Isolation and Molecular Characterization of Two Lectins from Dwarf Elder (Sambucus ebulus L.) Blossoms Related to the Sam n1 Allergen
2013

Pilar Jimenez
Nutrition and Food Science
Faculty of Medicine and CINAD (Center for Research in Nutrition, Food and Dietetics
Lucia Building-Science Park)
University of Valladolid
E-47005ValladolidSpain

Patricia Cabrero firstname.lastname@example.com.
Nutrition and Food Science
Faculty of Medicine and CINAD (Center for Research in Nutrition, Food and Dietetics
Lucia Building-Science Park)
University of Valladolid
E-47005ValladolidSpain

José E Basterrechea email@example.com.
Nutrition and Food Science
Faculty of Medicine and CINAD (Center for Research in Nutrition, Food and Dietetics
Lucia Building-Science Park)
University of Valladolid
E-47005ValladolidSpain

Jesús Tejero firstname.lastname@example.com.
Nutrition and Food Science
Faculty of Medicine and CINAD (Center for Research in Nutrition, Food and Dietetics
Lucia Building-Science Park)
University of Valladolid
E-47005ValladolidSpain

Damian Cordoba-Diaz firstname.lastname@example.com
Pharmacy and Pharmaceutical Technology
Faculty of Pharmacy and IUFI (Institute of Industrial Pharmacy)
OPEN ACCESS Toxins2013
Complutense University of Madrid
Madrid E-280405 1768Spain

Tomas Girbes
Nutrition and Food Science
Faculty of Medicine and CINAD (Center for Research in Nutrition, Food and Dietetics
Lucia Building-Science Park)
University of Valladolid
E-47005ValladolidSpain

Isolation and Molecular Characterization of Two Lectins from Dwarf Elder (Sambucus ebulus L.) Blossoms Related to the Sam n1 Allergen

Toxins
5201310.3390/toxins5101767Received: 12 August 2013; in revised form: 16 September 2013 / Accepted: 30 September 2013 /* Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.com;Sambucus ebulusblossomslectinebulinricinribosome-inactivating protein
Sambucus species contain a number of lectins with and without antiribosomal activity. Here, we show that dwarf elder (Sambucus ebulus L.) blossoms express two D-galactose-binding lectins that were isolated and purified by affinity chromatography and gel filtration. These proteins, which we named ebulin blo (A-B toxin) and SELblo (B-B lectin)-blo from blossoms-were subjected to molecular characterization and analysis by MALDI-TOF mass spectrometry and tryptic peptide fingerprinting. Both lectins share a high degree of amino acid sequence homology with Sambucus lectins related to the Sam n1 allergen. Ebulin blo, but not SELblo, was highly toxic by nasal instillation to mice. Overall, our results suggested that both lectins would belong to an allergen family exemplified by Sam n1 and could trigger allergy responses. Furthermore, they raise a concern about ebulin blo toxicity.

Introduction

Berries from S. ebulus, also known as dwarf elder, have been consumed since the Neolithic Age. In fact, it has been reported that berries from S. ebulus have been consumed as food or medicine since 5000 years ago in Italy and France [1,2]. To date, a number of compounds that display pharmacological activities, such as flavonoids, vitamins and lectins, have been isolated from Sambucus spp. [3][4][5][6][7]. Medicinal applications of Sambucus spp., like antiviral, anti-inflammatory and healing activities, have been described, and in some cases, the responsible molecules have been identified [8,9].

Lectins are one of the most studied Sambucus bioactive compounds [10]. Lectins (i) are proteins with sugar-binding ability and usually sugar specificity; (ii) some have cell-agglutinating activity; (iii) are ubiquitous in nature; and (iv) are found in all kinds of organisms [11]. In particular, they are very important in plant foods, since they may interact with the gastrointestinal mucosa, thereby changing the profile of bacterial interaction and may promote changes in glycoprotein expression on the mucosa surface that may result in changes in nutrient uptake [12]. A number of lectins with protein synthesis inhibitory activity, known as ebulins, have been isolated from different parts of Sambucus ebulus, like fruits (ebulin f), leaves (ebulin l) and rhizomes (ebulins r1 and r2) [13][14][15]. Ebulin toxins are known as type 2 (two chains) ribosome-inactivating proteins, or RIPs, since they have two chains, an A chain with N-glycosidase activity (E.C.18.104.22.168) on 28S rRNA, which is responsible for the irreversible inactivation of ribosomes, and a B chain with D-galactose-specific lectin activity [5,16,17]. S. ebulus also contains lectins of the B-B type with D-galactose-specific activity only, like SELld in leaves [17], SELlm in shoots [18] and SELfd in fruits [19,20].

Since ebulin shares amino acid sequences with Sam n1 isolated from blossoms, pollen and berries of S. nigra [21], we put forward the hypothesis that they and the related lectins devoid of translational inhibitory activity might be members of a broad family of structurally-related proteins (Sambucus lectins) of which Sam n1 allergen would be one of the family members. As a consequence, in a first approach to increase our knowledge on Sam allergens present in flowers that could trigger allergy and toxicity, we undertook studies aimed at investigating whether these kinds of lectins are present in dwarf elder blossoms. In the present research, we report two lectins structurally related to those found in fruits. We named them ebulin blo and SELblo. Ebulin blo was found to be a toxin of type A-B, highly toxic to mice by nasal instillation, while SELblo was found to be a lectin of type B-B devoid of acute toxicity comparable to ebulin blo.

Results

Isolation of Dwarf Elder Blossom D-Galactose-Specific Lectins

The SDS-PAGE of extracts prepared from dwarf elder blossoms revealed that they contain two major proteins in the zone of apparent Mr between 55,000 and 70,000 ( Figure 1). These proteins were found also in early green fruits and early fruit stalks. We presumed that these proteins could be forms of the lectins previously reported in well-developed green fruits [16]. The isolation of these lectins was performed as reported recently to avoid the water-soluble mucilage that binds to the chromatography resin [19]. The procedure involves the first step of affinity chromatography on acid-treated Sepharose 6B, and the bound protein was eluted with lactose. That protein was subjected to a second chromatography on Superdex 75, which resolved two protein peaks, as seen in Figure 2. Fractions of the peaks were collected to avoid cross-contamination. The major peak accounted for SELblo and the other one for ebulin blo. The ratio of SELblo to ebulin blo in blossoms was higher than that reported for ebulin f in early and late green fruits [19].

Molecular Characterization by SDS-PAGE

Molecular characterization was performed first by SDS-PAGE in the absence or the presence of 2-mercaptoethanol (2-ME). As shown in Figure 2, both proteins, which we named ebulin blo (the smallest) and SELblo, moved as single and well-defined protein peaks in the absence of 2-ME, with apparent Mr close to 60,000 and 68,000, respectively. Both proteins were very close to those found in the extracts of green fruits [19]. In the presence of 2-ME, both proteins dissociated to yield two subunits of different apparent Mr, in the case of ebulin blo, presumably an A-type with an apparent Mr of 30,000, a B-type chain with an apparent Mr of 34,000 and, in the case of SELblo, two apparently identical B-type chains of 38,000. As control for electrophoresis, ebulin f and SELfd were run together with the lectins of blossoms. Both proteins agglutinated human red blood cells (O + ), and that activity can be inhibited by these two sugars and was unaffected by D-glucose and D-mannose at concentrations up to 100 mM.

Molecular Characterization by Mass Spectrometry

Mass spectrometry analysis of both lectins revealed values of mass 63,225 m/z and a calculated pI of 5.56 for ebulin blo, mass 68,432 m/z and a calculated pI of 5.54 for SELblo. In order to get the best identification of these proteins, tryptic peptides were prepared. As shown in Figure 3, both proteins yield different peptides, which suggests significant differences. Some of tryptic peptides from each lectin were sequenced by mass spectrometry.  Figure 4 shows the sequences of tryptic peptides from both blossom lectins aligned with the sequences of the ebulin l precursor from S. ebulus leaf (gi/13171073) and the SNAld precursor from S. nigra leaf (gi/32892180). Mascot search revealed that the sequences obtained from ebulin blo peptides fit with those of ebulin l in both the A and B chains ( [14]; NCBI BLAST databank). Additionally, the sequence of SELblo peptides fit with those of SNAld. Furthermore, some peptides from the allergen, Sam n1, share a large amino acid sequence homology with the blossom lectins. . Alignment of amino acid sequences of some tryptic peptides of blossom lectins with ebulin l and SNAld. Boxed are sequences found in tryptic peptides from the allergen Sam n1 of S. nigra pollen. In red are coincident sequences. Highlighted in yellow and grey are tryptic peptides obtained from ebulin blo and SELblo, respectively.
Ebulin l 1 MRVVKAAMLY LHIVVLAIYS VGIQGIDYPS VSFNLAGAKS TTYRDFLKNL Ebulin blo 1 SNAld 1 MRVVAAAMLC LYIVVLAICS VGIQGIDYPS VSF----------------- SEL blo Ebulin l 51 RDRVATGTYE VNGLPVLRRE SEVQVKNRFV LVRLTNYNGD TVTSAVDVTN Ebulin blo 51 VATGTYE VNGLPVLR SNAld 51 -------------------------------------------------- SEL blo 51 ------------------------------------------------------ Ebulin l 101 LYLVAFSANG NSYFFKDATE LQKSNLFLGT TQHTLSFTGN YDNLETAAGTE Ebulin blo 101 SNLFLGT TQHTLSFTCN YDNLETAAGTR SNAld 101 -------------------------------------------------- SEL blo 101 Ebulin l 151 RRESIELGPN PLDGAITSLW YDGGVARSLL VLIQMVPEAA RFRYIEQEVR Ebulin blo 151 R ESIELGPN PLDGAITSLW YDGGVARSLL VLIQMVPEAA R SNAld 151 -------------------------------------------------- SEL blo Ebulin l 201 RSLQQLTSFT PNALMLSMEN NWSSMSLEVQ LSGDNVSPFS GTVQLQNYDH Ebulin blo 201 SNAld --------------------------------------------- SEL blo Ebulin l 251 TPRLVDNFEE LYKITGIAIL LFRCVATKTT HNAIRMPHVL VGEDNKFNDG Ebulin blo 251 SNAld 251 ----------------------------CK

Toxicity of Dwarf Elder Blossom D-Galactose-Specific Lectins to Swiss Mice

The instillation of 5 mg/kg body weight of ebulin blo dissolved in water to both nasal cavities promoted a 30% reduction of body weight after nine days, which recovered afterwards ( Figure 5). The reduction in weight was accompanied by the death of some animals five days after the treatment, reaching 50% mortality 14 days after application. Due to the clarity of the effect, the number of animals used was minimal. For comparative purposes, the effect of the intraperitoneal administration of 5 mg/kg body weight of ebulin f on the survival of mice has been included.

The same test was performed with SELblo, the major protein isolated from blossoms of S. ebulus, and as could be expected, this B-b lectin did not cause toxicity in the assayed animals (data not shown). The instillation of 2.5 mg/kg body weight of ebulin blo to female Swiss mice did not trigger deaths, but promoted toxicity revealed by a loss of weight, which reached 10% in seven days. This relative body weight loss was maintained until the recovery of the animals. Preliminary histological analysis indicates that ebulin administration orally promoted congestion in lungs (data not shown).

Discussion

Blossoms, early fruits and stalks contain lectins very similar to those present in fruits and in leaves [14,19]. By mass spectrometry, ebulin blo is slightly higher than ebulin f, while SELblo has the same Mr as SELfd. By SDS-PAGE analysis, both ebulin blo and ebulin f moved in gels with the same apparent Mr. The discrepancies in apparent Mr values determined by SDS-PAGE between the native protein and the summation of their subunits could be due to the different conformational states of the polypeptides when they are linked together in the native protein or when they are separated into their subunits. Ongoing experiments on intrinsic and aniline-naphthalene-sulfonate (ANS)-dependent fluorescence analysis indicated that ebulin f has a more flexible three-dimensional structure than SELfd, which facilitates locking-unlocking changes (data not shown).

Both blossom lectins are D-galactose-binding proteins, which enables their isolation by affinity chromatography on acid-treated Sepharose 6B. The sequences of some tryptic peptides revealed a high amino acid sequence homology with the allergen, Sam n1, and other toxic and non-toxic Sambucus lectins [19].

According to our laboratory safety rules, we handled ebulin blo always in a water solution, since we suspected that ebulin blo powder would form aerosols and, therefore, could be highly toxic by inhalation, like ricin [22,23]. The administration of ebulin blo to mice was done under mild anesthesia to avoid undesired head and body movements and to ensure that all of the ebulin blo instilled was administered. Ebulin blo displays a dose-dependent powerful biological activity, as shown by its toxicity on the experimental animals when administered by nasal instillation. In this sense, a relative body weight loss between 20% and 30% was associated with animal death. After 4-5 days of the intraperitoneal administration of ebulin f, a related ebulin isolated from fruits, a relationship between body weight reduction and animal death (data not shown) was also found. The target for ebulin f in these conditions was the small intestine, which was very affected, and, in particular, the Lieberkühn crypts, which almost disappeared [24]. Oral ingestion of ebulin f was also highly toxic, but less than by intraperitoneal administration [18]. Therefore, the possibility that the toxic effects of ebulin blo triggered by nasal instillation would be through the swallowing of part of the ebulin blo instilled and transferring it to stomach, enough to trigger toxic effects, like those observed previously [19,24], cannot be ruled out. However, this is improbable, due to the concentrations used and the observed effects.

Some of the toxicity differences between ricin and ebulin would be related to the distinct intracellular traffic pathways followed by both proteins. Ricin has been shown to follow a retrograde transport from endosomes to the trans-Golgi network and, finally, transferring to the rough endoplasmic reticulum [25,26]. In contrast, ebulin, like nigrin, follows a pathway that goes from endosomes to lysosomes, where they are degraded and the inactive products expelled out of the cell [27,28]. The concentration-dependent accumulation of ebulin and nigrin in endosomes would favor the non-specific translocation to the cytosol and reaching the ribosomes. The unfolded protein response reported for ricin as an important factor contributing to its toxicity [29] should be also taken into account for ebulin.

Dwarf elder grows wild and abundantly in many temperate areas and requires no care. The isolation of lectins from any part of the plant even in raw form is relatively easy, due to its high concentration in the plant. Therefore, and as has been reported for ricin [30], from our point of view, ebulin blo at a rather high concentration can promote a serious toxicity nasally and orally and could also be considered as a bioweapon. Further work will approach the toxicity of raw preparations of these lectins, their potential neutralization by food matrices and liquids, the histologic effects in the main organs and the allergenicity of pollen, raw and purified lectins.

Experimental Section

All common chemicals and biochemical compounds were of the highest purity available, and most of them were purchased from Sigma-Aldrich (Sigma-Aldrich Química S.L., Tres Cantos, Madrid,). Sinapinic acid, trifluoroacetic acid and the α-cyano-4-hydroxy-transcinnamic acid matrix were purchased from Sigma-Aldrich. Isoflurane was purchased from Esteve Veterinaria (Barcelona, Spain). The chromatographic supports for protein isolation were purchased from GE Healthcare Europe GmbH (Barcelona, Spain). The affinity chromatographic support was acid-treated Sepharose 6B and was prepared by treatment of Sepharose 6B beads with 0.1 N HCl at 50 °C for 3 h and extensive washing with Helix ® Millipore water (Millipore Ibérica S.A.U., Madrid, Spain) [19]. Dwarf elder (Sambucus ebulus L.) blossoms were harvested from Barruelo del Valle (Valladolid, Spain) in early July and stored frozen at −20 °C until use. The previously prepared acrylamide (37.5%)-bisacrylamide (0.8%) mixture was obtained from Amresco (Solon Ind., Solon, OH, USA).

Isolation of D-Galactose-Binding Lectins from S. ebulus Blossoms

Two-hundred grams of frozen blossoms were minced and ground in a mortar to obtain a paste material, which was extracted overnight with 800 mL of extraction buffer (280 mM NaCl containing 5 mM sodium phosphate-pH 7.5). That raw extract was strained through cheesecloth, and the fluid was then centrifuged at 7500 g for 45 min at 4 °C, after which the supernatant was centrifuged again at the same speed for 30 min and removed and stored at 4 °C until use. The remaining plastic material with a mucilaginous consistency was removed by filtration through two layers of common laboratory filter paper. The filtered extracts were chromatographed through acid-treated Sepharose 6B (AT-Sepharose 6B) to obtain D-galactose-binding proteins, as reported previously for the lectins of dwarf elder fruits [16]. Seven-hundred and fifty milliliters of extract were applied to a XK50 (5 × 15 cm) column (GE Healthcare Europe GmbH, Rosselló i Porcel, Barcelona, Spain) containing 200 mL of freshly prepared AT-Sepharose 6B (GE Healthcare Europe GmbH, Rosselló i Porcel, Barcelona, Spain) equilibrated with 0.28 M NaCl and 5 mM Na-phosphate (pH 7.5) buffer. Then, the column was washed with the same buffer until the A 280 reached values close to 0, and the protein fraction not retained by the AT-Sepharose 6B column was discarded. The retained protein fraction was further eluted with the same buffer containing 0.2 M lactose. Fractions of 10 mL were collected, and those containing proteins were pooled and concentrated with an Amicon system using a Y10 membrane (Millipore Ibérica S.A.U., Madrid, Spain) membrane down to 5 mL. The concentrated protein was applied to a Superdex 75 (26/60; GE) column equilibrated with 400 mM NaCl and 5 mM Na-phosphate (pH 7.5) buffer and was eluted with the same buffer at 2.5 mL/min. The first 70 mL were discarded, and then, fractions of 2.5 mL were taken and their A 280 measured. Those fractions containing protein were pooled, dialyzed against water and, finally, concentrated as above to reach concentrations of 2.5-4 mg/mL. Finally, the protein solutions were divided in aliquots of 0.1 mL and stored frozen at −20 °C. The purity of the proteins was assessed by SDS-PAGE.

Isolation of Crude Extracts

In some cases, small extract preparations (10 g of material extracted with 100 mL of extraction buffer) of blossoms, early green fruits and early fruit stalks were prepared for comparison of their SDS-PAGE protein profiles. They were obtained just as for the preparation of purified lectins described above, except that they were not subjected to chromatography. After, they were concentrated five-fold by ultracentrifugation with Millipore Amicon ultra 0.5 centrifugal filter units (Ultracel ® 10K, Millipore Ibérica SAU, Madrid, Spain) and stored at −20 °C until use.

SDS-Polyacrylamide Gel Electrophoresis of D-Galactose-Binding Lectins from S. ebulus Blossoms

The analysis of proteins by SDS-PAGE [31] was performed with the MiniVE (GE Healthcare Europe GmbH, Rosselló i Porcel, Barcelona, Spain) system using 4% stacking gel and 15% polyacrylamide separation slab gels. The protein samples in the loading buffer (62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol and 0.025% (w/v) bromophenol blue) were incubated for 5 min in a boiling water bath. When added, 2-mercaptoethanol (2-ME) was at 3.75%. Twenty microliters of sample buffer containing the protein sample were loaded into each well, and electrophoresis was carried out at 20 °C and 25 mA per gel, using a buffer containing 25 mM Tris-HCl (pH 8.3), 192 mM glycine and 0.1% (w/v) SDS. Gels were then stained overnight with mild shaking with a solution containing 1% (w/v) Coomassie Brilliant Blue R-250 (Sigma-Aldrich Química S.L., Tres Cantos, Madrid), 50% (v/v) methanol and 10% (v/v) acetic acid, and further, the stain was removed by shaking gels with a solution containing 5% (v/v) methanol and 7% (v/v) acetic acid.

Mass-Spectrometry Analysis of the Lectins

Mass spectrometry analysis of protein was carried out as described recently [19]. Briefly, 1 µL of sample (3.8 µg/µL) was spotted onto the sample target plate and allowed to air dry at room temperature. Then, 0.5 µL of a saturated solution of sinapinic acid (Sigma) in 30% acetonitrile and 0.3% trifluoroacetic acid (TFA) were added to the dried protein spots and, again, allowed to air dry at room temperature. Then, analyses in the MALDI-TOF/TOF mass spectrometer operating in positive linear mode (accelerating voltage of 20,000 V) were conducted. An external mass spectrum calibration was performed using BSA as the standard.

Tryptic Fingerprinting and Database Search

Tryptic fingerprinting was performed as described previously [19]. The samples were reduced with 10 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate for 30 min at 37 °C and subsequently alkylated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate for 15 min in the dark. Finally, samples were digested with 1:20 sequencing-grade trypsin (Roche Molecular Biochemicals, Sant Cugat del Valles, Barcelona, Spain) in 25 mM ammonium bicarbonate (pH 8.5), overnight at 37 °C. After digestion, 1 µL was spotted onto a MALDI target plate and allowed to air dry at room temperature. Then, 0.5 µL of a 3 mg/mL of α-cyano-4-hydroxy-transcinnamic acid matrix in 50% acetonitrile were added to the dried peptide digest spots and, again, allowed to air dry at room temperature. MALDI-TOF MS analyses were performed with a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems MDS Sciex, Toronto, Canada) operated in positive reflector mode (accelerating voltage of 20,000 V). Mass spectra were obtained (Nd:YAG laser at 355 nm, 40 shots/sub-spectrum for 800 Total shots/spectrum) by reflector positive mode. Mass spectra were internally calibrated using peptides from the auto-digestion of trypsin. Proteins identified ambiguously by peptide mass fingerprinting were subjected to MS/MS sequencing analyses. Thus, from the MS spectra, suitable precursors were selected for MS/MS analyses with CID in (atmospheric gas was used) 1 KV ion reflector mode and precursor mass; windows ±2 Da. For protein identification, the non-redundant NCBI Viridiplantae (Green Plants) database (1,716,557 sequences) was searched using MASCOT 2.3 through the Global Protein Server v3.6 from Applied Biosystems.

Toxicity of Dwarf Elder Blossom D-Galactose-Specific Lectins

Ebulin blo or SELblo dissolved in water up to the required concentrations adjusted for the mice weights were administered in 60 µL to Swiss mice by nasal instillation under mild isoflurane anesthesia to ease mice handling. Controls with the same volumes of water did not affect survival and body weight. Owing to the clarity of the results, only seven animals per group were used. Treatments, experiments and euthanasia of mice were conducted according to the European Communities Council guidelines (2010/63/EU) concerning the protection of experimental animals.

Conclusions

Two lectins, which we named ebulin blo and SELblo, have been found in S. ebulus blossoms. Ebulin blo (mass 63,225 m/z), like the ebulin present in leaves and fruits, are composed of two polypeptide chains of apparent molecular masses of 28,000 (A chain) and 35,000 (B chain) linked by a disulfide bond. SELblo (mass 68,432 m/z) is devoid of apparent toxicity and has two identical subunits (B chains) of an apparent molecular mass of 36,000 linked by a disulfide bond. Both lectins share amino acid sequence homology with the elderberry allergen, Sam n1. The nasal instillation of a 5 mg/kg body weight of an ebulin blo solution in water triggers lethal toxicity in mice, within five days after the administration. We believe that the administration as an aerosol could be even more toxic than in solution. The toxicity of ebulin blo raises concern due to its potential as a bioweapon, like ricin. More studies should be carried out in order to ascertain the toxicological relevance in that context and to develop analytical procedures for its detection, potential prevention and therapeutics.

Figure 1 .
1(a) Picture showing blossoms, early green fruits and early green stalks of S. ebulus. (b) SDS-PAGE of raw extracts of blossoms, early green fruits and early green stalks; 20 µL of extract concentrated by ultrafiltration were loaded into each well.

Figure 2 .
2(a) Affinity chromatography of raw protein extract on acid-treated Sepharose 6B. (b) Chromatography of affinity-bound protein on Superdex 75. (c) SDS-PAGE of proteins purified from S. ebulus blossoms. The amounts of protein per well were ebulin f (11.5 µg), SELfd (11.1 µg), ebulin blo (9.5 µg) and SELblo (10.8 µg). The markers were, from top to bottom: SNAI (Mr 136 kDa), BSA (Mr 68 kDa), Ng b (Mr 58 kDa), ovalbumin (Mr 45 kDa), SNAIV (Mr 30 kDa) and trypsin inhibitor (Mr 20 kDa). 2-ME, 2-mercaptoethanol.

Figure 3 .
3Mass spectrometry profiles of tryptic peptides from ebulin blo (a) and SELblo (b).

Figure 4
4Figure 4. Alignment of amino acid sequences of some tryptic peptides of blossom lectins with ebulin l and SNAld. Boxed are sequences found in tryptic peptides from the allergen Sam n1 of S. nigra pollen. In red are coincident sequences. Highlighted in yellow and grey are tryptic peptides obtained from ebulin blo and SELblo, respectively.

Figure 5 .
5Effects of the instillation of ebulin blo in Swiss mice on the evolution of survival (a) and relative body weight loss (b). Dashed line and circles: instillation of 2.5 mg/kg body weight of ebulin blo; continuous line and squares: instillation of 5 mg/kg body weight of ebulin blo; dotted line: intraperitoneal administration of 5 mg/kg body weight of ebulin f.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
AcknowledgmentsThis research was supported by Junta de Castilla y León (Grupo de Excelencia GR106 and Convenio-Consejería de Sanidad) and UVa-GIR funding to T.G. The proteomic analysis was carried out in the Proteomics Facility UCM-PCM, a member of ProteoRed network.Conflicts of InterestThe authors declare no conflict of interest.
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Reproductive Biology and Endocrinology Tissue engineering and cell based therapies, from the bench to the clinic: The potential to replace, repair and regenerate

William L Fodor firstname.lastname@example.com
Address: Center for Regenerative Biology and Department of Molecular and Cell Biology
University of Connecticut
06250-4243StorrsCTUSA

Reproductive Biology and Endocrinology Tissue engineering and cell based therapies, from the bench to the clinic: The potential to replace, repair and regenerate
BioMed Central Page 1 of 6 (page number not for citation purposes) Review * Corresponding author
The field of Regenerative Biology as it applies to Regenerative Medicine is an increasingly expanding area of research with hopes of providing therapeutic treatments for diseases and/or injuries that conventional medicines and even new biologic drug therapies cannot effectively treat. Extensive research in the area of Regenerative Medicine is focused on the development of cells, tissues and organs for the purpose of restoring function through transplantation. The general belief is that replacement, repair and restoration of function is best accomplished by cells, tissues or organs that can perform the appropriate physiologic/metabolic duties better than any mechanical device, recombinant protein therapeutic or chemical compound. Several strategies are currently being investigated and include, cell therapies derived from autologous primary cell isolates, cell therapies derived from established cell lines, cell therapies derived from a variety of stem cells, including bone marrow/mesenchymal stem cells, cord blood stem cells, embryonic stem cells, as well as cells tissues and organs from genetically modified animals. This mini-review is not meant to be exhaustive, but aims to highlight clinical applications for the four areas of research listed above and will address a few key advances and a few of the hurdles yet to be overcome as the technology and science improve the likelihood that Regenerative Medicine will become clinically routine.

Introduction

Many diseases and or physical defects due to injury result in the loss of specialized cells within organ systems and lead to organ system dysfunction. For example, Parkinson's disease results in the progressive loss of dopaminergic neurons within the substantia nigra region of the brain leading to motor deficits that result in dystonia and dyskinesia. Injuries, such as meniscal tears and spinal cord injury, can also result in the degeneration and loss of tissue leading to physical defects that can affect normal behavior. Additionally, insulin dependant diabetes mellitus (IDDM), multiple sclerosis (MS) and other autoimmune disorders lead to a loss of tissue that disrupts normal metabolism and bodily functions. The potential to treat these conditions with cell-based therapies holds promise for tissue/organ repair with the ultimate goal to regenerate and restore normal function. Several cell types will be discussed and are defined as; tissue specific differentiated cells, such as chondrocytes; progenitor cells isolated from specific tissues, such as bone marrow stem cells or neural stem cells; and embryonic stem cells that are derived from the inner cell mass of the developing blastomere.

Autologous Cell Therapy

Tissue specific differentiated autologous cells (as opposed to autologous progenitor cells, see below) harvested from an individual, cultured ex vivo to expand, and reintro-duced into a second site for repairing damaged tissue with "self" is ideal from an immunologic perspective. Several pre-clinical models as well as clinical applications are currently being explored and include chondrocytes for cartilage repair [1][2][3][4][5][6][7][8][9][10][11][12], keratinocytes and/or dermal fibroblasts for burn and wound repair [13][14][15][16][17], myocytes for myocardial repair [18][19][20][21][22][23], retinal pigment epithelial cells for age related macular degeneration [24][25][26][27] and Schwann cell transplantation to restore myelin in CNS lesions. [28][29][30][31][32]. The two most developed autologous cell therapies that have advanced from the laboratory to the clinic involve the repair of cartilage using autologous chondrocytes and the treatment of burns with autologous cultured keratinocytes.

Grande et. al., first demonstrated that autologous chondrocyte cultures could be utilized to repair articular cartilage defects in the rabbit knee [11,12]. Subsequently, this technique has been applied to the clinical treatment of articular cartilage defects [1,4,7,33] and has now evolved into an FDA approved therapy supplied by Genzyme Biosurgery http://www.fda.gov/cber/approvltr/ autogen082297L.htm. Genzyme Biosurgery has also developed an autologous keratinocyte culture procedure and currently markets Epicel ® as a treatment for burn victims http://www.genzymebiosurgery.com/prod/burn/ gzbx_p_pt_burn.asp. Although repairing "self" with "self" is attractive and doesn't require immunosuppressive drug therapy for graft maintenance, there are limitations related to the harvesting of tissue and expanded tissue culture. Typically, harvesting the original source tissue from the patient requires a surgical procedure which minimally is a biopsy, but could also require a large resection of the tissue. The concern is causing a second site defect that leads to pain, discomfort or a deficit that effects behavior, hopefully to a lesser extent than the original. The ability of the adult tissue to expand in tissue culture to generate sufficient numbers of cells is also a potential limitation. Additionally, primary cell cultures can become senescent or dedifferentiate during the culture period. Another limitation is that this type of therapy is only amenable to tissues that can sustain surgical harvesting and ex vivo culturing, emphasized by the fact that only two autologous cell therapies have achieved FDA approval for the medical market.

Autologous progenitor cells harvested from an individual and used for "self" tissue repair is also immunologically ideal. The most widely used source of adult progenitor cells are derived from bone marrow. The mesenchymal compartment within the bone marrow has the capacity to differentiate into many cell and tissue types given the appropriate growth conditions [34]. Early studies using bone marrow stromal cells for tissue repair focused on the repair of bone defects [35] however, more recent studies have applied bone marrow progenitor cells to repair a variety damaged tissue types, including cartilage [36][37][38], myocardium [39,40], liver [41], spinal cord injury [42][43][44] and most recently diabetes [45]. However, these differentiation studies are still in the experimental stage. The potential to differentiate "self" progenitor cells into a variety of tissues is extremely promising for the field of Regenerative Medicine, however continued experimentation is necessary to understand the differentiation processes and to be able to reproducibly guide these cells into the appropriate tissue, prior to clinical application.

Autologous peripheral blood or autologous bone marrow stem cells are currently used clinically, but not from a commercial stand point. A current search of the FDA web site http://clinicaltrials.gov resulted in 108 studies involving autologous peripheral blood or autologous bone marrow stem cells as a treatment therapy, 96 of which were related to cancer treatments. None of the trials are related to stem cell differentiation followed by transplantation.

Allogeneic Tissues and Cell Lines

The use of allogeneic tissue for transplantation is clinically routine due to the development of immunosuppressive drug therapies, such as cyclosporine, FK506 and rapamycin. The use of engineered tissue and specialized cell lines for the treatment of disease and injury is more recent and will also require immunosuppression unless engineering strategies are utilized to make the tissue resistant to immune destruction or through tissue processing to reduce immunogenicity. As is the case for autologous cell therapies, the furthest advances are in the area of connective tissue replacement, cartilage and skin. [46][47][48] Currently, Apligraft ® (Organogenesis, Inc) is used as a dermal replacement for chronic wounds and is composed of neonatal foreskin kerotinocytes and dermal fibroblasts [49]. Although earlier studies demonstrated that Langerhan's cell-free epidermal skin cultures were rejected following transplantation [50], Apligraft tissue appears to be uniquely non-immunogenic due to the processing of the tissue [51,52] and represents an exception to the need for immunosuppression during allogeneic transplantation. A similar product, Dermagraft ® is also available from Smith & Nephew.

Another interesting allogeneic cell type harvested from cadaveric sources for the treatment of Parkinson's disease are allogeneic cultured retinal pigment epithelial cells that are encapsulated to provide an immune barrier [53]. Titan Pharmaceuticals, Inc has advanced this research into the clinic and is currently conducting a Phase IIb clinical trial with initial positive results [54].

Allogeneic cell lines are also being developed as a source of cells for regenerating damaged tissue due to disease.

The human NT2 cell line was shown to differentiate and develop into neurons in rodent stroke models [55,56] and is currently being tested in clinical trials for the treatment of stroke by Layton Biosciences, Inc [57,58]. Patients receiving the NT2 cell grafts do receive immunosuppression to inhibit immune rejection of the graft [57,58].

The development of allogeneic engineered tissues for commercial purposes has similar limitations as commercial autologous cell therapies with the added complication of immune rejection. Encapsulation, tissue processing, tolerance induction and/or genetic modification will be necessary unless the patient population is receptive to and the severity of the disease warrants the use of immunosuppressive drugs.

Allogeneic stem cells

Allogeneic bone marrow transplantation is used clinically to treat hematologic disorders and cancer, but as is the case for autologous bone marrow transplantation, not from a commercial, tissue engineering standpoint. New clinical trials are focusing on the use of peripheral blood stem cells and specific subsets of bone marrow stem cells for these indications (for example [59,60]). Searching the FDA clinical trial data base identified 117 trials that utilize allogeneic stem cells or bone marrow in the treatment regime. Sorting through those trials, 99 were specific for stem cell therapy. As for autologous bone marrow or peripheral blood stem cell therapies, most were for cancer indications, 84/99. The data base included 9 trials utilizing allogeneic stem cells for anemia/hematologic disorders, 2 trials are designed to treat metabolic storage diseases and there are single trials for each of the following; Granulomatous Disease, HIV patients not responsive to highly active antiretroviral therapy, Mycosis Fungoides and Sezary Syndrome and allogeneic bone marrow rejection http://clinicaltrials.gov. Virtually all of these therapies are in combination with some form of immunosuppression and none of the trials are related to stem cell differentiation followed by transplantation.

The discovery and isolation of neural stem cells from fetal [61][62][63][64] and adult human brain [65,66] is a significant development in the area of neural cell differentiation that has led to the possibility of producing specialized cells for the treatment of neurologic disorders, such as Parkinson's disease and spinal cord injury. Many groups have studied the differentiation of neurospheres into neuron, glial, and astrocyte lineages, however the work from Goldman's group [66,67] sets a president for selection strategies that are most relevant to the field of tissue engineering. Nunes, et. al., and Keyoung et. al., showed that transfected/transduced specific promoter constructs driving green fluorescent protein (GFP) can be used to select specific neural progenitor cells by flow cytometry [66,67]. Expanding on these techniques could lead to the eventual development of a commercial application of neural stem cells by reproducibly selecting the desired neural phenotype. Demonstrating that specific lineages can be selected genetically with drug selectable or fluorescent expression plasmids lays the ground work for further selection schemes and further genetic modifications, such as modifying the immune response leading to a universally accepted source of human neural tissue for transplantation.

Although stem cells from adult tissues have more plasticity than originally thought, they typically are limited in their capacity to generate all possible tissue and cell types. Stem cells derived from the inner cell mass of the early embryonic blastocyst (ES cells) can proliferate indefinitely [68] and can give rise to virtually any cell type [69]. The development of human embryonic stem cells [70] has raised the possibility that an unlimited supply of human tissue could be generated from ES cells and that these tissues could be used to replace and repair damaged tissue in any organ system. It should be noted that although these human cells are referred to as ES cells, they cannot be qualified as a true "ES cell" which is defined by the ability to contribute to the germ line during embryonic development. ES cells from other species are tested in this manner, however it is ethically unfeasible with human ES cells. Having qualified the definition of a human ES cell, several studies have demonstrated that human ES cells retain the capacity to differentiate into a variety of tissues, including neuronal cells, myocytes, adipocytes and hematopoietic cells [71][72][73][74][75][76][77]. The challenge now is to be able to direct differentiation or select for the desired phenotype and to develop these therapies in the absence of immunosuppression, similar to the strategies taken by the field of xenotransplantation. The ability to genetically modify ES cells is a great advantage and could be used to overcome both the directed differentiation/selection hurdle as well as the immune response hurdle.

Xenotransplantation

In the ongoing search for a reliable source of tissue to replace lost cells, tissues and organs, research in the area of xenotransplantation (cross species transplantation) has grown tremendously in the last 20 years. Overcoming the immunologic hurdle of cross species transplantation as well as the problem of cross-species pathogen infectivity, i.e., xenozoonosis, are the scientific challenges facing the field [78,79]. The ability to genetically modify species such as the pig through transgenesis and nuclear transfer, to express human genes and to mutate detrimental genes expressed in pig cells [80][81][82][83][84][85][86] still holds promise for engineered tissues and organs for human transplantation. The production of galα1,3gal transferase null transgenic pigs [84] represents a significant development towards eliminating both hyperacute and acute vascular rejection and may lead to extended survival of pig organs in old world primates, including humans, in combination with standard triple drug immunosuppressive therapy.

Interestingly, there have been a series of pig-to-human xenotransplantation clinical experiments for the treatment of diabetes [87,88] and FDA approved clinical trials for the treatment of neurologic disorders using outbred pig tissue [89][90][91]. Although there was some evidence of cell engraftment in both indications [87,90], no efficacy was established due to the transplant [87,89]. To date, a Phase I clinical trial was completed using transgenically engineered pig livers to detoxify the blood of fulminant hepatic failure (FHF) patients via extracoporial perfusion [92], however there is yet to be an FDA approved transgenic animal tissue for use in human transplantation.

Although the theoretical risk of xenozoonosis is a risk and represents a significant psychosocial issue, several studies investigating the possibility of cross species infectivity, including a retrospective analysis of 160 human transplant recipients exposed to porcine tissues have yet to reveal transmission of porcine viruses to humans or primates in vivo [93][94][95][96]. The prospect of xenotransplantation is still relevant to solid organ and islet transplantation and with FDA oversight, animal as well as patient monitoring, the risks associated with xenozoonosis will be overcome.

The Future

The challenges associated with stem cell differentiation, tissue engineering, and xenotransplantation are manyfold in each of the respective fields, however one of the biggest challenges beyond the science and biology is the development of an FDA approved product from any of the developmental areas discussed above. The regulatory issues and points to consider documents are in the early stages of development by the FDA in collaboration with expert review panels and will continue to change as the technology advances into clinical applications. The experiences of Genzyme Biosurgery, Organogenesis, Diacrin, Layton Biosciences, Titan Pharmaceuticals and other tissue engineering companies will benefit the field as a whole.
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