extfieldsofstudy: Medicine Biology

text:
Acetylation of p65 at lysine 314 is important for late NF-κB-dependent gene expression

Background NF-κB regulates the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation and survival. We have earlier reported that p65, a subunit of NF-κB, is acetylated in vitro and in vivo at three different lysines (K310, K314 and K315) by the histone acetyltransferase p300. Results In this study, we describe that site-specific mutation of p65 at lysines 314 and 315 enhances gene expression of a subset of NF-κB target genes including Mmp10 and Mmp13. Increased gene expression was mainly observed three hours after TNFα stimulation. Chromatin immunoprecipitation (ChIP) experiments with an antibody raised against acetylated lysine 314 revealed that chromatin-bound p65 is indeed acetylated at lysine 314. Conclusions Together, our results establish acetylation of K314 as an important regulatory modification of p65 and subsequently of NF-κB-dependent gene expression.

Background
The inducible transcription factor NF-B has an important function in regulating immune and inflammatory responses, apoptosis, cell proliferation and differentiation and tumorigenesis [1][2][3]. NF-B is induced in almost all cell types by different extracellular stimuli causing the activation of an enormous array of target genes [4]. The NF-B transcription factor family comprises NFKB1 (p50/p105), NFKB2 (p52/p100), p65 (RelA), c-Rel and RelB, which form homo-and heterodimers. The most abundant, best-studied and "classical" form of NF-B is a heterodimer consisting of the two subunits p50 and p65. In most unstimulated cells, NF-B is found as inactive transcription factor complex through its physical association with one of the several inhibitors of NF-B (IB) [5]. This family of IB's includes IBα, IBβ, IBγ, IBε (p105/p50, C-terminus), p100/p52 (C-terminus), IB-R and Bcl-3. Virtually all cell types show NF-B responses where the activity of NF-B is specifically regulated at multiple levels [1,3,6]: the level of protein synthesis, the existence of at least 12 different NF-B dimers, the interaction of these dimers with specific IBs and their subcellular localization, post-translational modification of these dimers in the cytoplasm and the nucleus, differential accessibility of B sites in various promoter and enhancer, differential binding to B's response elements due to different affinities, and cell type and stimuli specific interaction with a combination of cofactors.
NF-B is subject to a variety of post-translational modifications (e.g., phosphorylation [7], ubiquitination [8] or prolyl-isomerisation [9]) that modulate its activity. Phosphorylation of the p65 subunit by the PKAc, MSK1 and PKCζ kinases enhances its interaction with the coactivator p300/CBP and stimulates the NF-B transcriptional activity [7,[10][11][12], while dephosphorylation of p65 by the phosphatase WIP1 negatively affected the interaction with p300 [13]. It has recently been shown that p65 and p50 are reversibly acetylated by p300 and PCAF [14][15][16]. Chen et al. identified lysine residues (K) 218, 221 and 310 of p65 as acceptor sites for p300 acetylation. Kiernan et al. identified K122 and 123 in p65 as acetylation sites modified by both p300 and P/CAF. A recent report presented the TGF-β1-mediated acetylation of p65 at K221 in vitro and in vivo enhancing the induced activation of NF-B by bacteria [17]. We recently confirmed acetylation on K310 and provided further evidence for acetylation of p65 on K314 and 315, two novel acetylation sites [18]. Genetic complementation of p65 knockout (-/-) cells with wild type and acetylation-deficient mutants of p65 revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by TNFα was affected by acetylation on these residues. Micro array analysis of these cells treated with TNFα for only 45 minutes identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of p65 [18]. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of p65 regulates the specificity of NF-B dependent gene expression.
Here, we extended the gene expression analysis to three hours after TNFα stimulation and identified genes, which are higher expressed by mutating K314 and 315. ChIP experiments with antibodies directed against acetylated K314 revealed that this lysine is indeed acetylated when p65 is bound to chromatin. Together our results provide evidence that acetylation of K314 is important for the regulation of NF-B-dependent gene expression in vivo.

Mutation of p65 K314/315 regulates TNFa-induced NF-B-dependent gene expression at 3 hours
We provided earlier evidence that acetylation of p65 at K310, 314 and 315 is important for the expression of a defined subset of genes [18]. These earlier studies provided a first glance of the functional relevance of p65 acetylation, since gene expression was measured only after 45 minutes of TNFα stimulation. In order to know if the requirement for site-specific acetylation is maintained for the same genes after longer exposure to TNFα, and to identify possible new genes regulated through p65 acetylation, we decided to extend our analysis to 3 hours of stimulation. For this, we used p65 (-/-) mouse embryonic fibroblasts (MEFs) complemented with acetylation-deficient mutants, where the target lysines for acetylation were mutated to arginines. These cells were described and extensively characterized previously [18]. p65(-/-) cells complemented either with wild type p65, an empty plasmid as control (pTV), the acetylation-deficient double mutant K314/315R or the triple mutant KTR (K310/314/315R), were stimulated by TNFα for 3 hours and total RNA was isolated in three independent replicates from these cells. RNA was amplified, labeled and hybridized to the Agilent Whole Mouse Genome Array. After statistical analysis of the expression profiles, differentially expressed genes were identified ( Fig. 1A-B, and Tables 1 and 2). We focused only on genes that required p65 for their proper induction, which were identified by comparing gene expression profiles from wild type and pTV cells. The majority of differentially expressed genes in KTR mutant were strongly downregulated compared to wild type cells, suggesting that acetylation of p65 at these residues is also an important modification for the expression of the extended NF-B-dependent gene expression (Fig. 1A). In contrast, experiments with the double K314/315R mutant revealed that the majority of genes were slightly upregulated after 3 hours compared to wild type cells (Fig. 1B).
Gene expression of Mmp10 and Mmp13 is enhanced when K314/315 are mutated We subsequently investigated the induction kinetics of several genes by real-time RT-PCR in the complemented cell lines stimulated by TNFα for different time points (between 20 and 360 minutes). Selection of these genes was based on their inducibility by TNFα, as well as their dependency on p65 and their regulation by acetylation of K314/315. Mmp13 and Mmp10 represented genes that were upregulated in the micro array experiments of K314/315R but not affected in KTR expressing cells. In contrast, Cfb and Mpa2l represented genes that were not affected in cells expressing K314/315R, but KTR, suggesting that acetylation of K310 is important for these genes. Overall, the absolute mRNA levels of Mmp10 and Mmp13 were strongly and significantly increased upon time (with a maximum value at 6 and 3 and hours, respectively) in cells expressing the K314/ 315R mutant ( Fig. 2A, B and Additional file 1: Supplemental Table S1), while the expression levels of Cfb and Mpa2l were not affected in the same cells (Fig. 2C, D and Additional file 1: Supplemental Table S1), corroborating our micro array results. Gene expression analysis in cells expressing the KTR mutant indeed confirmed that K310 is important for Cfb and Mpa2l, but counteracts the effect of K314/315R for Mmp10 or Mmp13 expression. Interestingly, the dependency on K310 acetylation for Cfb and Mpa2l was already observed when the basal expression levels were analyzed (0 hour time point).

Characterization of antibodies raised against acetylated lysine 314 and 315 of p65
To further assess the functional relevance of p65 acetylation in vivo, we generated different antibodies raised against the acetylated K314 or K315. All raised antibodies recognized their specific p300-mediated acetylated residues on recombinant p65 acetylated in vitro (Fig. 3A) or on over expressed p65 acetylated in vivo (Fig. 3B).
To confirm that endogenous p65 is indeed acetylated at the indicated lysines, immunoprecipitation experiments with the antibodies raised against acetylated K314, K315 or against p65 were performed. The antibody anti-acK314 immunoprecipitated p65 only in cells complemented with wild type p65 in a TNFα-dependent manner (Fig. 3C). No p65 was immunoprecipitated in  (Fig. 3D), suggesting, that endogenous p65 is indeed acetylated at K314 upon stimulation with TNFα. Unfortunately, anti-acK315 was not specific enough to recognize endogenous acetylation at K315.
Chromatin-associated p65 is acetylated at lysine 314 From the above selected genes only Cfb was already described to contain a B site in its promoter [19]. We therefore searched for putative B sites within the DNA sequence 1 kB upstream of the transcription start site (TSS) of the selected genes. Bioinformatic analysis identified several putative B sites in the promoters of Mmp10 and Cfb, one site in the promoter of Mpa2l, but none for Mmp13 (Fig. 4A). Mmp13 was thus not further investigated. ChIP studies in p65(+/+) MEFs stimulated with TNFα for 20 and 180 minutes revealed that p65 was recruited to the promoters of Cfb and Mpa2l in a stimulus-dependent manner, while no enrichment was observed in p65(-/-) MEFs (Fig. 4B). These recruitments were promoter-specific, since p65 occupancy to promoter of glucagon, a negative control, was not induced upon TNFα stimulation. IP-10, a known NF-B target gene with very well characterized B sites at its promoter, served additionally as a positive control. Unfortunately, no p65 enrichment could be observed to the B sites of Mmp10 (data not shown), indicating that p65 would activate this gene through other B sites or other transcription factors.    Interestingly, ChIP experiments using the anti-acetyl K314 antibodies showed that chromatin-associated p65 is indeed acetylated at K314 on all analyzed genes (Cfb, Mpa2l and IP-10) (Fig. 4C), although the expression of these genes was not affected by mutating K314/315 ( Fig.  2C and 2D). Furthermore, the detected amount of recruited p65, which was acetylated at K314, increased over time (comparing values at 180 min and 20 min). Unfortunately, the antibody raised against acetyl K315 was not able to immunoprecipitate p65 under the tested conditions (data not shown). Together, these experiments identified Mpa2l as a novel NF-B target gene through the recruitment of p65 to its promoter and provide strong evidence that chromatin-bound p65 is indeed acetylated at K314.

Discussion
We have previously shown that p65 acetylation plays an important role in regulating NF-B-dependent transcription of a subset of genes. Our current results confirm those earlier findings by identifying new genes differentially regulated in the acetylation-deficient mutants compared to wild type cells. Remarkably, only a few genes identified after 3 hours TNFα stimulation overlapped with the genes identified in our previous study after 45 minutes TNFα stimulation (data not shown). We also observed that TNFα does not induce NF-B activity in the complemented MEFs as efficiently as in the parental p65(+/+) MEFs used for ChIP (data not shown). Therefore, although our complemented MEFs are useful to address the role of p65 acetylation in vivo, we might have identified only part of the genes influenced by p65 acetylation with the micro array screen.
Cfb was previously suggested to be a direct target of NF-B by EMSA experiments [19]. Our ChIP assays confirmed that p65 is recruited to Cfb promoter in a TNFα-dependent manner in vivo and further identified Mpa2l as a novel NF-B target gene. Although Mmp10 and Mmp13 were differentially expressed in acetylationdeficient mutant cells compared to wild type cells, we failed to detect p65 recruitment to those promoters. One explanation could be that p65 is recruited to the promoter region of these genes with a different kinetics than the one we investigated here. Alternatively, p65 could bind to a regulatory element located far away from the promoter to regulate transcription, as has been Figure 3 Characterization of specific antibodies against p65 acetylated at K314 or 315. (A) Purified recombinant p65 wild type and the acetylation-deficient mutants were incubated with recombinant p300 in the presence (+) or absence (-) of acetyl Co-A. Proteins were resolved on SDS-PAGE and analyzed by western blot using the indicated antibodies. (B) HEK 293T cells were transfected with p65 wild type or mutants, with (+) or without (-) p300 co-transfection. Acetylation of p65 at specific lysines was assessed by western blot using the specific antibodies. * indicates a non-specific band. (C and D) p65 was immunprecipitated from untreated or TNFα-stimulated cells using the indicated antibodies. Immunoprecipitated proteins were separated by 10% SDS-PAGE and subsequently analyzed by western blot using an anti-p65 antibody. * indicates IgG-band.
shown for several NF-B target genes [20][21][22]. NF-B is known to activate the expression of many transcription factors and their regulators [4]; consequently, a third possibility is that p65 directly induces the expression of a protein that regulates the expression of these genes. Along this line, NF-B has been shown to directly activate the expression of the transcription factor Elk-1, which in turn induces Mmp13 gene expression [23].
Interestingly, the stimulatory effect of mutating K314/ 315 was lost when additionally mutating K310, suggesting that K310 acetylation is not affected by the lack of K314/315 acetylation, and that the modification of K310 would counteract the effect of K314/315 at the particular tested genes. Furthermore, the effect of mutating K314/315 on gene expression was more obvious after 3 hours of stimulation compared to early time points (e.g. 20 minutes), suggesting that acetylation of these lysines might more likely affect termination of gene expression than its induction itself. Here, we provide for the first time evidence that endogenous, chromatin-bound p65 is indeed acetylated at K314 upon stimulation with TNFα. Unfortunately, acetylation of K315 could not be confirmed due to the lack of a specific acetylation-dependent antibody that recognizes endogenous proteins. Interestingly, we observed acetylation of p65 K314 at promoters of genes whose expression was not affected by mutating K314/315 (e.g. IP-10). Thus, it could well be that p65 is acetylated upon recruitment of p300 at promoters of many genes, but that the expression of only some of these genes is affected by p65 acetylation at distinct lysines.
The observation that mutation of K314/315 increases expression of certain genes (e.g. Mmp10 and Mmp13) suggests that acetylation of K314 and possibly K315 might represses gene expression. A possible molecular mechanism is that acetylated lysines at p65 create docking sites for bromodomain-containing proteins which need to be recruited to promoters to modulate transcription [24]. On the other hand, we can currently not exclude that lysines 314 and/or 315 are modified by another post-translation modification (e.g. methylation, [25]) and that mutating these residues would thus affect gene expression independently of acetylation. However, provided the observed acetylation of chromatin-bound p65 at K314, acetylation is more likely to compete with another lysine modification, possibly resulting in crosstalk between different modifications [25]. Indeed, a recent study reported that these same two lysines (K314/ 315) can be methylated by Set9 to induce ubiquitinylation of NF-B and subsequently terminate gene expression [26]. Thus, acetylation at K314 and possibly K315 could prevent methylation-mediated repression of target genes and thus avoid methylation-induced termination of p65-dependent gene expression. A direct evidence for methylated p65 at K314/315 bound to chromatin is however still missing. Whether the same genes are regulated by both post-translational modifications should be further addressed. Alternatively, both post-translational modifications might regulate distinct set of NF-Bdependent genes and thus not influence each other.

Conclusions
Together, our results establish acetylation of K314 as an important regulatory modification of p65 and subsequently of NF-B-dependent gene expression.

Tissue culture
Complemented p65(-/-) NIH 3T3 MEFs stably expressing p65 wild type or the acetylation-deficient mutants were generated by lentiviral complementation as previously described in [18]. Briefly, HEK 293T cells were transfected with the packaging plasmid, the envelope plasmid and pTV-myc-RelA/p65 wild type, RelA/p65 K/ R mutants or the control pTV vector. p65(-/-) MEFs were infected with the viral supernatant and split into selective medium (2.5 μg/ml Blasticidin) after thirty-six hours. Expression of recombinant proteins was confirmed by western blot analysis. Pools of cells were used for further analysis. They, as well as the p65(-/-) and (+/+) parental NIH 3T3 MEFs kindly provided by A. Beg [27], were maintained in DMEM supplemented with 10% FCS, 100 units/ml penicillin/streptomycin (Gibco) and non-essential amino acids (Gibco). HEK 293T cells were maintained in DMEM supplemented with 10% FCS and 100 units/ml penicillin/streptomycin.

Plasmids
Plasmids for the mammalian overexpression of human p65 wild type and mutants K310R, K314/315R and KTR in HEK 293T cells were described elsewhere [18]. Briefly, pph-CMV-Km-RelA/p65 wild type, previously described in [28], was used as template vector for the generation of the p65 mutants by site-directed mutagenesis according to the QuickChange protocol (Stratagene). All introduced mutations were confirmed by sequencing.

Generation of recombinant proteins
The recombinant proteins were expressed by baculovirus in Sf21 cells using either the FastBac or the Bac-PAK systems (Clontech). His-tagged proteins were purified over Ni 2+ -beads (ProBond, Invitrogen).

Micro array
Complemented cell lines p65 wild type or acetylationdeficient mutants were starved overnight before either left untreated or stimulated with 30 ng/ml TNFα for 3 hours. Total RNA from three biological replicates per sample was isolated at different days using the 'Total RNA isolation mini kit' (Agilent Technologies). RNA quality was measured on the 2100 Bioanalyzer (Agilent Technologies). Microarray experiments were performed using 'Whole Mouse Genome (4 × 44 K) Oligo Microarray Kit' (Agilent Technologies) and 'One-color micro array-based gene expression analysis' (Agilent Technologies) following the manufacturer's protocol. Cy3-labeled cRNA was purified with the RNeasy kit (Qiagen). Dye incorporation was assessed with the ND-1000 Spectrophotometer (NanoDrop Technologies). Per sample, 1.65 μg cRNA from each of the three biological replicates was hybridized to independent arrays according to the manufacturer's protocol. Hybridized slides were scanned with the Agilent DNA Microarray scanner and quantified using the Agilent Feature Extraction software. The data analysis was performed using 'Rosetta Resolver® Gene Expression Data Management and Analysis System' (Rosetta Biosoftware). Briefly, data was processed and normalized with default settings. Then, lowsignal genes with signal intensities < 0.1 were filtered out. Differential expression between two conditions was assessed based on the average ratio and significance. All genes with expression ratios < 0.556 and >1.8, and a pvalue < 0.05 were selected to generate the tables of significantly regulated genes. These sequence data have been submitted to the GEO database http://www.ncbi. nlm.nih.gov/geo under accession number GSE15196.

Gene expression by quantitative RT-PCR
Complemented MEFs were starved overnight before treatment with 30 ng/ml recombinant TNFα for different time points. Total RNA was isolated from at least two biological samples at different days with the 'Total RNA isolation mini kit' (Agilent Technologies). RNA quantity was assessed with the ND-1000 Spectrophotometer (NanoDrop Technologies). 2 μg RNA was subsequently retro-transcribed using the 'High-capacity cDNA reverse transcription kit' (Applied Biosystems) following the manufacturer's protocol. Real-time PCR was performed using the Rotor-Gene 3000 (Corbett Life Science, now Qiagen) and TaqMan assays from Applied Biosystems for the following genes: Cfb, Mpa2l, Mmp10, Mmp13, Rps6 and CanX, according to Applied Biosystems' protocol. The last two genes were used as internal controls to normalize for RNA input. RNA from at least two biological replicates per sample was measured and analyzed with REST [29]. Each experiment was run three independent times, the mean value and ± SD was calculated and blotted into graphs. A 2-tails, paired t-Test was performed with the log of the values to additionally calculate the p-values.

Acetylation assays in cells
HEK 293T cells were transfected with expression plasmids for p300 and either myc-tagged p65 wild type, the acetylation-deficient mutants or an empty vector, using the calcium phosphate precipitation method. After 23 hours, cells were treated with 10 ng/ml human TNFα for 30 minutes. Then, whole cell extracts were prepared and 40 μg protein was analyzed by SDS-PAGE and western blot.
Additional file 1: Gene expression analysis of Mmp10, Mmp13, Cfb and Mpa2l with quantitative RT-PCR. The mean value of three independent runs relative to wild type unstimulated is shown, as well as ± SD and p-values. P-values were calculated comparing data of each cell line with wild type. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11-22-S1.DOC ]


extfieldsofstudy: Biology Medicine

text:
Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.

Introduction
In the last 25 years, essential progress has been made in prophylaxis against hepatitis B (HB) using one of the most effective and cheapest recombined vaccines (Goldstein and Fiore 2001). However, one third of the worldwide human population is still suffering from HB and the number of hepatitis B virus (HBV) chronic carriers is constantly growing, mainly in developing countries (Kao and Chen 2002). Insufficient medical infrastructure causes delays or even failure of mass-scale HB vaccination programs. Thus, Tomasz Pniewski and Józef Kapusta contributed  reliable and easily implementable prevention methods are desirable. Oral vaccines, including plant-based formulations, are considered as alternatives or supplements for standard injection vaccines, since they have the potential for simplified vaccination procedures against pathogens penetrating through mucosal membranes or blood (Aziz et al. 2007;Mestecky et al. 2008).
Research on the development of an oral plant-based vaccine against the HBV and other pathogens has been conducted for over 15 years. The small HBV surface antigen (S-HBsAg) was expressed in tobacco, potato, lettuce, and other plants, as well as in tissue and cell suspension cultures. Anti-HBs immune response triggered by the oral administration of plant material containing S-HBsAg was shown both in mice (Kapusta et al. 1999;Richter et al. 2000;Kong et al. 2001;Gao et al. 2003;Joung et al. 2004) and in humans (Kapusta et al. 1999(Kapusta et al. , 2001Mason et al. 2003;Thanavala et al. 2005). However, despite some successes, reproducible and long-lasting oral immunization by feeding proved to be extremely difficult in terms of the control of the immunization regime, raw plant material storage, and its inconvenient administration, which all might potentially lead to tolerance induction (Kirk et al. 2005). Moreover, previously used plants containing S-HBsAg exhibited resistance to antibiotics, which limits their application only to laboratory experiments.
We present here an approach to oral vaccines based on the low-dose administration of lyophilized tissue, derived from lettuce expressing S-HBsAg at a high level and resistant to glufosinate-derived herbicides, as the only selection marker. Lettuce was chosen as a vaccine producer since it represents a plant naturally free of harmful substances, adaptable for cultivation in various conditions, and simple for processing into formulas of a potential oral vaccine. Freeze-dried material containing S-HBsAg without exogenous adjuvants, administered to mice under a controlled regime, induced a mucosal and peripheral humoral response above the nominal protective titer. Lyophilized material, both a powdered semi-finished product or converted into tablets, preserved the antigen for at least one year. The presented results can facilitate the development of efficient, durable, and convenient plant-based oral vaccines for prophylaxis against HB and other diseases.

Construction of the plant transformation vector
The coding region of S-HBsAg of 681 bp in length was amplified on a template of the plasmid pHB321 containing the complete genome of HBV subtype ayw4 (GenBank accession no. Z35716), using the following primers introducing BamH I and Pst I restriction sites, respectively: 5'-GGATCCATG-GATCCATGGAGAACATCACATCAG-3' (forward) and 5'-CTGCAGTAGAGCTCTTAAATGTATACCCAGAGA-3' (reverse). The amplified fragment was cloned into the pMG2A plasmid, a derivative of pUC18 containing the 35 S RNA CaMV promoter (p35S) and nopaline synthase terminator (NOSt). The expression cassette p35S-SHBsAg-NOSt was cloned into the EcoR I and Hind III sites of pGPTV-BAR (Becker et al. 1992), replacing the GUS gene. The yielded vector pKHBSBAR ( Fig. 1), carrying the bar gene conferring resistance to glufosinate, was introduced into Agrobacterium tumefaciens EHA105 via electroporation.

Lettuce transformation, regeneration, and cultivation
Agrobacterium tumefaciens was cultured and prepared for explant inoculation as previously described (Pniewski and Kapusta 2005), but Agrobacterium was grown until OD 550 ≈ 1.0. Seeds of lettuce (Lactuca sativa L.) cv. Syrena were sterilized for 12 min with 20% v/v chloric bleach supplemented with 0.01% v/v Tween®20, washed 5-6 times with distilled water, and germinated on solidified 0.8% w/v agar (Serva) in the semi-shade at 25°C for 2-3 days. Cotyledons were then excised, inoculated in the Agrobacterium suspension for 10-15 min, dried briefly, and transferred onto the LR1 medium consisting of MS salts and vitamins, 3% w/v sucrose, 0.2 mgL −1 of 6-benzylamminopurine, and 0.05 mgL −1 of naphthaleneacetic acid and co-cultured for 2 days in darkness at 25°C. Explants were transferred once a week onto fresh LR1 selection medium, i.e., supplemented with 2.5 mgL −1 of glufosinate (Sigma) and 300 mgL −1 of Timentin. Calli with adventitious buds were transferred onto the LR2 selection medium consisting of SH salts, B5 vitamins, 3% w/v sucrose, 0.5 mgL −1 of kinetin, and 0.5 mgL −1 of zeatin. Developed plantlets were excised and Fig. 1 Organization of T-DNA of the binary plasmid pKHBSBAR. S-HBs, coding sequence of small surface antigen of HBV; bar, coding sequence of phosphinothricin acetyltransferase; p35S, CaMV 35 S promoter; pNOS, nopaline synthase promoter; NOSt, nopaline synthase terminator; g7t, g7 terminator; LB and RB, left and right T-DNA border sequences transferred onto a rooting selection medium LR3, consisting of half the amount of SH macroelements, but the full amount of SH microelements, B5 vitamins, and 3% w/v sucrose. All media were adjusted to pH 5.7 and solidified with 0.8% agar (Serva). In vitro cultures were conducted under 4,000 lx of light intensity, 16/8-h photoperiod, and at a 25/18°C temperature regime. Rooted plants were transferred into soil and grown in a greenhouse under 15-20 klx light intensity, 16/8-h photoperiod, and at a 22/16°C temperature regime. Harvested seeds were surface-sterilized and germinated as above on a half-strength MS medium with 2.5 mgL −1 of glufosinate. Seedlings of progeny plants were transferred into soil and cultivated as described above.

PCR and Southern blot analysis
Genomic DNA was extracted from leaves in a small-scale procedure according to McGarvey and Kaper (1991). Polymerase chain reactions (PCRs) were performed in a PTC-200 MJ Research thermal cycler, in a volume of 25 μl containing approximately 100 ng of plant genomic DNA, 10 pM of each of the above-mentioned primers, 0.2 mM of each dNTP, 3.5 mM MgCl 2 , and 1 unit of Taq DNA polymerase (Invitrogen). The PCR temperature profile was as follows: initial denaturation at 94°C for 4 min, then 35 cycles of denaturation at 94°C for 1 min, annealing at 68°C for 45 s, elongation at 72°C for 1 min, and final extension at 72°C for 5 min. The molecular weight of PCR products was estimated by agarose gel electrophoresis using the 200 bp DNA Ladder (MBI Fermentas).
DNA for Southern hybridization was extracted according to Doyle and Doyle (1990), cut with the EcoR I, run on 0.75% agarose gel, and then blotted onto a positively charged membrane (Roche). The probe specific to the S-HBs sequence was prepared by PCR using a DIG DNA Labeling Kit (Roche), hybridized according to the manufacturer's protocol, and signals were detected on an X-ray film using CSPD (Roche) as a chemiluminescent substrate.

TEM
Lettuce leaf tissue was prepared for transmission electron microscopy (TEM) according to the standard technique with slight modifications (Kocjan et al. 1996). Ultrathin sections of approximately 70 nm in thickness were obtained using an UltraCut S microtome (Leica Microsystems) and observed under a JEM1200EXII transmission microscope (Jeol).

Western blot of S-HBsAg
Leaf pieces, approximately 50-60 mg in weight, were ground in 300 μl of PBS buffer with 0.5% v/v Tween®20, then Laemmli buffer with DTT was added and samples were incubated at 65°C for 15 min. Samples were run under denaturing conditions (12% w/v polyacrylamide gel with 0.1% w/v SDS) and then blotted onto a nitrocellulose membrane (Roche). The blot was blocked with 3% w/v BSA in TBS buffer, then incubated with 1:4,000 TBSdiluted anti-SHBsAg rabbit serum (University of Gdańsk, Poland) or 1 μg/ml mAb C86132M (Biodesign). The 1:10,000 TBS-diluted anti-rabbit or anti-mouse wholemolecule polyclonal HRP-conjugated antibody (Sigma) was used as a secondary antibody. The molecular weight of protein bands, visualized after a reaction with the DAB substrate (Sigma), was estimated using a protein marker (MBI Fermentas).
ELISA assay of S-HBsAg in lettuce leaves, lyophilized plant tissue, and derived tablets Samples were ground in a mortar on ice in a gradually added extraction buffer consisting of PBS pH 7.4, 10 mM Na 2 SO 3 , 2% w/v PVP 40000 , 0.2% w/v BSA, and 1% v/v Tween®20. Crude extracts were centrifuged at 10,000 rpm at 4°C for 10 min. The ratio of extraction buffer volume to the sample weight was 25:1 for leaf and 200:1 for lyophilized tissue or tablet.
The quantitative enzyme-linked immunosorbent assay (ELISA) was performed according to typical guidelines with details specific for the S-HBsAg assay set as follows. A Microtiter MaxiSorp (Nunc) microplate was coated overnight at 4°C with 0.5 μg/ml mAb C86132 (Biodesign) in carbonate buffer pH 9.6 and then saturated for 2 h at 25°C with 5% w/v PBS-fat-free milk. Typically, extracts were added to PBS-filled wells and serially diluted 1:100-1:800 for leaf samples or 1:800-1:6,400 for lyophilized tissue or tablet samples. Afterwards, wells were incubated with 1:4,000 anti-SHBsAg polyclonal rabbit antibody (B65811R, Biodesign), then with 1:20,000 anti-rabbit IgG γ-specific biotin-conjugated mAb (Sigma), followed by 1:4,000 AP-conjugated ExtrAvidin® (Sigma). The reaction with pNPP substrate (Sigma) was developed at 25°C for 1 h and absorbance was measured at 405 nm using a Bio-Rad Microplate Reader, Model 680. The S-HBsAg concentration (microgram per gram of fresh or dry weight, μg/g FW or μg/g DW, respectively) was calculated as the arithmetic mean with standard deviation (SD) from three (lettuce leaves) or ten (lyophilized tissue and tablets) repeated assays, using Microplate Manager 5.2.1 software (Bio-Rad), according to the standard curve for unadjuvanted S-HBsAg (University of Ulm, Germany) in the negative control extract.
As a reference to the above polyclonal ELISA test for the total content of S-HBsAg, plants were screened using an Auszyme® Monoclonal Diagnostic Kit (Abbott) for the virus-like particle (VLP)-assembled antigen. The samples were extracted as above and assayed according to the provided protocol.

Preparation of lyophilized plant material and tablets
Leaves from lettuce lines of S-HBsAg content ≥10 μg/g FW were frozen in liquid nitrogen, then partially crumbled and freeze-dried (BETA 1-16, CHRIST®) in vacuum at 0.2 mbar and −30°C, for 36-72 h. Lyophilized tissue was powdered in a coffee-mill chilled on ice and stored at room temperature with silica gel, tightly sealed. For tablet preparation, the material was mixed with different fillers and binding agents. Finally, tablets were prepared from 2:1:1 w/w/w mixture of tissue powder with lactose (Meggle) and 10% w/v polyvinylpyrrolidone (BASF) in methylene chloride (Merck). The mixture was granulated into grains of 1 mm in diameter. Granules were mixed with 2% w/v magnesium stearate and then formed in a stamping press (Korsch) into tablets.

Mouse immunization
Mouse immunization studies were conducted with approval no. 35/2004 of the Local Bioethical Commission at the Medical University of Warsaw. Separate groups of 6-8-week-old 10 BALB/c mice were immunized with powdered lyophilized plant tissue containing S-HBsAg or the purified and alumadsorbed yeast-derived antigen from a commercial vaccine Engerix B® (GlaxoSmithKline) at 30-or 60-day intervals. A single dose of VLP-assembled S-HBsAg was 100 ng, suspended in 100 μl of PBS, and administered by gavage intubation. Directly before immunization, VLP-S-HBsAg was assayed by the Auszyme® kit to deliver an appropriate amount of the lyophilized plant material (ca. 9 mg/dose). Control mice were given lyophilized non-transgenic tissue or PBS every 60 days.
ELISA assay of anti-HBs antibodies and statistical analysis of immune response Blood and feces were collected 5 days before (pre-immune) and 10 days after each immunization. Sera were collected from blood centrifuged at 3,000 rpm at 4°C for 10 min. Antibodies were extracted from feces by suspending samples in five volumes (w/v) of PBS and incubation on ice for 15 min, twice repeated grinding and incubation on ice for 15 min, and shaking and centrifugation at 14,000 rpm at 4°C for 10 min. Anti-HBs antibodies (mIU/ml) were assayed twice using a Hepanostika® kit with quantitative standards (Organon Teknika, Boxtel) and analyzed statistically (the variance for one-way classification with repeated measurements, the Tukey and Duncan tests, p<0.05) using Statistica 6® software (StatSoft).

S-HBsAg expression in glufosinate-resistant transgenic lettuce plants
The efficiency of lettuce transformation amounted to 13%, i.e., 25 verified (out of 28) PCR-positive for S-HBs sequence, individual glufosinate-resistant transgenic lines were regenerated from 190 explants. The S-HBs sequence was detected by PCR in ≥75% of the 25-35 progeny (T1) plants of each analyzed line (Fig. 2a), and in most lines, Southern blot analysis confirmed 1-2 integration sites of T-DNA (Fig. 2b).
The primary (T0) transformants significantly differed regarding the total S-HBsAg expression (20 ng to 60 μg/g FW), hence, they were distinguished into three expression groups, i.e., of 'low' antigen level ≤5 μg/g FW, 'medium' 5-10 μg/g FW, and 'high' ≥10 μg/g FW (Fig. 3). The S-HBsAg content was maintained or increased in plants of the T1 generation for most lines of the 'low' or 'medium' groups. Significant fluctuations and decrease of antigen expression or some phenotype alterations, e.g., retarded growth or a low number of seed sets, were observed only for a few lines, mostly from the 'high' group. Finally, 12 lines of stable S-HBsAg content ≥5 μg/g FW were selected for further research, including 'high' lines designed for lyophilization. The content of S-HBsAg formed in tertiary structures, i.e., displaying immunodominant epitope 'a' when properly folded and assembled into VLPs, was estimated using an monoclonal Auszyme® kit (Abbott), being approximately 60-85% of the total values calculated by the polyclonal ELISA (Fig. 3). The expression of S-HBsAg assembled into dimers, both non-glycosylated (48 kDa, p48) and putative glycosylated (54 kDa, gp54), was also confirmed by Western blot analysis (Fig. 4). VLPs formed by plant-expressed S-HBsAg were directly observed in leaf cells using TEM (Fig. 5a, c), while they were not detected in non-transgenic lettuce (Fig. 5b). Observed plant VLPs were 17-22 nm in diameter, characteristic of native or yeast-derived purified VLPs (Fig. 5d).

Mouse immunization with lyophilized plant tissue containing S-HBsAg
Several trends in local and systemic humoral immune responses were observed after the oral delivery of 100 ng/ dose of VLP-assembled S-HBsAg, both plant-derived in powdered lyophilized tissue (ca. 9 mg/dose, coming from plants of ≥10 μgS-HBsAg/g FW) or the purified and alumadsorbed antigen (Fig. 6). Anti-HBs antibodies, both secretory IgA (S-IgA) in feces (Fig. 6a) and total serum antibodies (Fig. 6b), were induced at similar levels in all individual mice of particular groups, as demonstrated by low SDs. Immune response was clear and statistically significant for each immunization protocol, but more efficient for the longer schedule. The final levels of anti-HBs antibodies were similar for reactions induced by the plant-derived or purified antigen administered every 30 days, but for the 60-day schedule, plant-derived antigen elicited a higher immune response, especially in the mucosa. The systemic response was lower than that in the intestinal mucosa, approximately 3-4 times lower after priming and almost 2 times lower after boosting. However, serum anti-HBs antibodies reached the level above the cut-off value of 10 mIU/ml, accepted as the minimal protective titer.
Preparation of a plant-derived prototype for orally administered vaccine Lettuce leaves, harvested from T1 plants of S-HBsAg content ≥10 μg/g FW, were freeze-dried and ground into powder and stored at room temperature with a desiccating silica gel. This semi-product was mixed with different fillers and binding agents, e.g., mono-and oligosaccharides, starch, cellulose derivatives, gelatine, Arabic gum, inorganic compounds, etc. Finally, the 2:1:1 w/w/w mixture of the powdered plant tissue with lactose as the filler and a 10% w/v polyvinylpyrrolidone/methylene chloride as the binding agent met the required powder density and consistency suitable for the preparation of standard tablets, i.e., 10×3-mm in size and 500 mg in weight, with appropriate durability, friability, dissolution time, and possibility of room temperature storage (Fig. 7a). The level of total S-HBsAg in the lyophilized material reached 5 mg/g FW. However, the mean content of VLPstructuralized antigen was 11 μg/g of lyophilized tissue and 5 μg/g or 2.3 μg/unit of tablets, i.e., approximately half that of the original lyophilized material due to the added ingredients. The antigen level remained stable, i.e., without significant fluctuations, after one year of storage in dry air at room temperature, both in lyophilized tissue and in tablets (Fig. 7b). Fig. 3 Expression of total and VLP-assembled S-HBsAg in lettuce plants of T0 and T1 generation. Plants of T0 generation were divided into three expression groups, depending on the total S-HBsAg level calculated in μg/g of fresh weight (FW): low <5 μg/g FW, medium 5-10 μg/g FW, high >10 μg/g FW. The data show values calculated as the arithmetic mean and standard deviation (SD) from three assays for parental or 25-35 progeny plants, determined by polyclonal ELISA for total antigen or by the monoclonal Auszyme® diagnostic kit (Abbott) for VLP-assembled antigen

Discussion
Glufosinate-resistant lettuce expressing S-HBsAg as an initial material for plant-derived prototype oral vaccine against HBV The presented transformation and regeneration procedure made it possible to obtain independent glufosinate-resistant lettuce line plants with a 13% efficiency. The value is lower than 70% or 31% (Enomoto et al. 1990;Curtis et al. 1994) but comparable to 6% or 10% reported previously (Michelmore et al. 1987;Torres et al. 1993). The selection system based on the marker gene bar (Fig. 1), conferring resistance to strongly acting glufosinate, probably reduced the transformation rate, but, on the other hand, it guarantees high probability (90%, see Results section) of regeneration of authentic transformants in comparison to kanamycin (Mohapatra et al. 1999). The obtained 25 plant lines were analyzed to select 12 lines characterized by a normal phenotype, stable inheritance of the S-HBs transgene assembled by plant-expressed S-HBsAg. a VLPs in the endoplasmic reticulum (ER) and the nuclear envelope (Ne) of transgenic lettuce cell, magnification 3.6 × 10 4 , bar=100 nm. b Nuclear envelope (Ne) of non-transgenic cell, magnification 3.6 × 10 4 , bar = 100 nm. c VLPs inside ER vesicles, enlarged section of panel a), magnification 7.2 × 10 4 , bar=50 nm. d Yeast-derived purified VLPs in Engerix B®, magnification 7.2 × 10 4 , bar = 50 nm (Fig. 2), and expression of the antigen at a maintained and satisfactory level of 5-20 (and more) μg of S-HBsAg/g FW, both in T0 and T1 generations (Fig. 3). The observed average S-HBsAg expression level was similar or higher in comparison to the 0.05-16 μg/g FW reported in previous papers (e.g., Kapusta et al. 1999;Richter et al. 2000;Kong et al. 2001;Gao et al. 2003;Sojikul et al. 2003;Joung et al. 2004;Shulga et al. 2004;Huang et al. 2005), reaching 60 μg/g FW in some plants. The expression of native antigen forming tertiary structures was confirmed, since the Auszyme® or other diagnostic kit assays require specific binding of a monoclonal antibody to a displayed immunodominant epitope 'a' of S-HBsAg assembled into VLPs (Jolivet-Reynaud et al. 2001;Sojikul et al. 2003). Protein bands of 48 and 54 kDa, revealed by Western blot analysis, correspond to unmodified and glycosylated S-HBsAg dimers (Fig. 4). Presumably, the dimers are extremely durable and could be observed under standard denaturing conditions, since highly hydrophobic S-HBsAg is known to dimerize immediately post-translation, and units are additionally cross-linked by multiple disulfide bridges (Bruss 2007). The dimers indicate the first step of VLP selfassembly (Bruss 2007;Patient et al. 2007), observed also in plant cells (Joung et al. 2004;Shulga et al. 2004;Huang et al. 2005). Consequently, VLPs in lettuce were structurally identical to the yeast-derived particles utilized for vaccination (Fig. 5), similarly to previous observations in plant (Kong et al. 2001) and mammalian cells (Patient et al. 2007).
In addition to the relatively high S-HBsAg expression level, the presented transgenic lettuce represents some benefits as an oral vaccine producer. The glufosinateresistant lettuce conforms to GMO safety regulations and health requirements, since it is free of an antibioticsresistance marker gene. The cultivation of lettuce does not require special conditions and can be adapted to various local needs. Disadvantageously, the amount of harvested material is substantially lower than from typically exploited Fig. 6 Anti-HBs secretory IgAs (S-IgAs) in feces extract (a) and total serum antibody response (b) to oral immunization with 100 ng/dose of VLP-assembled S-HBsAg. Mice were immunized with lyophilized plant tissue (ca. 9 mg dry weight/dose) or purified antigen, and delivered at 30-or 60-day timings. Non-transgenic lyophilized tissue or PBS were delivered to control mice at a 60-day timing. Anti-HBs antibody level (mIU/ml) responses are expressed as arithmetic means with standard deviations (SD) from results for 10 BALB/c mice at pre-immune (5 days before prime) and 10 days after each immunization, determined twice by a Hepanostika® kit (Organon Teknika, Boxtel). Statistically homogenous or significantly distinct responses for S-IgAs or total serum antibodies are marked by identical or different letter indexes, respectively (Tukey and Duncan tests, p<0.05, Statistica 6®) tobacco or potato. However, lettuce is naturally free of harmful alkaloids, etc., and the antigen concentration can be increased by lyophilization. Lettuce leaves are easy to be freeze-dried in comparison to tomatoes, bananas, tubers, or carrot. Moreover, though lyophilization is necessary, S-HBsAg expression level in leaves is many times higher than in naturally dry seeds (Sunil Kumar et al. 2006).

Oral immunization with lyophilized plant tissue containing S-HBsAg
Potential plant-based oral vaccines require suitable antigen formulations and immunization protocols to ensure antigen stability through the alimentary tract and a balance between immune response and oral tolerance (Holmgren and Czerkinsky 2005;Mestecky et al. 2007).
The presented results show that a relatively low dosage, i.e., 100 ng of S-HBsAg VLPs, and an extended interval, up to 60 days between oral prime and boost, induced mucosal and systemic efficient immunization with a small amount of lyophilized tissue, ca. 9 mg (Fig. 6). The level of S-IgAs was somewhat higher than serum antibodies, similarly to some previous observations (Shchelkunov et al. 2006;Jiang et al. 2007). Response of the gut-associated lymphoid tissue (GALT) to the antigen administered solely orally could surpass periphery response, since GALT interacts with such antigens as the first line of the whole immune system and also mucosal membranes limit deeper antigen penetration (Czerkinsky et al. 1993), but on the other hand, mucosal immunity can broaden the protection range against penetrating pathogens (Isaka et al. 2001;Brandtzaeg 2003). Nevertheless, the antigen delivered on the described schedule elicited a statistically significant mucosal and systemic response in comparison to the preimmune and the control, finally reaching a titer ≥10 mIU/ml of anti-HBs total serum antibodies (Fig. 6b), which is considered as the minimal protective response (International Group 1988;Brocke et al. 2005). A low S-HBsAg dosage induced significant and similar immune responses in individual mice, as demonstrated by low SDs (Fig. 6), probably due to the fact that the GALT is a tuned immune milieu, where even small amounts of antigens organized into VLPs or other tertiary structures are recognized and processed (Mestecky et al. 2007). Conse-The GALT determines adaptive immunity versus unresponsiveness depending on antigen structure dosage and duration of its exposure (Mowat 2003;Pamer 2007). Accordingly, low antigen dosage applied in the study was probably conducive to local and systemic immune response, while oral tolerance can be induced by high dosage and/or repeated frequent antigen delivery (Peng et al. 1989;Friedman and Weiner 1994).
Other reports on oral immunization with S-HBsAg or other antigens corroborate the positive effect of a low Fig. 7 a, b Plant-based prototype of anti-HBV vaccine specimens. a Preparation of plant-derived materials. Panels: left lettuce plants expressing ≥10 μg S-HBsAg/g FW; middle powdered lyophilized plant tissue as a semi-finished product for the preparation of various formulas of an oral vaccine; right tablets of 10×3 mm in size and 500 mg in weight, containing a specific dose of S-HBsAg. b The level of total and VLP-assembled S-HBsAg in powdered lyophilized tissue and 1 g and 1 unit of tablets, freshly prepared and stored for one year. S-HBsAg content was calculated in μg/g of dry weight (DW) as an arithmetic mean and standard deviation (SD) from ten polyclonal ELISA or monoclonal Auszyme® assays antigen dosage and extended administration on immune response. A 5-ng dose of alum-adsorbed S-HBsAg administered orally to mice over 8 weeks induced a significantly higher response than a 500-ng dose delivered over 2 weeks (Kapusta et al. 2010), or the response was higher and reached the ≥10 mIU/ml protective level in human volunteers fed with lettuce on days 1 and 60 (Kapusta et al. 1999) than in those fed on days 1, 7, and 30 (Kapusta et al. 2001) with 1-2 μg of S-HBsAg per person. Accordingly, low nanogram doses of S-HBsAg elicited immune response both in the mucosa and the periphery, while higher doses stimulated tolerance (Kostrzak et al. 2009); primary humoral response was triggered at an oral delivery of 30 ng of fusion antigen TBI-HBsAg to mice and the successive boost by DNA immunization only slightly increased the level of anti-HBs antibodies (Shchelkunov et al. 2006). The efficacy of oral immunization conducted at a low, even nanogram, dosage and at extended timing was also confirmed for plant-expressed CT-B (Arakawa et al. 1998) or LT-B (Lamphear et al. 2002;Beyer et al. 2007). Also for dietary allergen ovalbumin, low dosage elicited immune response, while suppression was induced by high-dose early boosting (Franco et al. 1998).
Plant-associated S-HBsAg, delivered on the reported schedule and without exogenous adjuvants, elicited an immune response at the nominally protective level, although the antibody titer was several times lower than that usually triggered in cases of S-HBsAg parenteral administration (Brocke et al. 2005) or previous oral immunization trials using microgram doses of the antigen. However, in those experiments, it was necessary to combine the oral delivery of (in most cases) CT-Badjuvanted S-HBsAg with parenteral (prime or boost) vaccination (Richter et al. 2000;Kong et al. 2001;Gao et al. 2003;Mason et al. 2003;Joung et al. 2004;Huang et al. 2005;Thanavala et al. 2005). A similar trend of high immune responses following parenteral-oral abundant antigen delivery was also observed for other antigens, e.g., Norwalk virus, rabies glycoprotein, or plague. On the other hand, parenterally-orally administered micrograms of the alum-adsorbed antigen, but unadjuvanted with CT-B (Kong et al. 2001), or the purified and encapsulated antigen adjuvanted with bile salt or CpG oligos (Borges et al. 2007;Shukla et al. 2008), induced immune response comparable to the present results. Immune reaction at the level 10-30 mIU/ml requires to be enhanced though. However, toxinderived adjuvants are not approved for use in humans, hence, any GALT immunostimulator must be meticulously explored to eliminate possible hazardous side-effects (Brandtzaeg 2003).
On the other hand, it might be supposed that some phytochemicals could synergistically affect the immunogenicity of plant-expressed antigens as in the case of presented results or plant-expressed CT-B (Jiang et al. 2007). Medicinal plants are known for their natural immunostimulating or anti-microbial activity due to such secondary metabolites as lectins, saponins, and others. Lettuce does not contain significant amounts of these substances though, except terpenoids in latex sap, characteristic for the Asteraceae family. Moreover, some commonly occurring plant components have adjuvant properties, e.g., unmethylated CpGs motifs of DNA (Wang et al. 2002) or carotenoids, etc. Presumably, plant tissue together with the above-mentioned compounds protected and increased the immunogenicity of S-HBsAg in lettuce, since the reaction elicited by plant-associated antigen was somewhat higher than that induced by the purified and alum-adsorbed antigen (Fig. 6). Hence, it might be carefully assumed that raw edible plants can be used at the same time as an oral vaccine producer and, at least partially, as a source of endogenous adjuvants too.
A plant-derived durable prototype for an orally administered vaccine against hepatitis B Although oral immunization based on the consumption of raw transgenic plants containing S-HBsAg was demonstrated to induce an immune response in mice (Kapusta et al. 1999;Richter et al. 2000;Kong et al. 2001;Gao et al. 2003;Joung et al. 2004) and humans (Kapusta et al. 1999(Kapusta et al. , 2001Mason et al. 2003;Thanavala et al. 2005), immunization by feeding encounters logistical and medical contraindications (Kirk et al. 2005). Antigen instability in stored plant tissue and splitting of an initial dose of 'edible vaccine', both fresh and dry, into lower undefined subdoses, repeatedly exposed to GALT, and adsorbed as a dietary component (Swarbrick et al. 1979), subsequently is conducive to tolerance induction. Moreover, potential plantbased oral vaccines considered for mass immunization against HB and other diseases (Aziz et al. 2007;Mestecky et al. 2008) should be simple to administer. Expectations for plant-derived oral vaccines can be met by the application of freeze-dried plant materials, which was initiated in recent years in immunization experiments with monomeric and oligomeric antigens, such as CT-B (Huang et al. 2005), LT-B (Rosales-Mendoza et al. 2009), measles hemagglutinin (Webster et al. 2006), and others.
Here, a novel prototype plant-based anti-HBV vaccine in tablet form prepared from freeze-dried lettuce highly (≥10 μg/g FW) expressing VLP-assembled S-HBsAg is proposed (Fig. 7a). The administration of durable powdered freeze-dried plant tissue made it possible to concentrate and standardize antigen content throughout vaccine doses and would facilitate delivery of the antigen in a size-reduced specimen under a controlled regime (Figs. 6 and 7). However, during lyophilization, an at least 90% decrease of S-HBsAg VLPs content was recorded when compared to fresh tissue (Fig. 3) and that observed in freeze-dried tissue (Fig. 7b), considering that the dry weight is 5-10% FW. Advantageously, the low dose of VLPs applied in a 'single shot', avoiding extended exposure in sub-portions, appeared to be sufficient to induce local and systemic humoral response at an adequate protection level (Fig. 6). However, potential industrial production of the oral vaccine would require the optimization of freeze-drying parameters to effectively retain the VLPs.
The tablet form of a potential vaccine specimen seems to be feasible to produce, durable in storage even at room temperature, and easy to use, which is especially important in regions with an underdeveloped medical infrastructure. A compact tablet form made it possible to concentrate the dose of antigen assembled into immunogenic VLPs (2.3 μg/unit) in a relatively small weight and volume (see Results section), while the same antigen dose would have to be contained in a 4-5 times bigger capsule or other formula containing loose powder. Due to the powdery structure, high hygroscopicity, and low density of milled lyophilized tissue, the tablet filler, i.e., lactose, and the binding agent, i.e., polyvinylpyrrolidone, had to be established experimentally from among standard components to facilitate the compression of tablets of proper parameters (see Results section). Moreover, tablets, as well as lyophilized tissue, are characterized by the stability (95-98%) of S-HBsAg content for at least one year of room temperature storage (Fig. 7b). Tablets indicate a direction for the development of a compacted prototype oral vaccine, convenient for use and distribution, and may facilitate human immunization trials. It could be assumed that data acquired on immunization efficacy will contribute to further tablet optimization to enhance immune response, e.g., with the addition of adjuvants, immunomodulators, etc.

Conclusions
Research on a plant-derived vaccine against hepatitis B virus (HBV) requires further detailed studies on mucosal and systemic immune response and possible tolerance induction. Parameters of oral vaccination, such as dosage, timing, adjuvants, antigen formulation, immunization routes, etc., will have to be accurately adjusted. The presented results and other reports allow to carefully presume that immunity, probably balanced with tolerance, may be induced by an appropriate immunization protocol, based on a calibrated, extended delivery timing of generally low doses of lyophilized tissue containing small surface antigen of HBV (S-HBsAg). Lettuce or other plants efficiently expressing native S-HBsAg, resistant to herbicides and innately free of harmful substances, could be an initial material for the preparation of durable and reliable oral vaccine as tablets or other formulas. The reported results constitute a basis for the elaboration of an alternative or supplementary oral vaccine, easily implementable for HB immunization programs.


s2fieldsofstudy: Biology Medicine

text:
Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

Background Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation. Methods Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry. Results The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml). Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively. Conclusion Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.

Background
Follicular fluid is the microenvironment in which the cumulus-oocyte complex matures and somatic cells (granulosa and theca cells) proliferate and differentiate. It was suggested that the follicular fluid originates from both somatic cells, which produce factors related to their metabolic activity [1] and from plasma which enters the extra-vascular spaces, as well as the antrum of follicles [2]. Furthermore, it contains a number of soluble factors implicated in various stages of follicular development. The study of its components may contribute, at least in part, to our understanding of the mechanisms underlying this process.
In mammalian species, the preovulatory maturation of the oocyte and follicle is under the control of gonadotropin hormones synthesized by the anterior pituitary gland. In most species, the sudden surge in luteinizing hormone (LH) induces morphological and functional changes in the ovulatory follicle that result in granulosa and theca cells differentiation in preparation of follicle rupture and corpus luteum formation, in expansion of cumulus cells, and in the resumption of meiotic maturation of the oocyte. Some coordination between these events is required to achieve the production of a mature oocyte suitable for fertilization. This should also be essential for subsequent embryonic development and formation of a corpus luteum capable of supporting early pregnancy.
In the bitch, the hormonal peaks of follicle stimulating hormone (FSH) and LH are observed before ovulation [3] and LH levels reach 9 ng/ml (2-22 ng/ml; [4]). Nevertheless, the ovarian physiology in the bitch compared to that of other mammals exhibits at least three major uncommon features. Firstly, the endogenous LH peak is very lengthy since it lasts 48-72 h [3,4], and preovulatory luteinization is observed in dogs, with an increase in plasma progesterone level prior to the LH peak [3,4]. Secondly, around 40% of the follicles in the canine ovary are poly-oocytic. Some may contain as many as 17 oocytes [5,6]. But the most striking feature of canine ovaries is that oocytes are ovulated at an immature (GV; prophase I) stage and reach the fertilizable metaphase II stage only after maturation for [56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72] h within the oviduct [7,8]. Several mechanisms may explain the later feature: the preovulatory canine oocytes, in contrast to those of other mammals, may be incompetent for resumption of meiosis by themselves [9] or the canine follicular cells may be are unable to transduce the ovulatory signal to the oocyte. It is also possible that the follicular fluid either does not contain the adequate factors for achievement of oocyte maturation or contains meiosis inhibitory substances. Nevertheless, at the present time, no information is available in the literature about the composition of the canine follicular fluid.
It should be noted that in vitro technologies such as oocyte maturation (IVM) or fertilization (IVF) and subsequent embryo development are particularly difficult to obtain in canine. Actually, the rate of success in IVM is still very low (10-20%) and a high rate of polyspermy is observed during IVF [10]. These disappointing results may be related to the immaturity of the canine oocytes collected for in vitro studies. It has been speculated that these oocytes, even if collected in vivo at the pre-LH or post-LH stages, may be incompetent for IVM and IVF [9]. Indeed, even when preovulatory (post-LH) oocytes collected in vivo are cultured in vitro, the IVM rate remains quite low (maximum 32%; [11]). A better understanding of the factors required by the canine oocytes to complete their growth and resume meiosis in vivo, as well as putative meiosis inhibitor factors, would be of help in developing an adequate IVM medium, and might thus, to overcome the main limiting factor of assisted reproduction techniques in this species.
In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation. For this purpose, we first determined the levels of steroids (17beta-estradiol and progesterone) present in the canine follicular fluid in vivo, before and after the LH surge, then investigated the protein composition of the follicular fluid during the preovulatory phase, with special emphasis on the potential effect of LH. We also attempted to characterize specific differences in protein composition of follicular fluid and plasma in order to identify proteins that accumulate or are missing in canine follicular fluid. These proteins may hold the key to the reproductive process and further evaluation may be useful in the search of potential biomarkers of follicle/oocyte quality, or as factors responsible of the canine ovarian features.

Animals and monitoring of oestrous cycles
Thirty-two Beagle bitches (3.3 +/-0.5 years old) from our experimental kennel were included in this study. Ovarian cycles were assessed by blood progesterone assays and by vaginal smears coupled with Harris-Shorr staining and eosinophilic indexes determination [12]. For progesterone assays, the blood samples were collected at the cephalic or jugular vein on lithium heparinate, centrifuged and plasma was stored at -20°C until use. Plasma progesterone was assayed by enhanced chemi-luminescence Elecsys kit (Roche Diagnostics, Meylan, France). Intra-assay and inter-assay coefficients of variation were less than 2% [8].
Progesterone assays were carried out every other day as long as the percentage of superficial cells remained below 80% and daily once that percentage exceeded 80% ("oestrus" vaginal smears). As soon as the progesterone level reached 0.5 ng/ml, blood was collected 3 times a day and plasma was stored at -20°C until assayed for LH concentration. Once the progesterone concentration exceeded 2 ng/ml, ovarian transabdominal ultrasonography (ultrasonograph HDI 3500, probe 7.5 MHz; ATL, Philips Systèmes Médicaux, Suresnes, France, 0.19 mm in resolution) was carried out two to three times a day to assess follicular growth, as previously described [8].
Bitches were assigned a posteriori to one of two experimental groups: in the pre-LH group (n = 16 bitches), bitches had an oestrus vaginal smear, no LH rise was detectable in plasma samples and follicles larger than 2 mm were visible by ultrasonography. In the post-LH group (n = 16 bitches), bitches displayed an oestrus vaginal smear, LH peak had occurred and antral follicles were still visible at ultrasonography, demonstrating that ovulation had not started yet.
This protocol was approved by the Ethics Committee of the "Alfort National Veterinary School" (France).

Collection of follicular fluids and plasmas
Ovariectomies were performed during the preovulatory phase using a conventional surgical procedure [13]. The ovaries were collected from excised ovarian bursa. On gross morphological evaluation, visible follicles were counted and measured [14]. All follicles larger than 2.5 mm were punctured individually with stretched Pasteur pipettes. The collected follicular fluids were observed under a stereomicroscope to recover the cumulusoocyte-complexes. Evaluation of the cumulus mucification was used in parallel to the LH assay, to confirm the classification of canine follicles in pre-LH (no mucification) or post-LH (mucification occurred) stage. Only follicular fluids free from blood contamination were kept and were stored after centrifugation (10 min at 5000 g) to eliminate cells and other contaminants. Fluid supernatants were immediately frozen at -20°C. Peripheral blood was also collected at the time of ovariectomy, centrifuged, and plasma samples were stored under the same conditions as follicular fluids, until further analysis.

Steroid hormones assays in canine follicular fluids and plasmas
Follicular fluids were individually assayed for progesterone and 17beta-estradiol by radioimmunoassay (RIA), without prior extraction. Progesterone assay was performed as previously described [15,16]. 17betaestradiol was assayed with the kit [125I] E2 DIASORIN (Stillwater, Minnesota, USA). For assays in plasma, the steroids were extracted before either assay.

Protein assay and 2D-PAGE analysis of canine follicular fluid and plasma
In order to study the effect of the LH surge on the protein profile of canine follicular fluid, follicular fluids were collected from follicles larger than 4.5 mm at pre-LH stage (n = 4 from 3 bitches) and at post-LH stage (n = 4 from 4 bitches) and were analyzed by 2D-PAGE. To compare the 2D-PAGE protein patterns of follicular fluid and plasma, samples of 4 bitches from pre-LH group were considered; follicular fluids (n = 4) being collected from follicles between 4.5 and 5.5 mm. All follicular fluid samples used for 2D-PAGE were free from blood contamination. All the chemicals and materials used for proteomic studies were purchased from Bio-Rad (Marne-la-Coquette, France), unless otherwise indicated. The total protein content of crude canine follicular fluid and plasma samples was determined using a commercial protein assay kit (DC Protein Assay) with bovine serum albumin as standard (Pierce, Rockford, Illinois, USA) according to manufacturer's instructions. 2D-PAGE analysis of canine follicular fluids was performed as previously described by Fahiminiya et al. [17]. Briefly, 100 micrograms of proteins were diluted in a hydration solution that was absorbed actively into ReadyStrip™ IPG (11 cm Immobilized pH Gradient IPG strips, pH range 3-10). After protein separation by isoelectric focusing (IEF), strips were equilibrated for 15 min with SDS-PAGE equilibration buffer, in order to resolubilize the proteins and to reduce disulfide bonds. In a second step, the same equilibration buffer containing 2.5% w/v iodoacetamide was used for 15 min to block free sulfhydryl groups. IPG strips were then subjected to SDS-PAGE (10%). The 2D gels were stained with a mass spectrometry compatible silver nitrate staining [18].

2D-PAGE image analysis
Stained gels were scanned and digitalized at 16-bit resolution using ImageScanner (Amersham Pharmacia Biotech, GE Healthcare Europe GmBH, Orsay, France). The resulting TIFF images were analyzed with Progenesis software (version 2008; Nonlinear Dynamics Ltd, Newcastle upon Tyne, UK), as previously described by Fahiminiya et al. [17].

Identification of proteins from 2D-PAGE by mass spectrometry
Stained protein spots were excised from the gels, cut into small blocks and digested with trypsin, as previously described by Shevchenko et al. [18]. The digested peptide fragments were sequenced by nano-LC-MS/MS (nanoscale capillary liquid chromatography-tandem mass spectrometry) using ion trap mass spectrometers (GE Healthcare, Europe GmBH, Munich, Germany). The Ettan MDLC controlled by UNI-CORNTM software (GE Healthcare, Europe GmBH, Munich, Germany) was used for desalting and separation of tryptic peptides prior to online MS and MS/MS ion trap analyses. Each sample was desalted using a precolumn (Zorbax 300-SB C18 trap column, 300 μm i.d × 5 mm, Agilent Technologies, Massy, France). Peptides were separated on a C18 column (Zorbax 300-SB C18, 75 μm i.d × 150 mm, Agilent Technologies) using a flow rate of 300 nL/min and applying a gradient of buffer A (0.1% formic acid) and buffer B (0.1% formic acid/ 84% acetonitrile). Eluted peptides were analyzed online with LTQ Linear Ion Trap Mass Spectrometer (Thermo Electron, San Jose, CA, USA). Each scan cycle consisted of one full scan mass spectrum (m/z 400-2000) collected in centroïd mode followed by three MS/MS events in centroïd mode. For CID spectra (MS2), the isolation width was 2 m/z units and the normalized collision energy was 40%. Dynamic exclusion was activated during 30 s with a repeat count of 1.
The acquired MS/MS spectra were automatically searched against the non-redundant Uniprot SwissProt database using the TurboSEQUEST (Thermo Finnigan, San Jose, CA, USA) program in the BioWorksTM 3.1 software suite (Thermo Finnigan, San Jose, CA, USA). The databank search parameters allowed 2 tryptic missed cleavage and included oxidized methionine (+16 Da) and carbamidomethylation on cystein (+57 Da) as variable modifications. The output data were evaluated in term of Xcorr magnitude up to 1.7, 2.2 and 3.5 for charge states 1+, 2+ and 3+ respectively and a minimum of two peptides. In the case of identification with only one peptide, MS/MS spectrum was visually verified.

Statistics
For steroid analysis, results are expressed as mean +/-SEM. Results were considered significant when P < 0.05. Effects of the ovarian stage (pre-LH and post-LH) on steroid content were analyzed by a t-test. Effects of follicle size on steroid contents and relationship between plasma and follicular fluid steroids were analyzed with a linear regression and Pearson's correlation coefficient (software SAS, Cary, NC, USA). For proteomic analysis, means of spot volumes in each group of samples were compared after normalization, using the ANOVA statistical package included in SameSpots. Only differences with P < 0.01 for random occurrence were considered to be significant, and only spots that were statistically different were selected for analysis.

Follicle features and steroids content in canine follicular fluid and plasma
The diameter of follicles ranged between 2.5 mm (limit of collection) and 6.5 mm in the pre-LH group (72 follicles), and between 2.5 mm and 7 mm in the post-LH group (73 follicles). Out of them, 9 follicles were found poly-oocytic (7 at the pre-LH stage and 2 at the post-LH stage, all between 4.5 and 6.5 mm in diameter). The volume of follicular fluid collected was between 2 and 45 μl at the pre-LH stage and between 2 and 70 μl at the post-LH stage. Follicular fluid volume was correlated with the follicle diameter (r = 0.63 p < 0.01 and r = 0.54 p < 0.01 at the pre-LH and the post-LH stage, respectively).

17beta-estradiol
17beta-estradiol concentration was determined in 72 follicular fluid samples collected at the pre-LH stage and in 73 follicular fluids collected at the post-LH stage (Figure 1).
At the post-LH stage, the mean intrafollicular concentration of 17beta-estradiol was 48.3 +/-4.4 ng/ml (ranging from 4.1 to 161.5 ng/ml). No significant correlation between follicle size and 17beta-estradiol concentration was observed (p = 0.96). The decrease in intrafollicular 17beta-estradiol concentration after the LH surge was highly significant (p < 0.001). In poly-oocytic follicles, the level of 17beta-estradiol was in the same range of concentration (14.6 and 27 ng/ml).
In order to better characterize the steroid content in dogs, plasma levels of 17beta-estradiol at the time of ovariectomy were compared to intrafollicular levels. The mean plasmatic 17beta-estradiol concentration was 53.5 +/-4.1 pg/ml at the pre-LH stage and 49.0 +/-9.6 pg/ ml at the post-LH stage (p > 0.05). Intrafollicular 17beta-estradiol levels (ng/ml) were between 9884 and 26969 times higher than plasma levels (pg/ml) at the pre-LH stage and from 84 to 8670 times higher than  plasma levels at the post-LH stage. As shown in Figure 2, intrafollicular and plasmatic 17beta-estradiol concentrations tended to be correlated at the pre-LH stage (p = 0.06).

Progesterone
Progesterone was assayed in 68 follicular fluid samples collected at the pre-LH stage and in 73 follicular fluid samples collected at the post-LH stage (Figure 3). Actually, progesterone was not assayed in 4 follicles < 4 mm at the pre-LH stage because the volume of follicular fluid collected was too small. Those non-assayed follicles were 2.5 mm (n = 3) and 3 mm (n = 1) sized and originated from 4 different bitches.
At the post-LH stage, the intrafollicular concentration of progesterone was 11690.2 +/-693.6 ng/ml (ranging from 245.4 to 28096.5 ng/ml) and no significant correlation between follicle size and progesterone concentration was detected (p = 0.22). In the single follicle with a diameter below 4 mm, the progesterone concentration in the fluid was dramatically lower (245 ng/ml) than that detected in follicles ≥ 4 mm (11849.5 +/-684.5 ng/ml). The increase in intrafollicular progesterone concentration after the LH surge was highly significant (p < 0.001).
The plasmatic levels of progesterone were significantly higher at the post-LH stage (9.80 +/-0.63 ng/ml) than at the pre-LH stage (3.9 +/-0.3 ng/ml; p < 0.05). Intrafollicular progesterone levels were between 100 and 1693 times higher than in plasma at the pre-LH stage and from 843 to 2606 times higher than in plasma at the post-LH stage. As shown in Figure 4, intrafollicular and plasmatic progesterone concentrations tended to be correlated at the post-LH stage (p = 0.07, Figure 4).

Proteomic analysis of canine follicular fluid and plasma
The 2D-PAGE methodology was used to characterize the protein profile of canine follicular fluid and to com- samples of 4 bitches from pre-LH group were considered; follicular fluids (n = 4) being collected from follicles between 4.5 and 5.5 mm. All follicular fluid samples used for 2D-PAGE were free from blood contamination.

2D proteins profile of canine follicular fluid
Slightly more than 1000 protein spots with molecular weight (Mr) between 10 and 200 kDa were detected by silver nitrate staining after IEF/PAGE (3.0 < isoelectric point pI < 10) separation ( Figure 5). Several protein spots were excised from the gel and analyzed by mass spectrometer for identification. Among them, 38 spots were identified, corresponding to 21 different proteins. The high-abundant proteins albumin (ALB) and immunoglobulin heavy chains (HV01 and HV02), appear as intense and large spots in canine follicular fluid protein patterns (indicated by dark rectangles in Figure 5). Albumin was identified in 10 of the collected spots, mainly as fragments (indicated by dark circles in Figure  5: 67 < Mr < 28; 5.07 < pI < 6.82; see Additional file 1, Table S1). Two other spots also identified as albumin most probably correspond to the native protein that has not correctly entered into the IEF gel (indicated by dotted rectangle in Figure 5). All other protein spots that have been identified in canine follicular fluid are shown in Figure 5 and their characteristics (Uniprot accession code (http://www.ebi.uniprot.org/index.shtml), Mr and pI are indicated in Table 1. Among them, 4 spots were identified as apolipoprotein A-1, and 2 spots as clusterin, complement factor B, inhibitor of carbonic anhydrase and haptoglobin. Most of the identified proteins are known to be secreted proteins, whereas 2 other are mostly known as cytosolic, such as actin and gelsolin, which are described as cytoskeleton proteins.
Potential effect of the LH surge on the protein profile of canine follicular fluid Computerized 2D protein pattern analysis was performed in order to compare 2D profiles at the two stages (pre-and post-LH) and to detect the differentially expressed proteins. We observed a high similarity in the 2D protein patterns before and after the LH peak, with only one group of spots and two singles that displayed significantly different levels of staining (indicated by rectangles in Figure 6A). The magnified images of the differential spots are presented in Figure 6B, and the statistical analysis of the quantified spot volumes (% volume) is shown in Figure 6C. The volumes of these spots are lower at the post-LH stage, by a magnitude of 2.5 (spot 123; Figure 6C) to 13 (spot 3; Figure 6C). Protein spots numbered 3, 5 and 14 corresponding to box 1 ( Figure 6A) were identified as complement factor B, which has previously been detected in human follicular fluid [19]. Comparison between canine follicular fluid and plasma protein patterns Follicular fluid and plasma samples from the same bitches (n = 4; pre-LH stage) were analyzed by 2D-PAGE. Computerized comparison of 2D patterns allowed detection of differentially stained protein spots between these two biological fluids ( Figure 7A). One group of spots (box 1) and a single spot (box 2) were found differentially expressed between plasma and follicular fluid, as demonstrated by the magnified images and quantified spot volumes presented in Figures 7B  and 7C, respectively. Spots from box 1 (spots n°8, 10, 13 and 20) were identified as Gelsolin. They were found at higher levels in plasma (7.8, 6.1, 5.8 and 5.1 fold increases, respectively) than in follicular fluid. Spot number 33 (box 2) was identified as a small isoform of clusterin which is in higher amount in follicular fluid than in plasma (3.8 fold increase).

Discussion
During follicular growth and maturation, the follicular fluid is the microenvironment of follicular cells and of oocyte. It contains substances presumably implicated in cell differentiation, rupture of the follicular wall, and gamete quality. One can reasonably expect that the determination of its steroid content and protein composition will contribute to a better understanding of ovarian physiology and of regulation of follicular growth and maturation. The canine species is characterized by several ovarian activity features that are not extensively described and well known yet, such as preovulatory luteinization; oocyte ovulated at GV (prophase 1) stage and poly-oocytic follicles. The present study was designed to characterize the steroid and, for the first time, the proteomic content of canine follicular fluid and plasma. These may help in the future to explain and to better understand the species specificities that  Table S1). All other identified proteins are named on the figure and their characteristics are shown in Table 1. NI: non-identified proteins are described in dogs. In our investigation, 2D-PAGE study of canine follicular fluid revealed few differentially expressed proteins between follicular fluid and plasma (gelsolin, clusterin), and between follicular fluid collected before or after the endogenous LH surge (complement factor B and some unidentified proteins).
As previously mentioned, one of the main peculiarities of the canine ovarian physiology is the preovulatory luteinization [3], which is clearly observed in our study, with a significant increase in plasmatic progesterone (nearly 2.5 fold) between the pre-and the post-LH stages. The plasmatic levels of progesterone, but also of 17beta-estradiol, found in our study are similar to the ones previously reported by others [20,21] and by our own group [4]. However, the most important question is not the plasmatic, but the intrafollicular levels of progesterone/17beta-estradiol, i.e. the level to which the oocytes are directly exposed. Almost no published data are available regarding the steroid content of canine follicular fluid. To our knowledge, a single study has been carried out earlier (Metcalfe,unpublished data [22]) in Labrador bitches at the post-LH stage, with no precise description of punctured follicle sizes (from 5 to 11 mm in diameter). Our study is the first describing of intrafollicular levels of 17beta-estradiol and progesterone in parallel to blood concentrations with precise characterization of follicular diameter and stage (pre-and post-LH stages) in the canine species.
As recorded previously in several mammalian species, concentrations of 17beta-estradiol and progesterone were found to be dramatically lower in blood than in follicular fluid. This may be due to the fact that in dogs like other species, the follicle is a major site of steroid synthesis, and that steroids are mostly secreted and concentrated in follicular fluid, before entering blood flow. Nevertheless, we found no data regarding the ovarian localisation and regulation of steroid synthesis and secretion in the canine species. We observed that the concentration of 17beta-estradiol in follicular fluid dropped significantly after the LH peak whereas that of The experimental position of identified spots is shown in Figure 5. 1 AC is an accession number from the UniProt database 2 Exp: Experimental; Theo: Theoretical; pI: Isoelectric point; Mr: Molecular weight; P N°: Peptid number; SC: Sequence coverage 3 Score is −10*Log (P), where P is the probability that the observed match is a random event 4 Sequence coverage (%) is based on number of peptide masses matched in UniProt databases progesterone increased significantly, which is consistent with findings in other mammals (sheep: [23]; pig: [24]; cow: [25]; human: [26]; horse: [27]). We can assume that this reflects either a lower/higher level of the enzymes involved in steroid synthesis in the follicle, or to an inhibition/activation of their activity, or may be linked to substrate availability. For example the decrease in intrafollicular 17beta-estradiol after LH may be due to a decrease in aromatase level or activity, or to a decrease in androgen availability. Additionally, the increase in intrafollicular 17beta-estradiol and progesterone concentrations coincident with the increase in  follicle size that we observed before the LH surge, may also be the result from steroidogenic enzyme regulation within each follicle size category. Progesterone levels recorded in the present study in canine follicular fluid are much higher than those found in sheep [28], pig [24], cow [25] and human [26]. This is probably linked to preovulatory luteinization observed in the female dog [3]. This result suggests that, in view of mimicking in vivo conditions prior to in vitro maturation of canine oocytes, one should use high level of progesterone in the culture medium [29]. Finally, the high rate of poly-oocytic follicles is a peculiarity of the ovarian physiology in dogs [5,6,30]. Recently, polyovular/poly-oocytic follicles have been reported in porcine to have a higher intrafollicular concentration of 17beta-estradiol and a lower concentration of progesterone than mono-oocytic follicles [24]. In our study, no relationship could be demonstrated between the presence of several oocytes within a follicle and 17beta-estradiol or progesterone intrafollicular levels, but the number of follicles examined was very limited.
The second objective of our study was to describe the protein composition of the canine follicular fluid during the preovulatory phase, with special emphasis on the potential effects of LH. First, we used 2D-PAGE to characterize the global protein profile of canine follicular fluid, and mass spectrometry to identify some of the proteins present in follicular fluid. By combining these two approaches, we identified 38 protein spots in the canine follicular fluid, corresponding to 21 different proteins. Our identification data are in agreement with earlier studies performed in human [31], bovine [32] and porcine [33] follicular fluids. Most of the identified proteins have previously been reported in plasma of various species [34,35], but in the present study, only follicular fluid free from blood contamination were used for the proteomic analysis. Indeed, the blood-follicular barrier is known to allow the free diffusion of molecules under 500 kDa [36]. Thus, the presence of such proteins in follicular fluid may be due to the vascular permeability of ovarian vessels during follicular growth and, at least for some of them, to local synthesis and secretion by follicular cells.
In this study, we were able to identify 11 of the protein spots in the canine follicular fluid as albumin or its fragments, and 2 as heavy chains of immunoglobulin. These two types of proteins (albumin and immunoglobulin) were reported earlier in human [31] and bovine [32] follicular fluids proteome studies. The existence of multiple forms of albumin with different isoelectric points could be due to chemical modifications of amino acid side chains [37]. Furthermore, this protein is known to function as carrier and transporter of proteins within blood and to bind physiologically important species such as lipid soluble hormones (steroid hormones), free fatty acids (apoprotein), calcium, ions (transferrin), and cytokines [38,39]. Intrafollicular albumin may participate in the transport of metabolites involved in follicular growth or in the transfer to the general circulation of some follicle specific products like steroids.
Another group of proteins that we identified in the canine follicular fluid is made up of acute phase proteins including fibrinogen gamma, haptoglobin, complement factors, apolipoprotein A-I, alpha-2-HS-glycoprotein, transferrin and retinol-binding protein 4, which are involved in inflammatory events [40,41]. Kim et al. [42] have recently shown that the level of fibrinogen gamma present in human follicular fluid may be a useful marker for the diagnosis of recurrent spontaneous abortion. Moreover, the impact of haptoglobin on women's fertility has been shown by Bottini et al. [43]. The transport of this protein into the antrum depends on the integrity of the blood-follicle barrier and might be associated with oocyte quality, possibly by interfering with the role of apolipoprotein A1 in cholesterol or vitamin E exchange between high-density lipoproteins and granulosa cells [44]. Jarkovska et al. [19] have recently showed the possible role of complement cascade and complement regulatory proteins in the maturation of the oocyte and in the development of its competence for successful fertilization.
Two types of apolipoproteins (APOA1 and APOA4) were identified in 5 protein spots on our 2D-PAGE of canine follicular fluid. These proteins are the major protein component of high density lipoprotein (HDL). APOA1 has been previously shown to be expressed in mouse granulosa cells and is regulated by progesterone receptor A (PGR-A) which is induced by LH [45]. Balestrieri et al. [46] demonstrated that haptoglobin in human follicular fluid inhibits the reverse transport of cholesterol to circulating blood by preventing the apolipoprotein A1 stimulation of the lecithin-cholesterol acyltransferase activity. These two proteins have already been observed in human follicular fluid [31].
Retinol and retinoids have been suggested to be essential for reproduction and to be involved in ovarian steroidogenesis, oocyte maturation and early embryonic development [47,48]. In the present study, a single isoform of retinol-binding protein was found in canine follicular fluid. This result is in accord with earlier observations by Anahory et al. [49], and in contrast with those of Angelucci et al. [31], who identified three different isoforms of retinol-binding protein in human follicular fluid. In canine follicular fluid, other isoforms of retinol-binding protein may be present but either spots have not been stained and collected, or spot analysis did not permit identification. Its presence in follicular fluid may be due to either passive filtration from serum across the blood follicular barrier into the follicular fluid, or to local synthesis [50].
In the present study, paraoxonase and transferrin were identified in canine follicular fluid. Both proteins are synthesized in the liver. Paraoxonase is bound and transported in plasma along with HDL. It functions as an antioxidant by preventing the oxidation of LDL (Low-density lipoproteins). Its presence has already been shown in human follicular fluid [31]. Transferrin is known as a circulating iron carrier protein, but is also produced in the testis and the ovary, where it acts as a growth factor, in addition to its role in iron endocytosis. The expression of transferrin was demonstrated in granulosa cells in human and mouse follicles [51]. In humans, the level of transferrin in the follicular fluid is highly correlated with the circulating level, suggesting that the contribution of local synthesis by granulosa cells during follicle maturation may be important.
There is plenty of evidence supporting the role of the LH surge in regulating numerous proteins implicated in ovulation and luteinization [52]. Although most of these proteins display a cellular localization, some have been localized in follicular fluid such as extracellular matrix glycoproteins [53,54], proteinases [55,56] and their inhibitors [57,58]. In the present investigation, we attempted to visualize and identify proteins in canine follicular fluid that may be modulated in response to the increase in circulating LH level. Computerized protein pattern analysis and comparison of pre-LH and post-LH canine follicular fluids demonstrated the presence of higher levels of complement factor B at the pre-LH stage. The reason why its expression decreases at post-LH stages in dog is not clear, and such a decrease has never been described previously. According to previous studies in other species, the activation of the complement system may cause a deficiency of free vascular endothelial growth factor (VEGF). As the LH surge can induce the expression of VEGF mRNA and/or protein in granulosa cells of various species [59][60][61], one may assume that the decrease in complement level at post-LH stage could help angiogenesis which is necessary for ovulation.
Two other protein spots that were demonstrated in our study with a lower expression at the post-LH stage could not be identified by mass spectrometry. This could be due to the low quantity of protein within spots, to the small number of cleavage sites for trypsin, which results in small number of peptides for identification. The presence of formaldehyde in the coloration method may also interfere with amine functions and modify the protein mass.
Because of the high permeability of the blood-follicle barrier, the follicular fluid content resembles that of plasma. In the present study, we hypothesize that comparison of 2D-PAGE proteins profiles of follicular fluid and plasma may reveal some proteins specific to the ovary. In this view, we demonstrated in the present study a higher level of clusterin in canine follicular fluid than in plasma. Our result is in accordance with that of Jarkovska et al. [19] who showed high levels of clusterin in human follicular fluid. Clusterin is a complement regulatory protein which plays an active role in inhibition of complement-mediated cell damage [62] and may also play a protective role in reproduction [63]. The high level of clusterin in follicular fluid might contribute to the inhibition of cytolytic activity of complementmediated membrane attack. Finally, we showed higher level of gelsolin in plasma than in canine follicular fluid. Circulating gelsolin is the secreted isoform of cytoplasmic gelsolin, which participates in the clearance of actin from general circulation [64]. This protein was identified earlier in human follicular fluid [65], and in the mouse ovary where it is predominantly found in the theca externa and in stromal cells [66]. In the ovary, Teubner et al. [66] proposed that gelsolin may play a role in contractile and morphogenetic processes that take place during follicular growth and ovulation via the modulation of the activity of the actomyosin ATPase. Nevertheless, intrafollicular gelsolin may also be related to actin clearance that takes place in follicular fluid and its lower concentration in canine follicular fluid compared to plasma could be explained by this function [64].

Conclusions
This study provides the first description of 1/intrafollicular levels of steroids (17beta-estradiol and progesterone) at the pre-LH and the post-LH stages in the canine species, and 2/the protein composition of follicular fluid during the preovulatory phase in the bitch. Our results show that in canine as in other species, the intrafollicular steroid content is related to the follicle size, and is regulated at the time of the LH surge. In contrast, intrafollicular progesterone levels are much higher than those found in other mammals, most probably due to intense luteinization in canine. Of note is the fact that in canine, intrafollicular steroid levels are largely higher than blood levels. Moreover, a combination of 2D-PAGE, computing image analysis and mass spectrometry led to the identification of 21 different proteins in canine follicular fluid. Most of them are functionally interesting and are expected to play an essential role in female reproduction. We demonstrated the regulation of intrafollicular complement factor B at the preovulatory stage, and the specific levels of gelsolin and clusterin in plasma and in follicular fluid. These proteins may play a role in follicle physiology and ovarian activity at the preovulatory stage, or may be involved directly or indirectly in the regulation of the meiosis resumption. Our observation may help in the future to explain and to better understand the species specificities that are described in dogs.

Additional material
Additional file 1: Supplemental Table 1: Characteristics of different fragments of albumin (ALB) and immunoglobulin heavy chain.