Immunomodulating properties of Argentine plants with ethnomedicinal use.
Five Argentine medicinal plants selected according to folk traditional or ethnomedical use, references and primary pharmacological screening; were chosen to elucidate their immunomodulating properties.
Dichloromethane, methanolic and aqueous extracts of the aerial parts of Achyrocline flaccida (A. flaccida), Eupatoriurn arnottianum (E. arnottianum) and Eupatorioum buniifolium (E. buniifolium), leaves of Lithraea molleoides (L. molleoides) and leaves and stems of Phyllanthus sellowianus (P. sellowianus) were analyzed to disclose their effects on murine normal and tumor cell growth as well as on complement hemolytic activity. Modulation of cell growth was evaluated by tritiated thymidine incorporation while inhibition of complement activity was measured on both classical and alternative complement pathways (CP and AP respectively). The results obtained show that most of the extracts exerted inhibitory effect on tumor as well as on mitogen activated normal spleen cell growth. On tumor cells, [IC.sub.50] ranged between 1-75 [mu]g/ml for most of the extracts with the exception of dichloromethane of L. molleoides and P. sellowianus which required concentrations higher than 100 [mu]g/ml to produce the effect . On mitogenic activated splenocytes, [IC.sub.50] ranged between <1 to 85 [mu]g/ml with the exception of methanolic extract of E. buniifolium or P. sellowianus which were not effective on ConA or LPS stimulated splenocytes respectively. Only E. buniifolium was active on murine normal splenocytes proliferation ([IC.sub.50] 0.5-1.5 [mu]g/ml). Finally, one (7%) of 15 extracts showed inhibition of complement activity on CP and 6 extracts (40%) presented moderate activity on CP. The dichloromethane extract of E. arnottianum was the most active ([IC.sub.50] 5 [mu]g/ml), although remarkable effect was also obtained with dichloromethane and methanolic extracts of P. sellowianus ([IC.sub.50] 11.2 and 17.3 [mu]g/ml respectively). Besides, 2 extracts (13%), dichloromethane extract of E. arnottianum and aqueous extract of P. sellowianus, showed moderate inhibition on AP.
Key words: Argentine plant extracts, immunomodulation, lymphocyte proliferation, complement system
Since ancient times, several diseases have been treated by administration of plant extracts based on traditional medicine (Pezzuto, 1997). Among Argentine medicinal plants, several species have been commonly used in folk medicine for the treatment of immunologic disorders. The possibility of finding new compounds possessing these activities led us to assay extracts of five of them.
Achyrocline flaccida Wein DC (Asteraceae). Commonly named "marcela", "marcela macho". Infusions of the aerial parts are used in the northern regions of Argentina as antispasmodic, febrifuge, stimulant, emenagogue, excitant and antihelmintic (Zardini, 1984). Previous research has described antibacterial and antiviral activities of different compounds present in this plant (Garcia et al. 1999).
Eupatorium arnottianum Griseb (Asteraceae), called "clavel" or "uoue" (Toba Indians). Aerial parts are used in locally folk medicine against gastric pains. Antimicrobial, antiviral and antinociceptive actions have been reported for different extracts of this plant (Clavin et al. 2000).
Eupatorium buniifolium H et Arn (Asteraceae) is a medicinal plant found in Northeastern and Central Argentina. Aerial parts are used as a tincture and for its hepatoprotective and disinfectant properties. Extracts of E. buniifoliun have been reported as having antiviral activity (Garcia et al. 1990).
Lithraea molleoides (Vell.) Engl. Anacardiaceae, known in Argentina as "chichita" or "molle de Cordoba", is a tree which grows in South America, especially in Argentina, Brasil and Uruguay. Decoctions of the leaves are used by rural people of these countries for its medicinal properties which include antiarthritic, haemostatic, diuretic, tonic effect and for the treatment of respiratory diseases (Ratera and Ratera, 1980). Besides, it causes allergic contact dermatitis (Munoz, 1990).
Phyllanthus sellowianus Muller Arg. (Euphorbiaceae), commonly known as "sarandi blanco", is a shrub that grows in northeastern Argentina, southern Brazil, Paraguay and Uruguay. The leaves and stem bark of P. sellowianus have been used extensively in traditional medicine in the form of infusions or decoctions as hypoglycemic, diuretic, laxative and as an antiseptic agent. Its use is so widespread that it has been incorporated to the National Argentine Pharmacopoea (6th Edition). In previous studies we found that an infusion (5% w/v) of the stem bark of P. sellowianus produced a significant hypoglycemic effect when administered orally to streptozotocin-induced diabetic rats (Hnatyszyn et al. 1997). A single oral administration of the infusion (400 mg/kg) produced a significant increase in the urinary excretion in rats. Besides, in murine studies on acute toxicity neither mortality nor neurobehavioral or autonomic profile changes could be observed (Hnatyszyn et al. 1999).
Despite the recent development of new immunosuppressants and anti-tumor drugs, less toxic and more widely applicable agents are needed (Zhang and Li, 2001). In this work we have investigated the biological effects of extracts of the Argentine plants describe above. Leukocytes proliferation and complement system inhibition were used to measure the effect on the immune system with the aim of searching for new active drugs.
* Materials and Methods
Plant material was collected in the central phytogeographic region of Argentine. Voucher specimens are kept at the Museum of Pharmacobotany "Juan A. Dominguez" School of Pharmacy and Biochemistry, University of Buenos Aires, under numbers A. flaccida (BAF 2983), E. arnottianum (BAF 3535), E. buniifo1ium (BAF 5067), L. molleoides (Munoz 1714) and P. sellowianus (BAF 3037).
Hepes, Concanavalin A from Canavalia ensiformis (Con A), lipopolysaccharide from E. Coli 026:B6 (LPS), Tripan blue and 2 mercapto-ethanol were obtained from Sigma Chemical Company (St. Louis, MO, USA), Dichloromethane (DME) and Methanol (M) from Merck (Darmstadt, Germany), RPMI, penicillin, streptomycin and foetal calf serum (FCS) from Gibco (Grand Island, NY) and L-glutamine from Life Technologies (Paisley, U.K.). Other chemical reagents were of commercially available analytical grade.
VSB: Veronal saline buffer, containing 5 mM veronal and 150 mM NaCl at pH 7.35, served as a stock solution for [VSB.sup.2+], containing 0.5 mM [Mg.sup.2+] and 0.15 mM [Ca.sup.2+], and for EGTA-VSB, containing 5 mM [Mg.sup.2+] and 8 mM ethylene glycol-bis-(2-aminoethyl)-tetra-acetic acid (EGTA). Chemicals were obtained from Sigma Aldrich Chemical Co (Madrid, Spain).
RPMI-C: RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 20 mM Hepes Buffer, 100 [mu]g/ml penicillin, 150 [mu]g/ml streptomycin and 5 mM 2-mercapto-ethanol.
Three types of extracts of different polarity were prepared as follows:
(1) Aqueous extract (AE): Infusion at 5% of powdered dry plant material during 20 min. After filtration and concentration, the extract was freeze-dried.
(2) Dichloromethane extract (DME): Maceration of 50 g of powdered dry plant material in 3 x 250 ml of [CH.sub.2] [Cl.sub.2], 24 h each at room temperature.
(3) Methanol extract (ME): Maceration of the marc of the former extraction in 3 X 250 ml of MeOH, 24 h each at room temperature.
The extracts were concentrated to dryness and kept at <4 [degrees]C.
Normal 2- to 4-month-old Balb/C mice of either sex were raised in the animal colony at the School of Pharmacy and Biochemistry, Buenos Aires University. Animals were housed in temperature-controlled rooms and maintained on Cargill pellets and water ad libitum.
Animal studies were conducted in accordance with NIH Guide for the Care and Use of Laboratory Animals. Animal experiments were approved by local ethical committee.
Normal murine splenocytes
Mice were killed by cervical dislocation and spleens removed aseptically. Single cells were prepared by mincing spleen fragments and pressing through a stainless steel mesh in RPMI-C. After washing three times, cells were resuspended in RPMI-C and cultured at 1 x [10.sup.6] cells/ml. Viability was determined by Trypan blue exclusion test. At least 200 cells were counted and the percentage of viable cells recorded.
The murine T-cell lymphoma (designated LB) arose spontaneously in a 6-month-old male Balb/C mouse. (Ruggiero et al. 1984) and was maintained by serial passages in the peritoneal cavity of syngeneic host (Hajos et al. 1996).
LB cells were obtained by intraperitoneal (ip) puncture under sterile conditions, then resuspended and maintained in RPMI-C medium at 1 x [10.sup.6] cells/ml. Viability was determined by Trypan blue exclusion test. At least 200 cells were counted and the percentage of viable cells recorded.
Rabbit and sheep erythrocytes
Fresh rabbit erythrocytes (RaE) were obtained from New Zealand white rabbits (Harlan Iberica, Spain) and maintained in Alsever's solution.
Fresh sheep erythrocytes were obtained from the School of Veterinary (Universidad Autonoma de Barcelona, Spain), maintained in Alsever's solution and sensitised with anti-sheep erythrocyte antibodies (ShEA), (ICN Pharmaceuticals Inc., USA) according to Klerx et al (Klerx et al. 1983).
Human pooled serum (HPS) was prepared in our laboratory from the blood of at least five healthy volunteers (extractions were done in the Medical Services of University of Barcelona). Human heat inactivated serum was obtained at 56 [degrees]C during 30 min. HPS and heat inactivated serum were stored at -80 [degrees]C until use.
Cell proliferation assay
The effect of each extract on cell proliferation was evaluated on LB tumour cells (5 x [10.sup.5] cells/ml) or on normal splenocytes (1 x [10.sup.6] cells/ml) alone or stimulated either with ConA (4 [mu]g/ml) or LPS (20 [mu]g/ml) (lipopolysaccharide from E. Coli 026:B6, Sigma Cehmical Chemical Company, St. Louis, MO, USA) according with Fernandez et al (Fernandez et al. 1998). Briefly, cells were cultured in RPMI-C in the presence of extract concentrations ranging from 1 to 100 (g/ml. Each extract was dissolved in the minimal amount of dimethyl sulfoxide (DMSO) and diluted with RPMI to obtain the indicated concentration. Control cells were treated with the same DMSO-RPMI mixture. After 24 h at 37 [degrees]C in a humidified atmosphere of 5% C[O.sub.2] in air, cultures were pulsed with one [mu]Ci of [H.sup.3]-thymidine/well (Du Pont, Nen Products, Boston, MA, USA) and maintained for an additional 18 h period prior to harvest. Incorporated [[H.sup.3]]TdR was measured in a Beckman liquid scintillation be ta counter, (MD, USA). Results were measured as the mean cpm of [[H.sup.3]]TdR incorporated in triplicate cultures. Each experiment was repeated at least five times. Stimulation index (SI) was calculated as:
SI = (experimental cpm - basal cpm /basal cpm) x 100
Control cells represent SI = 0%.
Extract concentration required for 50% inhibition of DNA synthesis ([IC.sub.50]) was calculated from plots of percentage of cell growth versus log of drug doses.
Viability was determined by Trypan blue exclusion test. At least 200 cells were counted and the percentage of viable cells recorded.
Inhibition of complement haemolytic activity
Classical and alternative complement pathway activities (CP and AP respectively) were determined in HPS. The tests were performed in 96 wells microtiter plates with ShEA and RaE as target cells for CP and AP, respectively (Klerx et al. 1983). Serial two fold dilutions of extracts were made in [VSB.sup.2+] (CP assay) or EGTA-VSB (AP assay) in U-bottom microtiter plates. Immediately thereafter, HPS in [VSB.sup.2+] (CP) or HPS in EGTA-VB (AP) were added to each well. After pre-incubation at 37 [degrees]C for 30 min, ShEA (4 x [10.sup.8]) (CP) or uncoated rabbit erythrocytes (1.15 x [10.sup.8]) (AP) were added. The plates were incubated at 37 [degrees]C for 60 min (CP) or 30 min (AP). Subsequently the plates were centrifuged for 2 min at 1250 x g. To quantify haemolysis, 50 [mu]l of the supernatant were mixed with 200 [mu]l water in flat-bottom microtiter plates and the absorption at 405 nm was measured with an
ELISA BIO RAD reader Model 550.
Controls in this assay consisted of similarly treated supernatants of erythrocytes incubated in water (100% haemolysis), in buffer (0% haemolysis) or in buffer supplemented with HPS (0% inhibition). Data were collected as mean of duplicates from 4 samples and [IC.sub.50] values were calculated.
The one-way analysis of variance (ANOVA) followed by Dunnet Multiple Test was performed using the Graph Pad PRISM [TM] software (Graph Pad Software Inc., San Diego, CA). * p values <0.05.
Cell proliferation assay
Different activity profiles were found among the plant extracts studied. Most of the analyzed extracts exerted inhibitory effect on tumor cells growth and also on ConA or LPS mitogen activated normal spleen cells (Con A or LPS-NMS). On tumor cells, [IC.sub.50] ranged between 1-75 [mu]g/ml with the exception of DME of L. molleoides and P. sellowianus in which concentrations higher than 100 [micro]g/ml were necessary to produce the effect. Furthermore, on mitogenic activated splenocytes, [IC.sub.50] ranged between< <1 to 85 [mu]g/m1 with the exception of E. buniifolium ME or P. sellowianus AE which were not effective on Con A-NMS or LPS-NMS respectively. Besides, only E. buniifolium was active on murine normal spleno-cytes proliferation ([IC.sub.50] 0.5-1.5 [mu]g/ml) (Table 1).
L. molleoides DME had significant stimulating effect on proliferation of normal murine splenocytes but inhibited proliferation of mitogen stimulated splenocytes and LB tumor cells. E. arnottianum DME was able to significantly stimulate NMS proliferation only at 10 [mu]g/ml, while all the fractions inhibited proliferation of tumor LB cells as well as mitogen activated splenocytes. E. buniifolium inhibited NMS proliferation and its inhibitions were more intense for normal cells than for tumor LB cells. Finally, P. sellowianus was only able to significantly inhibit Con A-NMS proliferation (Fig. 1).
Inhibition of complement hemolytic activity
The effect of the extracts on CP and AP of human complement is shown in Table 2. The E. arnottianum DME was the most active inhibitor on CP ([IC.sub.50] 5 [mu]g/ml) (Fig. 2a). Besides, the extracts that showed moderate inhibitory activity of complement system (anticomplementary effect) on CP were: DME, ME and AE of P. sellowianus (Fig. 2b); DME and AE of A. flaccida, and DME of E. buniifolium. Only DME of E. arnottianum (the most active on CP) and AE of P. sellowianus were moderate inhibitors on AP.
Since ancient times, all medicines have derived from natural sources, either plants or microorganisms. Gradually, the amount of knowledge about drugs increased and actually compounds from natural origin play a significant role in modern medicine. Examples include anticancer drugs as doxorubicine, vincristine and paclitaxel as well as immunomodulators as Cyclosporin A, FK506 and Sanglifehrin A used in organ transplantation (Clark, 1996; Zhang and Liu, 2001).
In order to determine the immunomodulatory actions of South American species with ethnomedical uses related to immunomodulating properties, we have studied extracts of different polarities from five plants selected on the base of their popular use and literature background.
The different extracts (DME; ME; AR) of A. flaccida, E. Arnottianum, E. buniifolium, L. molleoides and P. sellowianus were tested on normal murine or tumor leukocytes. As shown in our results, most of the extracts exerted inhibitory effect on tumor cells and/or mitogen activated normal spleen cells growth ([IC.sub.50] 1-85 [mu]g/ml). These results are in accordance with previous studies from our laboratories obtained with Argentine species, which were able to inhibit proliferation of activated leukocytes (Fernandez et al. 1998; Anesisi et al. 2001). Moreover, Uncaria tomentosa extracts and its fractions exerted a direct antiproliferative activity on MCF7, HL6O and Raji cells, while K562 cells were more resistant to the inhibition (Riva et al. 2001; Sheng et al. 2001). An antiproliferative effect was also found for the methanolic extracts (10 and 100 [mu]g/ml) of some medicinal plants used in Tanzanian traditional medicine evaluated in vitro on HeLa, HT29 and A431 human tumor cell lines (Kamuhabwa et al. 2000) in the same range concentrations tested by us.
When studying the effect on complement pathway, all the extracts were able to inhibit the classical complement pathway, being DME of E. arnottianum the most active ([IC.sub.50] 5 [mu]g/ml). Remarkable effect was also obtained with the DME and ME of P. sellowianus ([IC.sub.50] 11.2 and 17.3 [mu]g/ml respectively). However, on alternative pathway only the DME of E. arnottianum and AE of P. sellowianus were effective. These results showed a high anticomplementary activity when compared with other plant extracts. Since, complement system contributes to tissue damage in many clinical conditions; it is of interest to discover effective complement inhibitors to prevent adverse pathologic effects (Sahu and Lambris, 2000).
As can be seen the extracts of E. arnottianum are very effective in inhibiting either proliferation of mitogen stimulated splenocytes or LB tumor cells and complement activity of both CP and AP. Besides, it does not affect NMS proliferation.
Work is in progress by bioguided fractionation studies to led to the isolation and identification of the active compounds present in the most effective extracts and to elucidate the mechanism involved.
The chemical constituents which might be responsible for the immunomodulating activity of the plant extract are not yet identified, however, from Achyrocline satureioides and various
Eupatorium and Phyllanthus species water soluble polysaccharides with powerful immunomodulating potential have been isolated (Wagner et al. 1999). Comprehensive investigations on complement activating polysaccharides from plants have been carried out in Japan from Yamada's research group (Yamada and Kiyohara, 1999).
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Table 1 50% inhibitory concentration ([IC.sub.50]) of the different plant extracts on cell proliferation assays. A. flaccida E. arnottianum Extract DME ME AE DME ME AE NMS NMS+ConA 26.3 16.6 14.1 14.4 11.5 18.6 NMS+LPS 33.1 8.9 35.5 36.3 70.8 47.9 LB 15.5 36.3 30.2 1.2 13.2 10.7 E. buniifolium L. molleoides Extract DME ME AE DME ME AE NMS 0.54 1.52 1.27 NMS+ConA 3.5 NE 19.0 79.4 14.1 11 NMS+LPS 14.5 25.1 <1 >100 35.5 38.0 LB 29.5 17.4 47.9 >100 18.6 20.9 P. sellowianus Extract DME ME AE NMS NMS+ConA 83.1 52.1 52.5 NMS+LPS 79.4 >100 NE LB >100 75.9 50.7 Extract concentration ([micro]g/ml) capable of inhibiting 50% basal cell proliferation of each cell type was obtained with data from proliferation assays. DME - dichloromethanolic extract; ME - methanolic extract; AE - aqueous extract; NMS - normal mouse splenocytes; ConA - Concanavalin A from Canavalia ensiformis; LPS - lipopolysaccharide from E. Coli 026:B6; LB - Murine T-Lymphoma cells. Table 2 Inhibition of complement hemolytic activity results. Species Extract [IC.sub.50] ([micro]g/ml) CP AP A.flaccida DME 23.5 [+ or -] 1.4 NA ME 88.9 [+ or -] 4.5 NA AE 27.2 [+ or -] 0.9 NA E. arnottianum DME 5.0 [+ or -] 0.1 101.3 [+ or -] 1.5 ME 92.0 [+ or -] 2.7 NA AE 155.9 [+ or -] 9.0 NA E. buniifolium DME 44.1 [+ or -] 3.5 NA ME 66.7 [+ or -] 0.3 NA AE 58.3 [+ or -] 5.2 NA L. molleoides DME 86.1 [+ or -] 9.1 NA ME 69.2 [+ or -] 3.1 NA AE 59.0 [+ or -] 1.0 NA P. sellowianus DME 11.2 [+ or -] 1.8 NA ME 17.3 [+ or -] 3.3 NA AE 22.0 [+ or -] 2.2 280.6 [+ or -] 7.1 CP - classical pathway, AP - alternative pathway. NA - No activity (extract without activity at range of concentration assayed) (N = 4). DME - dichloromethanolic extract; ME - methanolic extract; AE - aqueous extract; [IC.sub.50]: Extract concentration required for 50% inhibition of complement haemolytic activity.
Financial support for this investigation was provided by SECYT-UBA B079, B081. This work is part of Proyecto XI, Subprograma Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED).
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T. Fernandez (1), P. Cerda Zolezzi (1), E. Risco (3), V. Martino (2), P. Lopez (2), M. Clavin (2), O. Hnatyszyn (2), S. Canigueral (3), S. Hajos (1,4), G. Ferraro (2,4), and E. Alvarez (1,4)
Catedras de (1.) Inmunologia/IDEHU y
(2.) Farmacognosia-IQUIMEFA, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina
(3.) Unitat de Farmacologia i Farmacognosia, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Espana
(4.) Member of Research Career, CONICET, Argentina
Dra. T. Fernandez, Catedra de Inmunologia- IDEHU, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, 4 Piso, Buenos Aires CP 1113, Argentina
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