• Ascaris;
  • Blomia tropicalis;
  • Dermatophagoides;
  • cross-reactivity;
  • tropomyosin


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background:  Analysis of cross-reactivity between the nematode Ascaris ssp. and dust mites, two important allergen sources in the tropics, will contribute in understanding their influence on asthma and atopy. The objective of this study was to investigate immunoglobulin E (IgE) cross-reactivity between Ascaris and two domestic mites in the tropics.

Methods:  Sera from 24 asthmatic patients were used in ELISA and immunoblotting IgE-binding inhibition assays using Ascaris, Blomia tropicalis and Dermatophagoides pteronyssinus extracts and the recombinants Blo t 10, ABA-1 and Blo t 13 as competitors. Identification of Ascaris allergens was confirmed by mass spectrometry (LC-MS/MS).

Results:  We detected at least 12 human IgE-binding components in Ascaris extract. Blomia tropicalis and D. pteronyssinus inhibited 83.3% and 79% of IgE-binding to Ascaris, while Ascaris inhibited 58.3% and 79.3% to B. tropicalis and D. pteronyssinus respectively. Mite tropomyosin inhibited 85% of IgE-binding to Ascaris. Affinity-purified human IgE to rBlo t 10 identified an allergen of 40 kDa in Ascaris extract, further confirmed as tropomyosin by LC-MS/MS. We found no evidence of IgE cross-reactivity between rABA-1 and any allergen component in mite extracts, including rBlo t 13.

Conclusions:  There is cross-reactivity between Ascaris and mites, determined by several allergens including tropomyosin and glutathione-S-transferase. In addition to its potential impact on asthma pathogenesis, Ascaris infection and mite allergy diagnosis relying on the determination of specific IgE could be affected by this cross-reactivity. ABA-1 has no cross-reactive counterpart in mite extracts, suggesting its usefulness as a more specific marker of Ascaris infection.

Allergy and asthma are phenotypes influenced by the environment. Mite sensitization is a risk factor for asthma, especially in tropical regions, where exposure to allergens from Blomia tropicalis and Dermatophagoides pteronyssinus is perennial (1–3). Some surveys have found that infection by Ascaris lumbricoides is a risk factor for asthma and atopy while others suggest the contrary (4). However, scarce information regarding cross-reactivity between allergens of mites and Ascaris is available, even though it could influence diagnosis of nematode infection and mite allergy and modify the natural history of mite-induced asthma. Analysis of this cross-reactivity will contribute to define, for example, the origin of the strong immunoglobulin E (IgE) responses to both sources in asthmatics (5, 6) and to find more specific reagents for diagnosis. Among potential cross-reactive proteins are tropomyosins, paramyosins, glutathione transferases, proteases and fatty acid-binding proteins (FABPs). Cross-reactivity with mite tropomyosins can be evaluated because some of them have been cloned (7–10). For example, tropomyosin Blo t 10 is an important allergen from B. tropicalis (8) and has 73% amino acid sequence identity with the A. lumbricoides tropomyosin (11), therefore, it is likely to be cross-reactive with the homologous counterpart in the extract.

ABA-1 (Asc s 1), an allergen of Ascaris ssp., is a member of the nematode polyprotein allergen/antigens and has fatty acid-binding properties (12, 13). It has been used as serological marker of Ascaris infection (14–16). However, although it has only been found in nematodes, there are no studies of cross-reactivity with mite’s FABPs (e.g. Blo t 13) or other allergens. The objective of this study was to evaluate the allergenic cross-reactivity between mite and Ascaris extracts and to identify allergens of that cross-reactivity using whole extracts and recombinant allergens.


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References


Sera of 24 asthmatic patients from Cartagena, a tropical city of Colombia were selected for preparing pools according to their IgE levels to Ascaris, D. pteronyssinus, B. tropicalis, rBlo t 10, rBlo t 13 and rABA-1 (Table 1). Asthma diagnosis was done as described previously (17). A pool of five sera from healthy nonallergic subjects was used as negative control. For correlation studies with rBlo t 10, 32 sera double positive to Ascaris and B. tropicalis were included. Subjects lived in an urban, nonindustrialized setting and belong to the lower three socioeconomic strata. The Bioethics Committee of the University of Cartagena approved the study and informed signed consent was obtained.

Table 1.   Immunological characteristics of sera
Serum pool A*
CodeAgeGenderIgE to Blomia tropicalis (kUA/l)IgE to Dermatophagoides pteronyssinus (kUA/l)IgE to Ascaris (kUA/l)Total IgE (lU/ml)
Serum pool B and C†
CodeAgeGenderIgE to B. tropicalisIgE to D. pteronyssinusIgE to AscarisTotal IgE (lU/ml)IgE to Blo t 10 (OD)
Serum pool D
CodeAgeGenderIgE to B. tropicalisIgE to D. pteronyssinusIgE to AscarisIgE to rBlo t 13 (OD)IgE to rABA-1 (OD)
  1. OD, optical density units.

  2. *The pool A was prepared with these nine sera and pool B.

  3. †Serum included in pool C.


Ascaris and mite extracts

Ascaris extract was prepared by acetone–saccharose precipitation method (18). As it is the source currently employed for in vitro testing because of its close relationship with A. lumbricoides, Ascaris suum extract was used. Adult worms were washed in sterile saline with penicillin and streptomycin and homogenized in 4 ml of 0.25 M saccharose per gram of parasite. Extraction was performed by shaking the homogenate in cold acetone and leaving it for 20 min in cold bath (−80°C); then the sediment was resuspended in cold acetone, shaked and kept for 1 h in cold bath. The sediment was resuspended in acetone and centrifuged for 15 min at 9500 g, lyophilized and reconstituted in phosphate-buffered saline (PBS) pH 7.2 in a proportion of 0.4 ml per initial volume of homogenate. After shaking in cold for 16 h, it was centrifuged for 1 h at 9500 g (4°C). The supernatant was dialysed for 24 h against PBS pH 7.2 in a 3500 cutoff membrane (Spectrum Medical Industries, Los Angeles, CA, USA). Protein concentration (1.8 mg/ml) was determined by Lowry method. The lyophilized extract was kept at −20°C until used. Dermatophagoides pteronyssinus and B. tropicalis extracts were produced as described previously (19). To evaluate the effect of IgE-binding cross-reactive carbohydrates, periodate oxidation of the extracts was performed under mild conditions (20, 21). Briefly, lyophilized extracts were diluted in sodium metaperiodate (Sigma, St. Louis, MO, USA) −0.05 M acetate buffer pH 4.5 to a final concentration of 10 mM, incubated for 1 h in the dark and then dialysed at 4°C.

Purified recombinant allergens: rABA-1, rBlo t 10 and rBlo t 13

cDNA encoding ABA-1 from A. suum was cloned into pGEX-1λT, and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (Invitrogen Corporation, Carlsbad, CA, USA). It represents a single A-type repeat with few amino acid differences from the ABA-1 of A. lumbricoides and fully immunologically cross-reactive (22). Expression was induced with Isopropyl β-d-thiogalactopyranoside (0.1 mM), 4 h at 37°C. The rABA1 was purified with glutathione–sepharose beads and digested with thrombin; the nonfusion-protein was dialysed and kept at −20°C. rBlo t 10 was expressed in E. coli (8). rBlo t 13 was also expressed as fusion protein with GST as described previously (23).

Specific IgE to extracts and recombinant allergens

Total IgE was determined by duplicate using an ELISA kit (RIDASCREEN, R-Biopharm, Germany) according to the manufacturer instructions. Specific IgE to mite extracts (5 μg/well), Ascaris extract, rBlo t 13, rBlo t 10 and rABA1 (1 μg/well) were determined by indirect enzyme-linked immunosorbent assay (ELISA) as described previously (17). Immunoglobulin E antibodies to mites and Ascaris extracts were also determined by ImmunoCAP (Phadia AB, Uppsala, Sweden).

ELISA inhibition

Pools were adsorbed with increasing concentrations of inhibitors. After adsorption for 10 h at 4°C, 100 μl were loaded into wells coated with the relevant antigen and the plates incubated overnight. Antibodies bound to solid phase were measured by ELISA and the results were expressed in percentage of inhibition, calculated as follows: per cent of inhibition = OD without inhibitor − OD with inhibitor/OD without inhibitor × 100. All assays included bovine serum albumin (BSA) as nonrelated inhibitor and GST as allergen control in experiments using Blo t 10/GST.

Immunoblotting inhibition

Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (20 μg/lane) in a Mini-Protean 3 Electrophoretic Cell (Bio-Rad, Hercules, CA, USA). Samples were loaded with 5%-β-mercaptoethanol for reducing conditions. Proteins were electro-transferred onto 0.45 μm pore size nitrocellulose membranes using the Mini Trans-Blot Cell (Bio-Rad) with buffer 25 mM Tris–HCl, 192 mM glycine, 20% MeOH (pH 8.3). Membranes were blocked with 3% BSA/PBS, washed with 0.1%TPBS (Tween-PBS) and incubated overnight with serum pool. Strips were washed and incubated with alkaline-phosphatase-labelled anti-IgE (1 : 500) in buffer 0.5% TPBS, 0.2% BSA and then washed with buffer 0.15 M NaCl 0.05 M Tris–HCl (pH 7.5). Allergenic components were revealed using freshly prepared p-nitro-blue-tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate dissolved in alkaline-phosphatase buffer (pH 9.5) during 3 min. For immunoblotting inhibition, strips were incubated with 2 ml of each pool, adsorbed (5 h) with 100 μg/ml of inhibitor. All assays included BSA as nonrelated control inhibitor and negative control serum. Estimation of molecular weight was done by calculating relative mobility according with migration of known MW standards (Broad Range Standards, Bio-Rad).

rBlo t 10 affinity-purified antibodies and immunoblotting

Affinity-purified IgE to rBlo t 10 was used to identify tropomyosin in Ascaris extract. Four microgram of allergen were electro-transferred to a nitrocellulose membrane and incubated overnight with a pool prepared with seven rBlo t 10 reactive sera, adsorbed with Schistosoma japonicum-purified GST. Antibodies bound to rBlo t 10 were eluted with 2 ml of 100 mM glycine (pH 2.5). After neutralization with 1 M Tris–HCl (pH 8.0), 4 mg/ml of BSA were added and the eluate was incubated with nitrocellulose strips containing Ascaris and mite extracts, rBlo t 10 and rBlo t 5.

LC-MS/MS peptide sequencing

Protein bands were excised and in-gel digested using the ProteoExtract All-In-One Trypsin Digestion Kit (Calbiochem, San Diego, CA, USA). Tryptic peptides were separated by reversed phase-capillary high-performance liquid chromatography (HPLC, Nanoease Symmetry 300TM trap column and Nanoease Atlantis dC18TM separating column; Waters, Milford, MA, USA) directly coupled to an ElectroSpray ionization-quadrupole-time of flight mass spectrometer (Q-Tof Ultima Global; Waters). Data acquisition and instrument control was done with the masslynx software V4.1 (Waters). The instrument was calibrated with the fragment ions of 22-Fibrinopeptide B (Sigma). Peptides were eluted with an acetonitrile gradient (Solvent A 0.1%v/v formic acid / 5% v/v acetonitrile, solvent B 0.1%v/v formic acid / 95% v/v acetonitrile; 5–45% B) and directly subjected to ionization and mass spectrometry. Data were acquired in the data-directed analysis mode in the mass range from 350 to 1900. Fragmentation time of the peptides was 4 s per ion, four ions were fragmented simultaneously. Peptide fragmentation data were acquired in the mass range from 50 to 1900. Survey and fragment spectra were analysed using the software Proteinlynx Global Server version 2.2.5 (Waters) with automatic and manual data validation. For sequence identification, Swiss-Prot and TrEMBL database were used.


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Human mite-allergic sera identify several IgE-binding components in Ascaris extract

Under reducing conditions, we detected 16 IgE-binding components in the Ascaris extract,  those of 40, 34 and 23 kDa being the strongest (Fig. 1A). The same pool identified 19 components in D. pteronyssinus extract (116, 102, 95, 90, 81, 66, 60, 54–55, 50, 43, 41, 37, 35, 33, 29–30, 18, 17, 14–15 and 13 kDa) and 21 components (136, 102, 97, 84, 75, 50, 46, 45, 41, 39, 36, 31, 28, 25, 22, 21, 19, 17, 15, 14 and 11 kDa) in B. tropicalis. Under nonreducing conditions, the pattern of IgE-binding in the Ascaris extract was slightly different, being 12 (200, 116, 83, 77, 60, 58, 40, 34, 33, 23, 15 and 10 kDa) clearly visible (Fig. 1A). This was also observed with mite extracts (data not shown) consistent with previously reported data (24).


Figure 1.  (A) Immunoblotting of Ascaris and mites using pool A (1 : 5). Ascaris extract under reducing (R) and nonreducing nonreducing (NR) conditions. Under reducing conditions bands of 200, 175, 144, 130, 100, 97, 70, 61, 53, 47, 40, 34, 33, 31, 23 and 15 kDa are visible in Ascaris extract. As, Ascaris; Dp, D. pteronyssinus; Bt, B. tropicalis; N, negative control serum. (B) Ascaris in solid phase, pool B, dilution 1 : 20, the species-specific allergen of 15 kDa is pointed by the arrow. The names of inhibitors are shown above each lane, NI: no inhibited.

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There is cross-reactivity between Ascaris and domestic mite allergens

Blomia tropicalis and D. pteronyssinus extracts inhibited 83.3% and 79% of IgE-binding to Ascaris (Fig. 1B), while Ascaris extract inhibited 58.3% and 79.3% of IgE-binding to B. tropicalis and D. pteronyssinus. We also analysed the inhibition of IgE-binding to Ascaris using pool C, obtaining 92.6% and 93% inhibition with D. pteronyssinus and B. tropicalis. Using pool B and periodate-treated extracts, B. tropicalis and D. pteronyssinus inhibited 68.4% and 50.6% of IgE-binding to Ascaris while Ascaris extract inhibited 63% of IgE-binding to mite extracts (data not shown).

There are several cross-reactive allergens in mite and Ascaris extracts

To investigate which allergens were cross-reactive we did immunoblotting inhibition assays. We observed the inhibition of a 40 kDa allergen and no inhibition of a 15 kDa allergen (Fig. 1C). Using pool C, there was total inhibition of IgE-binding to the 40 kDa allergen and other allergenic components (200, 97 and 23 kDa) of Ascaris by both mites (data not shown). To increase the IgE repertoire, we performed experiments using pool A. Under reducing conditions, mite extracts inhibited IgE-binding to allergens of 200, 175, 144, 100, 97, 61, 53, 40, 33 and 31 kDa (Fig. 2A). Under nonreducing conditions, mites inhibited IgE-binding to components of 200, 116, 77, 58 (only D. pteronyssinus), 40, 33 and 23 kDa, while components of 10, 13, 15, 34 and 60 kDa were not inhibited (Fig. 2B). The 200, 40, 33 and 23 kDa allergens were consistently inhibited by both mites in reducing and nonreducing conditions. The Ascaris extract partially inhibited the IgE-binding to 11 allergens of D. pteronyssinus (Fig. 2C) and to another 11 of B. tropicalis with total inhibition to those of 22 and 19 kDa (Fig. 2D). No inhibition of IgE-binding to allergens of 15 kDa or those ranging from 14 to 17 kDa was found.


Figure 2.  Immunoblotting inhibition using pool A (1 : 10), under (A) reducing and (B) nonreducing conditions. (C) Pool 1 : 50. Ascaris extract inhibited IgE-binding to allergens 116, 95, 90, 81, 66, 54–55, 37, 35, 33, 29 and 18 kDa, (D) Pool 1 : 20. Ascaris inhibits IgE-binding to 116, 102, 84, 77–75, 71, 43, 41, 33–34, 24, 22 and 19 kDa allergens.

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Tropomyosin is a cross-reactive allergen between Ascaris and domestic mites

A significant correlation between IgE levels to rBlo t 10 and Ascaris was found (Spearman = 0.61, = 0.0001). rBlo t 10 in solid phase showed 95% homologous inhibition, 71% with B. tropicalis, 72% with D. pteronyssinus and 37% with Ascaris (Fig. 3A). With Ascaris in solid phase, 91% homologous and 75.2% inhibition with rBlo t 10/GST was found (Fig. 3B). Most of the IgE reactivity was directed to the 40 kDa allergen in Ascaris extract, which was strongly inhibited by mites. Therefore, we performed immunoblotting inhibition by preadsorption with rBlo t 10-GST. The IgE-binding to the 40 kDa allergen was completely inhibited by Blo t 10-GST while the 34 kDa allergen remained unchanged (Fig. 3B). Additionally, using human-specific IgE to rBlo t 10 the 40 kDa allergen was detected in the extract, as well as the rBlo t 10-GST fusion protein while no signal was obtained with the nonrelated allergen rBlo t 5 (Fig. 3C). By tandem LC-MS/MS and subsequent database search, this allergen was confirmed as A. lumbricoides tropomyosin (TrEMBL accession number A8DKY0) with a match of 10 peptides and sequence coverage of 30.9% (Fig. 4).


Figure 3.  (A) ELISA inhibition using pool C and rBlo t 10 in solid phase. (B) Ascaris in solid phase. (C) Anti-tropomyosin IgE-binding components in Ascaris (lane 1); no related allergen rBlo t 5 (lane 2) and rBlo t 10/GST (lane 3). Preabsorption (PA) of pooled sera with rBlo t 10/GST decreases the intensity of IgE recognition of the 40 kDa allergen of Ascaris extract.

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Figure 4.  Coomassie staining of Ascaris suum extract showing the bands selected for in-gel digestion and analysis by LC/MS/MS. Coverage map obtained by the resulting sequenced peptides are shown for the tropomyosin (40 kDa) and ABA-1 (15, 13 and 10 kDa).

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Anti-tropomyosin antibody also recognized a 23 kDa allergen in the Ascaris extract, corresponding to GST, indicating that the adsorption of the pool was incomplete. This band was also recognized by pool C and inhibited by mite extract. By tandem LC-MS/MS, eight peptides of the 23 KDa allergen matched to GST from A. suum (P46436), resulting in a sequence coverage of 30.1%.

ABA-1 is not cross-reactive with mite allergens

We consistently found no inhibition of allergenic components of 15, 13 and 10 kDa. These Ascaris-specific allergens were all identified by tandem LC-MS/MS as fragments of ABA-1 (Fig. 4). To investigate whether this FABP is cross-reactive with any component in mite extracts, we analysed the IgE-binding inhibition by preadsorption with B. tropicalis, D. pteronyssinus and rBlo t 13. We observed total inhibition of IgE-binding when Ascaris and the homologous molecule were used as competitors, but, there was no inhibition with B. tropicalis, D. pteronyssinus or Blo t 13, suggesting that mite extracts do not contain ABA-1 epitopes and this allergen is not cross-reactive with the mite FABP, Blo t 13 (Fig. 5A). Immunoblotting inhibition with rBlo t 13 in solid phase showed total homologous inhibition and partial inhibition with its native equivalent in the B tropicalis extract, but no inhibition with Ascaris extract or rABA-1(Fig. 5B,C).


Figure 5.  Immunoblotting inhibition using whole extracts and purified Ascaris and mite FABPs. (A) ABA-1 in solid phase (1 μg/lane), (B) and (C) Blo t 13 in solid phase (1 μg/lane). The names of inhibitors are above each lane. B13, Blo t 13. All the experiments were done with pool D, double positive to ABA-1 and Blo t 13.

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  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We analysed the allergen cross-reactivity between the two most important allergenic mites in the tropics and the nematode Ascaris, using sera from mite-allergic patients living in a place where exposure to mites is perennial and naturally infection with this parasite is endemic. As a result, we found that (i) Ascaris extract has at least 12 human IgE-binding components; (ii) there is important cross-reactivity between the mites B. tropicalis and D. pteronyssinus and Ascaris; (iii) there are at least four allergens of Ascaris involved in this cross-reactivity, among them tropomyosin and GST and (iv) the allergen ABA-1 from Ascaris is not cross-reactive with components of mite extracts.

In Ascaris extract, we detected 16 IgE-binding components under reducing and 12 under nonreducing conditions. Thus, it is likely that the actual number of individual allergens is that found under nonreducing conditions, when allergens are less denatured. This number and recognition pattern is similar in Anisakis simplex under nonreducing conditions (25). However, it is necessary to analyse the frequency of sensitization at the population level to obtain information about the patterns of sensitization in patients and controls.

We found that cross-reactivity with Ascaris is high for both mites but it is important to consider whether this is related to cross-reactive carbohydrates. Our assays using periodate-oxidized extracts showed a low reduction in the level of cross-reactivity compared with the crude extracts, suggesting that carbohydrate groups are involved, but most of the cross-reactivity is because of protein epitopes, which is supported by our experiments using recombinant nonglycosylated allergens.

To identify the cross-reactive allergens, we did immunoblotting inhibitions using the same sera of ELISA experiments and mass spectrometry analysis. Under reducing conditions, mite extracts inhibited the same 10 allergens of Ascaris and the same seven allergens under nonreducing conditions. Four of them (200, 40, 33 and 23 kDa) were inhibited by mites in both conditions, suggesting that they are distinct molecules. Thus Ascaris extract has several mite-cross-reactive allergens, among them the 40 kDa allergen, which exhibited the highest intensity of IgE-binding. We demonstrated, for the first time, cross-reactivity between rBlo t 10 and this 40 kDa homologous allergen in the Ascaris extract, confirmed as Ascaris tropomyosin by LC-MS/MS. Our results are in agreement with the reported 40.9 kDa native tropomyosin of A. simplex (26) and to that described for A. suum (27). In mite extracts, we also observed allergens with the molecular mass of tropomyosins (7, 8), partially inhibited by Ascaris extract, as was described in cross-reactivity experiments between A. simplex and four mite species (25).

Cross-reactivity between mites and nematodes has been suspected for a long time and has only been described for D. pteronyssinus and A. simplex (25). However, cross-reactivity between mites and Ascaris had not been demonstrated, even though several studies have suggested a relationship between specific IgE to this nematode and atopy, allergic sensitization, wheezing, asthma and asthma severity (4, 28). In this regard, our findings have two important implications that deserve further analysis.

First, in surveys using IgE to Ascaris extract as a marker of infection, the results could be biased for cross-reactive allergens. The interpretation of serological tests for this infection using the nematode extract currently considers cross-reactivity with other nematodes (29, 30) but should also take into account cross-reactivity with mites. We found no components sharing IgE-epitopes with ABA-1 in mite extracts, suggesting that this molecule is more specific for evaluating the infection with Ascaris. Secondly, in a tropical environment, where parasites are common and people are co-exposed to mites, the cross-reacting allergens could provide a permanent boosting, promoting synthesis of high specific IgE levels and influencing the inception of allergy and the clinical evolution of asthma.

Most reports on cross-reactivity between mites and other invertebrates suggest that mites are the primary sensitizer (31). It is difficult to define this point using whole extracts and pooled sera, but our results support this idea. In most sera, IgE levels to mites are higher than to Ascaris (Table 1) and inhibitions were stronger when using mite extracts than Ascaris extract. However, prospective investigations are necessary to identify the source inducing the primary response.

In summary, we found cross-reactivity between Ascaris and domestic mites, contributing with data about allergens potentially involved in asthma and suggesting a more specific reagent for serological diagnosis of Ascaris infection.


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank all subjects for their voluntary participation in the study; Dr Carmen Vidal (Servicio de Alergias Hospital de Conxo, Santiago de Compostela, Spain) for performing the ImmunoCap assays and Dr Josefina Zakzuk (Allergy-Innovations, Germany) for her collaboration in the preparation of extracts. This work was supported by Colciencias Grant Number 093-2007. MWK has been supported by the Wellcome Trust and the Medical Research Council (UK). NA was supported by Fundemeb.


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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