7FIGURE 7FIGURE 7. Effect of Azithromycin treatment for 20 days on kinetic clearance of C. albicans

A los 20 das de tratamiento con azitromi-cina, tampoco se registraron diferencias signi-ficativas entre las rectas de regresin, con valoresde R2 de 0.987 0,0141 para los animalestestigos y 0.999 0.0346 para los tratados(Figura 7). En cuanto a las tasas de depura-cin, tambin aqu, a los 5 minutos, fue ma-yor el promedio de los animales testigos (80%)

que el de los tratados (61%), si bien la disper-sin de los datos impidi llegar a diferenciassignificativas.

4. DISCUSIN

La depuracin de carbn coloidal se hautilizado desde hace aos como una medidade la capacidadfuncional del sistema fagocti-co-mononuclear11,12, y sigue recurrindose a

ella en la actualidad13, 14. Algunos trabajos hanmostrado una correlacin entre este parme-

After 20 days of azithromycin treatment,no significant differences among the straightsof regression were observed. R2 values of 0.987 0,0141 for control animals and y 0.999 0.0346 for treated animals were obtained (Fi-gure 7). Similarly, with regard to clearancerates, at 5 minutes the highest averages wereobtained for control animals (80%) in compa-

rison with treated animals (61%). However,the dispersion of data did not produce signi-ficant differences.

4. DISCUSSION

The clearance of colloidal carbon has beenused for many years as a measure of the func-tional capacity of the phagocytic-mononuclearsystem11,12, and is still being used today 13, 14.

Some studies have shown a correlation bet-ween this parameter and the resistance to ex-


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tro y la resistencia a infecciones experimenta-les por bacterias y levaduras15. Sin embargo,el uso de microorganismos vivos constituyeuna aproximacin ms representativa de lacapacidad defensiva real del organismo.

La inmunidad innata se basa en la capaci-dad de determinadas clulas y molculas parareconocer estructuras comunes a amplios gru-pos de microorganismos, denominadas PAMPs(pathogen-associated molecular patterns)16.Se han identificado diversos PAMPs en lasuperficie microbiana: la murena bacteriana,el lipopolisacrido (LPS) y las lipoprotenasde bacterias Gram-negativas, los cidos lipo-teicoicos (bacterias Gram-positivas), los lipoara-binomananos de micobacterias, el zimosn de

levaduras, los glicoinositolfosfolpidos de Trypa-nosoma cruzi, son diversos ejemplos de PAMPssuperficiales17. La interaccin de los PAMPsde la superficie microbiana con sus recepto-res (PRRs, pattern recognition receptors) tienegran importancia en la fagocitosis. AlgunosPRRs estn ubicados en la superficie de clu-las fagocticas, e intervienen en la unin demicroorganismos como paso previo a la fago-citosis. Otros PRRs residen en molculas plas-mticas, pertenecientes a sistemas como elcomplemento, que se activan en presencia demicroorganismos, liberando opsoninas quefacilitan la fagocitosis. Este tipo de interac-ciones no se pueden reproducir con partculasinertes. Sin embargo, una limitacin inherenteal uso de microorganismos es la posible inter-ferencia de anticuerpos con capacidad opso-nizante, que podran estar presentes en algu-nos animales previamente al ensayo, comoconsecuencia de contactos subclnicos o dereacciones cruzadas con antgenos ambienta-les. Esta limitacin debe ser considerada cuando

se trabaja con microorganismos ubicuos comoE. coli18.

No hay antecedentes en la bibliografa sobrela comparacin de la depuracin in vivo depatgenos extra- e intracelulares. Nuestros datosmuestran divergencia entre las dos bacteriasintracelulares ensayadas, L. monocytogenes yS. enterica serovar Typhimurium. La depura-cin de L. monocytogenes fue similar a la deE. coli, mientra que S. enterica serovar Typhi-murium revel una notable resistencia a la

depuracin. Ambos patgenos intracelularesse caracterizan por su capacidad para sobre-

perimental infections produced by bacteria andyeast15. However, the use of live microorga-nisms constitutes a more representative approachto the determination of an organisms defencecapacity.

Innate immunity is based on the capacityof determined cells and molecules to recogni-se common to wide ranging groups of micro-organisms, known as PAMPS(pathogen-as-sociated molecular patterns)16. NumerousPAMPS have been identified on the microbialsurface: the murine bacteria, the lipopolysac-charides (LPS) and the lipoproteins of Gram-negative bacteria, lipoteichoic acids (Gram-positive bacteria), lipoarabinomannan frommycobacterium, zymosan from yeasts, glyco-

inositolphospholipids from Trypanosoma cruzi,are numerous examples of superficial PAMPS17.The interaction between PAMPS and the mi-crobial surface and their receptors(PRRs,pattern recognition receptors) is hig-hly important in phagocytosis. Some PRRs aresituated on the surface of phagocytic cells,and have a part to play in the binding ofmicroorganisms prior to phagocytosis. OtherPRRs reside in the plasmatic molecules, be-longing to such systems as the complement,which are activated in the presence of micro-organisms, liberating opsonins, which facili-tate phagocytosis. These types of interactionscannot be reproduced with inert particles.However, an inherent limitation in the use ofmicroorganisms is the possible interference fromantibodies possessing opsonizing capacity,which could be present in some animals befo-re testing, due to sub clinical contact or cros-sed reactions with environmental antigens. Thislimitation should be considered when workingwith ubiquitous microorganisms such as E.

coli18.There are no bibliographical precedents on

the comparison between in vivo clearance ofextra and intracellular pathogens. Our datashows differences between the two intracellu-lar bacteria tested, L. monocytogenes and S.enterica serovar Typhimurium. The clearan-ce of L. monocytogenes was similar to that ofE. coli, while S. enterica serovar Typhimu-rium showed a noteworthy resistance to clea-rance. Both intracellular pathogens are cha-

racterised by their survival capacity insidemacrophages, except when activated by cyto-


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vivir en el interior de macrfagos, exceptocuando estos estn activados por accin decitokinas. En principio, esta resistencia nodebiera afectar a la cintica de depuracin, esdecir, a la capacidad de los macrfagos fijos

de bazo e hgado para retirar bacterias de lasangre por fagocitosis, aunque las bacteriasintracelulares sobreviviran en ambos rganos,donde podran proliferar y formar abcesos. Sinembargo, en el caso de S. enterica serovarTyphimurium, parece que existe algn factorde virulencia adicional, que permite a la bac-teria evitar la fagocitosis. Este factor podraser la naturaleza de la cadena O del LPS, yaque se ha descrito que una cepa de S. entericaserovar Typhimurium con un polisacrido O

capaz de activar eficientemente la va alterna-tiva del complemento, es eficazmente depura-da de la sangre, en comparacin con otra cepacuya cadena O no es eficiente en la activa-cin del complemento, siendo esta ltima msvirulenta19. Podemos concluir que la depura-cin por fagocitosis in vivo se ve influencia-da por factores de virulencia concretos (comola naturaleza del LPS en el caso de bacteriasGram-negativas) en mayor extensin que porla condicin de patgenos extra- o intracelu-lares o la estructura de la pared (definida porla tincin de Gram).

La eleccin de C. albicans para medir elefecto BRM de la azitromicina se bas en losdatos de cintica de depuracin, as como ensu resistencia frente a este agente antibacte-riano, in vitro20 e in vivo21,22.

La azitromicina ha sido utilizada en trata-mientos de larga duracin, en enfermos defibrosis qustica (al menos 3 meses) y pan-bronquiolitis difusa (al menos 6 meses), conbuenos resultados clnicos atribuibles, al me-

nos en parte, a sus efectos antiinflamatorios23.Sin embargo, no hay suficientes evidenciasexperimentales que soporten claramente estahiptesis, dada la escasez de estudios sobreactividad BRM en este tipo de tratamientos.

Al igual que otros macrlidos, la azitromi-cina se concentra notablemente en el interiorde los fagocitos24, pero no hay concordanciarespecto a los posibles efectos de esta acumu-lacin en las funciones fagocticas. Los dis-tintos parmetros medidos en cada caso pue-

den ser responsables de estas discrepancias.La medida de quimioluminiscencia en neutr-

kins. In principle, this resistance should notaffect clearance kinetics, that is to say, thecapacity of fixed macrophages in the spleenand liver to withdraw bacteria from bloodthrough phagocytosis. However, these intra-

cellular bacteria would survive in both organs,where they could proliferate and form absces-ses. However, in the case ofS. entericaserovarTyphimurium, an additional virulence factorseems to exist, which allows the bacteria toavoid phagocytosis. This factor could be attri-buted to the nature of the LPS O chain, giventhat a strain of S. enterica serovar Typhimu-rium with an O polysaccharide that is capableof efficiently activating the alternative com-plement pathway, is efficiently cleared from

blood in comparison with a strain whose Ochain does not efficiently activate the com-plement, with the last of these being the mostvirulent19. In conclusion, it can be said thatclearance through in vivo phagocytosis is in-fluenced to a greater extent by concrete viru-lence factors (such as the nature of the LPS inthe case of Gram-negative bacteria), rather thanthe extra or intracellular nature of pathogensor the structure of the wall (defined by Gramstaining).

The decision to use C. albicans, as a meansto measuring the effect of BRM of azithromy-cin, was based on kinetic clearance data, aswell as its resistance to this antibacterial agentin both in vitro20 and in vivo21,22 conditions.

Azithromycin has been used in long termtreatment for patients presenting cystic fibro-sis (at least 3 months) and diffuse panbron-chiolitis, (at least 6 months), with good clini-cal results that may be attributable, at least inpart, to its antiinflammatory effects23. Howe-ver, there is insufficient experimental eviden-

ce to clearly support this hypothesis, giventhe scarcity of studies carried out on the roleof BRM activity in this type of treatment.

As in the case of other macrolides, azi-thromycin is concentrated to a noticeable degreewithin the interior of phagocytes24. However,there is no agreement as to the possible effectsof such an accumulation on phagocytic func-tions. The differing parameters measured ineach case may be responsible for these dis-crepancies. Chemiluminiscence in human neu-

trophiles was not altered by azithromycin treat-ments in ex vivo20,25 and in vitro25 tests.


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filos humanos no se alter por tratamientoscon azitromicina en ensayos ex vivo20,25 e invitro25. En cambio, se ha descrito un efectosupresor del antibitico ex vivo en la incor-poracin de bacterias fluorescentes medida por

citometra de flujo26 y una modulacin de losperfiles de citokinas en ensayos in vitro, coninhibicin de la respuesta de clulas T hel-per 1 e induccin de la respuesta de T hel-per 227. Por el contrario, la azitromicina po-tenci la fagocitosis y la capacidad microbicidade la lnea de macrfagos J774.128, y su ad-ministracin a enfermos tratados con ciclos-porina mejor la actividad fagoctica daadapor este agente inmunosupresor29. Reciente-mente, se ha sugerido que el tratamiento con

azitromicina podra tener un efecto bifsico,consistente en una inmunoestimulacin a cor-to plazo, seguida de la inhibicin de la res-puesta inflamatoria9. Este efecto antiinflama-torio ha sido confirmado experimentalmente:el tratamiento de ratones con azitromicinadurante 4 semanas inhibi significativamentelos niveles plasmticos de citokinas proinfla-matorias (factor necrosante de tumores alfa,interleukina 1 beta, interleukina 6) y xidontrico30. Dado que la respuesta inflamatoriase asocia con una estimulacin de la depura-cin de bacterias en sangre31, el efecto antiin-flamatorio de la azitromicina en tratamientosde larga duracin estara en concordancia conla depresin de la depuracin manifestada ennuestros resultados.

En conclusin, la cintica de depuracinde C. albicans inoculada por va intravenosaes un ensayo apropiado para medir los posi-bles efectos de agentes antimicrobianos sobrela fagocitosis in vivo. Su aplicacin al estudiode la actividad BRM del macrlido azitromi-

cina ha mostrado que el tratamiento durante10 y 20 das con este antibitico ejerce unefecto inhibidor sobre la capacidad de depu-racin del sistema fagoctico-mononuclear.

AGRADECIMIENTOS

K. Alkouwatli manifiesta su agradecimien-to al Ministerio de Enseanza Superior de Siria,por la financiacin de su estancia de investi-

gacin en la Universidad de Granada. Los au-tores agradecen su colaboracin al Dr. A. Mar-

Conversely, a suppressing effect of the ex vivoantibiotic on fluorescent bacteria measuredthrough flow cytometry, and a modulation ofthe cytokin profiles in in vitro tests has beendescribed26. This produced the inhibition of

the T helper 1 response and the inductionof the T helper 2 response27. On the otherhand, azithromycin promoted phagocytosis andmicrobicidal capacity of the macrophage lineJ774.128, and its administration in patients treatedwith cyclosporin enhanced phagocytic activi-ty damaged by this immunosuppressing agent29.It has been recently suggested that azithromy-cin treatment could have a biphasic effect,which would consist of short term immunos-timulation, followed by the inhibition of the

inflammatory response9

. This antiinflammatoryeffect has been confirmed experimentally: Azi-thromycin treatment in mice over a period of4 weeks was found to significantly inhibitplasmatic levels of proinflammatory cytokins(tumour necrosis factor alfa, interleukin 1 beta,interleukin 6) and nitric oxide30. Given thatinflammatory response is associated with thestimulation of blood bacteria clearance31, theantiinflammatory effect of azithromycin in longterm treatments would be consistent with thedecrease in clearance found in our results.

In conclusion, the kinetic clearance of in-travenously inoculated C. albicans is an appro-priate test for the measurement of the possibleeffects of antimicrobial agents on in vivophagocytosis. Its application to the study ofBRM activity of the macrolid azithromycynhas shown that treatment over a period of 10and 20 days with this antibiotic exerts an in-hibitory effect on clearance capacity of thephagocytic mononuclear system.

ACKNOWLEDGEMENTS

K. Alkouwatli would like to thank theMinistry of Higher Education of Syria for thefinancing of his research at the University ofGranada. The authors would like to thank Dr.A. Martnez-Brocal for his collaboration. Thisresearch work has been financed by Aid forYear on Year Activity awarded by the Depar-tment for Innovation, Science and Business

of the Junta de Andalusia to the CVI201 group.


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tnez-Brocal. La investigacin ha sido finan-ciada por la Ayuda para la Actividad Inte-ranual concedida por la Consejera de Inno-vacin, Ciencia y Empresa de la Junta deAndaluca al Grupo CVI201.

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MESA_001_Tec_estudio_fagocitosis[1]