To the Editor: In 1998, a new lineage of triple reassortant influenza A (H3N2) virus (TR-H3N2) with genes from humans , swine , and birds (polymerase acidic [PA] and PB2) emerged in the U.S. swine population. Subsequently, similar viruses were isolated from turkeys (Anas platyrrhynchos] and 1 from a northern pintail [Anas acuta] of 266 birds sampled) in north-central South Dakota. We report on the characterization of these TR-H3N2 viruses and hypothesize about their potential for interspecies transmission.5).Two of these isolates, A/mallard/South Dakota/Sg-00125/2007 (H3N2) and A/northern pintail/South Dakota/Sg-00126/2007 (H3N2), were recovered from the birds sampled in north-central South Dakota, 45°44′30′′N, 98°16′30′′W; 2 isolates, A/mallard/South Dakota/Sg-00127/2007(H3N2) and A/mallard/South Dakota/Sg-00128/2007(H3N2), were sampled at 45°46′30′′N, 98°15′30′′W. Viral RNA was extracted, reverse transcribed, and amplified; all segments were sequenced in entirety and submitted to GenBank under the identified virus names. Phylogenetic analysis showed significant nucleotide identities (99%–100%), differing only in 4 nucleotide positions: 1 each from PB1, PA, NP, and NS genes. Among 4 substitutions, 3 were nonsynonymous , and 1 (PB1) was synonymous. A1725G substitution in PB1 was identified in 2 isolates. C419T change was identified in 3 isolates , resulting in substitution of threonine by phenylalanine. Three isolates  carried an A at residue 149 of the NP gene (leading to S50N change) and 1 isolate (Sg-00128) had a G at that position (encoding serine). G809A change was present in the NP gene of 3 isolates . Genomes of the 4 isolates had high nucleotide and amino acid identities (>98%) with North American swine TR-H3N2 virus (A/swine/Iowa/533/99 [H3N2]). Phylogenetic analysis indicated that TR-H3N2 waterfowl and North American TR-H3N2 swine isolates belonged to a single cluster. The H3N2 subtypes from avian and swine isolates of our sequencing projects belonged to different clusters . DeducedInasmuch as we identified a swine lineage virus in waterfowl, we first investigated laboratory contamination by using trace back and history of swine virus isolations during the time the surveillance samples were processed. No H3N2 subtype were isolated from swine sources in the Minnesota Veterinary Diagnostic Laboratory during this period. Furthermore, phylogenetic analysis of all HA segment sequences from isolates obtained in that 4-month period confirmed no contamination. We then investigated whether an ecologic niche existed for potential exposure of waterfowl to pigs. We identified a swine herd near the wildlife refuge area where the waterfowl sampling occurred. Pigs were housed outdoors, and the owner of this swine herd reported that geese and ducks inhabit the water ponds/stock dams/slough area to which the pigs had access. Contact with the local veterinarian and the South Dakota Veterinary Diagnostic Laboratory indicated no recent reports of influenza A (H3N2) episodes in the swine herd. In addition, this herd was not vaccinated for swine influenza.The mode of transmission of swine-origin virus to waterfowl is not clear. In previously published cases, where swine influenza viruses have been identified in turkeys, the flocks were in close proximity to swine herds (1) showed that 10 of 19 samples reacted with all 4 waterfowl isolates; titers ranged from 10 to >640. Although low titers may have occurred because pigs were exposed to heterologous cross-reactive viruses, the high titers in most animals with positive serum samples suggest exposure to an influenza (H3N2) virus similar to that recovered from the waterfowl. Our data emphasize the need to investigate the possible role of waterfowl in the maintenance and transmission of influenza A viruses to humans and to lower mammalian species.In late 2008, serum samples were collected from this swine herd. Hemagglutination inhibition test (


Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells. Monoclonal antibodies (mAbs) are currently the fastest growing class of biotherapeutic molecules Tricistronic vectors that express LC, HC, and selection marker genes in one mRNA are able to alleviate the above problems of traditional vectors. When vectors get fragmented, the mRNA unit would be incomplete and no genes would be expressed. Internal ribosome entry site (IRES) elements, which have a length of several hundred base pairs, allow expression of multiple genes in one mRNA. When IRES elements are included between multiple open reading frames (ORFs), the first ORF is translated by the canonical cap-dependent mechanism while the rest are translated through a cap-independent mechanism in vivo gene therapy An alternative approach for co-expressing multiple genes in one mRNA is using 2A elements. 2A elements are much shorter than IRES, having only 60 to 80 base pairs. 2A linked genes are expressed in one single open reading frame and “self-cleavage” occurs co-translationally between the last two amino acids, GP, at the C-terminus of the 2A polypeptide, giving rise to equal amounts of co-expressed proteins In studies conducted using F2A for mAb expression, mAb quality has only been characterized by western blotting. Detailed quality characterization of mAb expressed using F2A has not been reported. Only one study has compared IRES and F2A for mAb expression in transient transfections in HEK293 cells 5 cells/mL in 25 mL fresh medium. Cell density and viability were measured using the trypan blue exclusion method on an automated Cedex counter .Parental DHFR-deficient CHO DG44 cells  were cultured in suspension using a protein-free medium consisting of HyQ PF  and CD CHO (Life Technologies) at a 1∶1 ratio and supplemented with 2 g/L sodium bicarbonate , 6 mM glutamine (Sigma-Aldrich), 0.1% Pluronic F-68 (Life Technologies), and 1% hypoxanthine and thymine (HT) (Life Technologies). Regular passaging was carried out every 3 to 4 days in 125 mL shake flasks  by diluting cells to 2×10Two tricistronic vectors, L-IRES-H and H-IRES-L , were ob6 cells were co-transfected with 2 µg of the appropriate mAb encoding plasmid and 0.2 µg of a green fluorescence protein (GFP) encoding plasmid pMax-GFP (Lonza). The GFP plasmid acted as an internal control to account for variations in transfection efficiency. The transfected cells were then resuspended with 2 mL of HT-containing protein-free medium in 6-well suspension culture plates . Transfections were carried out in duplicates. At 48 h post-transfection, cells and supernatant were collected to measure GFP fluorescence intensity using a FACS Calibur  and to measure the mAb concentration using an enzyme-linked immunosorbent assay (ELISA) respectively. ELISA was performed as previously described using capture antibody of affinity purified goat anti-human IgA+IgG+IgM (HC+LC)  and detection antibody of goat anti-human IgG (Fc specific) conjugated to alkaline phosphatase (Sigma-Aldrich) Transient transfection of DG44 cells were carried out using Nucleofector kit V and program U-24 on a Nucleofector I system . In each transfection, 2×107 CHO DG44 cells were transfected with 5 µg of BglII linearized mAb expressing plasmid. Transfection for each of the four vectors was carried out in duplicates. The transfected cells were each incubated in 2 mL HT-containing protein-free medium in a 6-well suspension culture plate for 24 h. Samples were next centrifuged at ∼100×g for 5 min and cell pellets resuspended in 15 mL HT- and protein-free medium in 125 mL shake flasks to select for stable transfectants. Selection required around 2 to 3 weeks. When the stably transfected pools recovered with viability above 95%, gene amplification was induced by passaging in medium containing 50 nM methotrexate (MTX) (Sigma-Aldrich). The amplification process also required 2 to 3 weeks. Productivity of stable transfection pools at 50 nM of MTX was determined in 125 mL shake flask batch cultures. Cells were seeded in 25 mL of medium at a cell density of 2×105 cells/mL. Growth was monitored every day until the end of culture when viability measurements dropped below 50%. Supernatant was collected at the end of culture and analyzed for mAb concentration using a nephelometric method on an IMMAGE 800 immunochemistry system . The IMMAGE 800 system utilized anti-human Fc region antibodies for IgG detection. The specific productivity (qmAb) was calculated as the mAb concentration divided by the integrated viable cell density (IVCD) which was determined based on the trapezoidal method.Transfections to generate stably transfected pools were also performed using Nucleofector Kit V and program U-24 on a Nucleofector I system (Lonza). 1×10Supernatants, which were collected from stably transfected pools at the end of culture, containing 10 ng of mAb as determined using the nephelometric method were mixed with NuPAGE sample loading buffer and reducing buffer (Life Technologies) and heated at 70°C for 2 min. Samples were loaded onto NuPAGE 4–12% Bis-Tris gel (Life Technologies) in MES buffer (Life Technologies). Precision plus protein dual color standards  were used as molecular weight ladder and to check for transfer to the membrane. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Life Technologies) using the iBlot system (Life Technologies). Membranes were blocked in 5% blocking milk (Bio-Rad Laboratories) in TBS with 0.1% Tween-20 (Bio-Rad Laboratories) for 1 h at room temperature and incubated overnight in HRP conjugated goat anti-human IgG Fc antibody  and HRP conjugated goat anti-human IgG Kappa LC . Detection was done using ECL Prime  and exposed on Lumi-Film Chemiluminescent Detection Film (Roche).mAb in the supernatant collected at the end of culture of stable transfection pools was purified by affinity chromatography using protein A column on a GE AKTA explorer 100  as previously described Protein A purified mAb in stable transfection pools generated using different tricistronic vectors were separated by SDS-PAGE. Prior to SDS-PAGE separation, 10 µg of each purified mAb sample was denatured by boiling in the presence of 2-mercaptoethanol (Sigma-Aldrich) at 95°C for 5 min in the loading buffer containing 0.063 M Tris-HCl at pH of 6.8, 10.5% glycerol , and 10% (w/v) SDS  and 0.1% bromophenol blue , and then ran at 15 mA for 90 min in a 10% T polyacrylamide gel (in-house created). Upon completion, the gel was immediately fixed and stained in coomassie blue composed of 0.3% (w/v) Coomassie Brilliant Blue R-250  in 45% methanol and 10% acetic acid, and then washed with 10% methanol and 5% acetic acid to remove the background. The gel was scanned on Imagescanner III  and the relative intensities of the protein bands for each lane were quantified using the ImageQuant TL 7.0 software  based on the peak heights of each generated line graph.LC-MS/MS analysis was performed on each visible protein bands observed on the coomasie blue stained SDS-PAGE page gel. The bands on the gel were excised and washed in 150 µL of 50% acetonitrile  solution containing 25 mM ammonium bicarbonate. The washing solution was removed and the excised gels were dried using the Savant Speedvac . Proteins within the excised gels were reduced by adding 25 µL of 20 mM DTT and incubated at 55°C for 60 min. Upon removal of the reducing agent, alkylation of the reduced proteins was facilitated by adding 25 µL of 55 mM iodoacetamide  and incubated at room temperature in the dark for 45 min. The excised gels were then washed repeatedly in 100 mM ammonium bicarbonate solution, followed by 100% acetonitrile. After drying the excised gels using the Savant Speedvac, 20 µL of trypsin  at 0.04 µg/µL in 25 mM ammonium bicarbonate solution was added to each excised gel sample. The enzyme-sample mixtures were incubated with agitation at 37°C overnight. Each digested sample was extracted by adding 20 µL of acetonitrile with sonication. Each acetonitrile solution containing digested proteins was transferred to a new clean vial and dried using the Savant Speedvac. Prior to injecting the samples onto the LC-MS/MS system, each sample was reconstituted in 5 µL of aqueous buffer containing 0.1% formic acid (Sigma-Aldrich) and 1% methanol (Fisher Scientific).LC-MS/MS analyses were performed on a Velos Orbitrap mass spectrometer  coupled to a nanoAcquity UPLC system  fitted with a 180 µm × 20 mm Symmetry C18 peptide trap (Waters) and a 75 µm × 200 mm BEH130 C18 1.7-µm particle size analytical column (Waters). Depending on the peptide concentration of each sample, 1 to 2 µL of peptide digest was injected. Peptides were resolved by applying a linear binary gradient from 2 to 35% solvent B at 300 nL/min over 60 min at 35°C, where solvent A and B were 0.1% (v/v) formic acid in Milli-Q water  and 0.1% formic acid in acetonitrile, respectively. To minimize sample carryover, a dedicated column wash run (10 to 95% B in 30 min) followed by column re-equilibration (2% B for 30 min) was performed prior to the next sample injection. The nano-ESI source was fitted with a 30-µm stainless steel nano-bore emitter (Thermo Fisher Scientific) with 1.7 kV applied near the tip. The MS instrument method used was the data-dependent acquisition mode that specified each orbitrap survey scan  to be linked to a maximum of 10 MS/MS events; each with maximum ion trap fill time of 25 ms and isolation window of 2 m/z. The threshold for triggering an MS/MS was set at 500 counts. The ion trap CID fragmentation employed an activation time of 10 ms, q value of 0.25 and normalized collision energy of 35%. Charge state screening was enabled, with unknown and singly charged states excluded. Dynamic exclusion was enabled with a list size of 500 and exclusion time of 60 s.Acquired LC-MS/MS data were processed by the Proteome Discoverer 1.3 software (Thermo Fisher Scientific) using the Sequest search engine. The peptide and fragment ion mass tolerances used were ±5 ppm and ±0.5 Da, respectively. The specified search parameters were carbamidomethylation of cysteine as fixed modification, oxidation of methionine as dynamic modification and tryptic digestion with 2 missed cleavages. Depending on the tricistronic vector configuration used , each saThe aggregation of protein A purified mAb collected at the end of culture of stable transfection pools was determined using size exclusion chromatography (SEC) coupled to a UV-visible detector and a dynamic light scattering detector. Analysis was carried out for duplicate pools generated using each tricistronic vector. The instrument setup consisted of a HPLC system  as previously described An anti-HER2 IgG1 biosimilar was first used as a model mAb to compare IRES and F2A for mAb expression level and quality. Four tricistronic vectors were designed to express the anti-HER2 LC, anti-HER2 HC, and DHFR under the control of one CMV promoter . LC and IRES and F2A were first compared for transient expression levels of anti-HER2 in 6-well plate cultures . As cap-Stable anti-HER2 expressions for the four vectors were next compared using shake flask batch cultures . SpecifiStably transfected pools generated using the four vectors also exhibited differences in growth . The L-I2LC2, LC2 dimer, and LC monomer, a sign that the L-IRES-H vector expressed LC in excess. Product from the H-IRES-L vector consisted of HC2LC2 complete IgG1 and HC2 dimers, providing evidence of expression of excess HC. Products from both F2A-mediated tricistronic vectors presented a smear with molecular weight greater than 100 kDa. The fractions above 150 kDa could correspond to mAb aggregates, and those around 150 kDa could be degraded IgG1 monomers, suggesting that mAb expressed from F2A was not stable. The two thin bands expressed from the L-F2A-H and H-F2A-L vectors with molecular weights of 100 kDa could be HC2, and the one band from the L-F2A-H vector with a molecular weight of 80 kDa could be a fusion protein of LC+F2A+HC.Supernatant collected at the end of culture of CHO DG44 stably transfected pools generated using each of the tricistronic vectors were first analyzed under non-reducing conditions to characterize the products being secreted . ProductWestern blotting analysis of reduced products in the supernatant was next performed to identify the different components in the IgG1 being produced using the various vectors . Two banIRESwt has three ATGs at the 3′ end, which are designated as ATG-10, ATG-11, and ATG-12 as described previously To characterize the protein more accurately than just using western blotting, the reduced protein A purified mAb samples from duplicate pools of each vector were separated on SDS-PAGE  and the H+HC), and fusion proteins of LC+F2A+SPH+HC, respectively. The four excised gel bands from the H-F2A-L vector with molecular weights of 25, 50, 55 and 80 kDa were LC with incorrectly cleaved signal peptide (SPL+LC), HC, SPH+HC+F2A, and SPH+HC+F2A+SPL+LC, respectively. No incorrect signal peptide cleavage was observed on LC and HC in the IRES generated pools. The four extra amino acids which might attach to the signal peptides during translation initiation at IRES ATG-11 did not seem to affect the signal peptide cleavage. In contrast, incorrectly cleaved signal peptides were detected in both LC and HC expressed downstream of F2A as indicated by the detection of SPL+LC and SPH+HC with signal peptide residues attached. This is possibly due to attachment of the extra P from F2A to the signal peptide.The excised gel bands from the L-IRES-H or H-IRES-L vector at molecular weights of 25 and 50 kDa were confirmed by LC-MS/MS analyses as LC and HC polypeptides, respectively. The excised gel bands from the L-F2A-H vector corresponding to molecular weights of 30, 50, and 80 kDa were LC and F2A fusion proteins (LC+F2A), HC with incorrect or uncleaved signal peptide (SPH+HC fusion protein. The rest were cleaved into similar abundances of LC+F2A (37%) and SPH+HC (42%) as expected. 16.5% of product expressed from the H-F2A-L vector was detected as SPH+HC+F2A+SPL+LC fusion proteins. The remaining was cleaved into products consisting of 32% HC, 12.5% SPH+HC+F2A, and 39% SPL+LC. The amount of SPL+LC was similar to the sum of HC and SPH+HC+F2A.Cleavage of F2A was less efficient in products expressed from the L-F2A-H vector than the H-F2A-L vector. Occurrence of LC+F2A+HC or HC+F2A+LC fusion proteins indicates cleavage failure at both furin and 2A recognition sites. Existence of LC+F2A or HC+F2A indicates successful cleavage at the 2A recognition site but cleavage failure at the furin recognition site. 21% of the LC and HC expressed from the L-F2A-H vector existed as a LC+F2A+SPAggregation of protein A purified anti-HER2 IgG1 mAb produced in stable transfection pools was analyzed and quantified using SEC coupled to a UV and a dynamic light scattering detector. As replicate pools gave identical chromatograms, only one representative UV chromatogram is shown for each vector . MoleculPrevious reports which utilized F2A for expressing mAb did not report any issues with 2A peptide cleavage under their tested conditions 2 dimer, resulting in falsely high mAb titers. By measuring mAb in crude supernatant with these two assays, F2A vectors appeared to give higher mAb expression than IRES vectors in both transient and stable transfections. However, after considering the product distribution of complete IgG1 monomers, aggregates and fragments detected during SEC analysis , HC signal peptide with MATT residues (MATT+SPH) and HC signal peptide with an additional P (P+SPH) were analyzed using the Kyte and Doolittle hydrophobicity index  at their signal peptide sequence as they emerge from the ribosome within the cytosol and are targeted to SRP receptors of translocon complex on the ER membrane for translocation into the ER ty index  [50]. MA2LC2 in the ER Incomplete cleavage of F2A had been observed when used with various fluorescent reporter proteins The L-IRES-H was the best vector design for expressing IgG1 mAb in CHO DG44 cells among the four versions of tricistronic vectors. While the F2A system is a promising design, issues with 2A processing and furin cleavage affected product yield and quality. Further optimization of the type of 2A peptide to be used, addition of GSG linkers and the furin cleavage sequence need to be performed Figure S1Peptide mapping of tryptic peptides of LC and HC expressed from the four tricistronic vectors. Protein A purified product from duplicated stable transfection pools of each vector were analyzed. Purified samples from each pool were reduced and separated on SDS-PAGE. The bands (refer ds refer  were exc(DOC)Click here for additional data file.


SLC22A1, PXR, ABCG2, ABCB1 and CYP3A5*3 genetic polymorphisms on imatinib mesylate (IM) pharmacokinetics in Asian patients with chronic myeloid leukemia (CML).This study aimed to explore the influence of 0h) and clearance (CL) were determined in the patients at steady state. Haplowalk method was applied to infer the haplotypes and generalized linear model (GLM) to estimate haplotypic effects on IM pharmacokinetics. Association of haplotype copy numbers with IM pharmacokinetics was defined by Mann-Whitney U test.Healthy subjects belonging to three Asian populations  and CML patients (n = 38) were enrolled in a prospective pharmacogenetics study. Imatinib trough , to be significantly associated with IM clearance (p = 0.013). Haplotype-specific GLM estimated that the haplotypes AGT and CGC were both associated with 22% decrease in clearance compared to CAC . Patients harboring 2 copies of AGT or CGC haplotypes had 33.4% lower clearance and 50% higher C0h than patients carrying 0 or 1 copy . Further subgroup analysis revealed SLC22A1 and ABCB1 haplotypic combinations to be significantly associated with clearance and C0h .Global haplotype score statistics revealed a SLC22A1-ABCB1 haplotypes may influence IM pharmacokinetics in Asian CML patients.This exploratory study suggests that  BCR-ABL gene which is then translated to the BCR-ABL protein with intrinsic tyrosine kinase activity that is critical to the development of CML Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder, characterized by the presence of the Philadelphia chromosome (Ph) that results from a balanced reciprocal translocation between chromosomes 9 and 22. Functionally, this translocation results in the formation of the safe haven that promises” favorable long-term outcomes in patients with CML However, despite significant progress in CML treatment, about 30–40% of the patients still fail to achieve major molecular response (MMR) at 18 months BCR-ABL mutations in vivoSuboptimal response and eventual treatment failure may be associated with several factors including the presence of et al. showed that in patients on IM doses less than 600 mg/day, 82% of those with low activity of hOCT-1, the major active influx pump for IM, failed to achieve MMR by 18 months, compared to only 17% of those with high hOCT-1 activity. Furthermore, the negative impact of low hOCT-1 activity may be overcome by escalating to high dose IM at 800 mg/day SLC22A1 (encoding for hOCT-1) expression or polymorphisms on IM pharmacokinetics have revealed rather heterogeneous results. High expression levels have been associated with favorable response in some studies SLC22A1 SNPs and response to IM has also been observed in some reports One plausible hypothesis to explain the wide variability may be a result of disparities in population pharmacokinetics and pharmacogenetics. Differences in activity, expression levels as well as functional single nucleotide polymorphisms (SNPs) of both efflux transporters  and uptake transporters  SLC22A1 SNPs identified on whole gene sequencing, as well as 11 candidate SNPs from ABCB1, ABCG2, CYP3A5 and PXR genes on IM pharmacokinetics were examined in a cohort of healthy subjects belonging to Asian populations from Singapore, consisting of Chinese, Malay and Indian ethnic groups as well as patients with Ph+ chronic phase CML.To address these issues, we investigated if differences in SNP frequencies of efflux and influx transporters, as well as drug metabolizing enzymes, would be able to account for inter-ethnic and inter-individual variation in IM trough levels. In this exploratory study, the effects of 89 All participants provided written informed consent for the study, which was approved by the ethics review committee of the National Cancer Centre, Singapore.BCR-ABL fluorescence in situ hybridization (FISH) studies. At least 20 metaphases were assessed for cytogenetic analysis. Peripheral blood BCR-ABL/total ABL ratio was obtained by using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Patient characteristics are summarized in A total of 210 healthy subjects belonging to Asian populations from Singapore, consisting of Chinese, Malay and Indian ethnic groups (n = 70 per group) were enrolled for this study. In addition, a total of 38 patients with Ph+ chronic phase CML  who received IM at the Singapore General Hospital from January 2001 to September 2007 were prospectively recruited. Ethnicity of the subjects was confirmed by careful screening and verified against their National Registry Identification Cards. Prior to commencing therapy, all patients were required to have a complete blood count including the white cell count differential, as well as standard biochemistry. Baseline investigations also included bone marrow evaluation for morphology, conventional cytogenetic analysis by G-banding, and BCR-ABL/total ABL ratio obtained from RT-PCR was also taken to indicate CCyR BCR-ABL/total ABL ratio was repeated every 3 months after treatment with IM. According to the definition of the international scale BCR-ABL/total ABL ratio. Treatment failure was defined as (1) inability to achieve any CyR at 6 months, (2) less than PCyR at 12 months or less than CCyR at 18 months, (3) disease relapse as indicated by a loss of CCyR or a confirmed one log increase of BCR-ABL/total ABL ratio, as well as (4) transformation to accelerated phase disease or blast crisis. In cases of treatment failure, tyrosine kinase domain mutation tests were indicated.Patients were monitored regularly for treatment response on an outpatient basis. A complete hematological response was defined as normalization of peripheral blood count and disappearance of splenomegaly, if present, within 3 months. To evaluate for cytogenetic response, a repeat bone marrow biopsy was performed 6 monthly after treatment with IM. Cytogenetic response was defined using standard convention with respect to the percentage of Ph-positive cells in the bone marrow as complete , partial  and minor (35–90%). A <1% SLC22A1 gene (UCSC RefSeq: NM_003057), spanning 40 kilobases, including 2 kilobases upstream and 1.5 kilobases downstream, on chromosome 6. Eleven candidate functional polymorphisms in ABCB1, ABCG2, CYP3A5 and PXR genes which were described in our previous publications CYP3A4 polymorphisms were observed very rarely in Asian population in our previous studies Genomic DNA was extracted from 3 ml of peripheral blood samples using the Gentra® Puregene® extraction kit  and stored at −80°C until analyzed. Pharmacogenetic analyses involved screening and genotyping of polymorphic variants in the entire 0h) were estimated via a validated high performance liquid chromatography (HPLC) method that was modified from a previously published technique g at 4°C for 10 min, and 20 µl of the supernatant was subject to HPLC analysis. Chromatographic separation was accomplished using a C8 column  with a mobile phase consisting of 20 mM potassium dihydrogen phosphate:acetonitrile . The lower limit of quantitation was 7.8 ng/mL. The calibration curve was linear over a concentration range of 7.8–8000 ng/mL. The within-day and between-day coefficients of variation were less than 10%. Imatinib clearance (CL) was calculated by the formula IM dosage/, whereby the IM dosage ranged from 300–600 mg and dosing interval was 24 hours. The final parameters used in the analyses on C0h and CL were normalized by IM dosage.Peripheral blood samples were taken from the patients 24 hours following their last IM dose. Steady state plasma trough concentrations of IM (C2) values. Tag-SNPs were identified by using Tagger program in Haploview software. Haplowalk method SLC22A1 and ABCB1 haplotype profiles on the prediction of IM pharmacokinetics. Imatinib trough level association with clinico-demographic characteristics and response parameters was evaluated with Mann-Whitney U test for categorical data and Kendall Tau correlation test for continuous data. All tests were performed using SPSS statistics software ver. 18 . All statistical evaluations were made assuming a two-sided test with significance level of 0.05 unless otherwise stated.The Hardy-Weinberg equilibrium between the genotypes was assessed using Fisher’s exact test. The Chi-square test was employed to assess the differences in genotype and allele distributions among the different groups in healthy subjects. Linkage disequilibrium (LD) analysis and LD block constructions were carried out using the software Haploview ver. 4.2  and quantified by |D′| and rho-square (r−2 (range: 17.9–49.5), respectively. All patients received IM for at least 18 months, starting at a daily dose of 400 mg. At the point of recruitment, 4 patients were on an escalated daily dose of 600 mg as they could not achieve major molecular response, and 2 were on an attenuated dose of 300 mg due to toxicity issues. Median follow-up time from the start of IM was 101.6 months (range 25.6–132.7). All patients achieved complete hematological response within 3 months of IM treatment. At 6 months, one patient had no CyR and was considered to have failed treatment, whereas the remainder had achieved minor CyR (n = 2), PCyR (n = 10) or CCyR (n = 24), or remained unknown (n = 1). At 12 months, no patients were identified to have achieved less than PCyR except for the one who had failed treatment earlier. At 18 months, all patients achieved CCyR except for 3 who were considered to have failed treatment. Of 28 patients with available data for molecular response at 18 months, 14 patients did not achieve MMR and were considered to have suboptimal response to IM treatment. In the course of follow-up post-IM therapy, one patient eventually relapsed at 56.5 months whilst another patient succumbed to blast crisis at 90.7 months. Upon further analysis, non-synonymous mutations were detected in the BCR-ABL kinase domain in these two patients - a double mutation at loci G250E and E255V in the former patient and a single mutation at Y253H in the latter patient. No mutations were detected in the remaining three patients. Overall, a total of five patients (13.2%) were considered to have failed treatment.The median age at diagnosis and BMI of the patients were 42 years (range: 16–69) and 24.4 kgmThe median time from diagnosis to IM plasma level testing was 71.5 months, and the median time from the commencement of IM therapy to plasma level testing was 58.4 months. Trough IM plasma levels were highly variable, ranging from 625 to 5271 ng/mL, with mean (±SD) and median levels of 1959 (±1073) and 1719 ng/mL, respectively. For patients on the standard IM dose of 400 mg (n = 32), the mean (±SD) and median levels were 1899 (±1054) and 1395 ng/mL, respectively. Patients on the escalated dose of 600 mg (n = 4) had higher mean (±SD) and median levels of 2785 (±1093) and 2384 ng/mL, respectively, although this was not statistically significant. Notably, we observed that out of 34 patients on ≤400 mg dose of IM, 31 (94%) had an IM trough concentration of >1000 ng/mL (range: 679–5272 ng/mL) . None ofSLC22A1 gene was first performed on healthy subjects of Chinese, Malay and Indian ethnicity. SLC22A1 gene structure and locations of these SNPs. Fisher’s exact test showed that SLC22A1 genotype frequencies at all loci conformed to Hardy-Weinberg equilibrium except for a single intronic SNP IVS1+556G>A (rs62440864) in the Malay population (p<0.001). Of these 89 SNPs, 20 were found to be monomorphic in Chinese, 9 in Malays and 11 in Indians. Six SNPS  were found to be highly polymorphic in all ethnic groups . Statistically significant interethnic differences in the genotypic distributions among three ethnic groups were detected for 34% (n = 30) of the polymorphisms (p<0.016).Complete screening of the orphisms , p<0.016SLC22A1 gene region. Tight linkage was observed amongst the following SNPs across all ethnicities: IVS5-61G>A (rs2282142) with 1022C>T (rs2282143), IVS2-687G>T (rs3798175) and IVS4+1028A>G (rs3798170) ; IVS2+97G>A (rs4646273) and IVS2+797C>G (rs4646274) with IVS4+1040T>G (rs3798169) and IVS7-1368C>T (rs1867350) ; IVS2-461C>T (rs3798174) with IVS2-99C>T (rs3737088), IVS4+886C>G (rs3798172) and IVS7-1053C>T (rs7766568) ; IVS2-257C>T (rs4646275) with IVS4+886C>G (rs3798172), IVS4-98G>A (rs4646276) and IVS7-1053C>T (rs7766568) ; IVS2-1010T>G (rs614890) with IVS4+597A>G (rs594709)  located in the region spanning introns 2–7; 5′-upstream region SNPs were in moderate linkage with intronic SNPs, -1756_-1755insT and -1620T>C (rs9457840) with IVS2-687G>T (rs3798175), IVS4+1028A>G (rs3798170) and IVS5-61G>A (rs2282142) ; −1795G>A (rs6935207) with IVS1-207T>C (rs9457841) and IVS1-43T>G (rs4646272) ; introns 8 and 9 polymorphisms, IVS8+1322G>T (rs644992) with IVS8+2698A>G (rs637841) ; IVS8+5101T>C (rs7750592) with IVS8-4331A>G (rs9347386)  and IVS8-1803C>T (rs4709401) with IVS8-1793C>T (rs4709402) and IVS9+43C>T (rs2297374) . A detailed LD pattern is shown in SLC22A1 region for each ethnic group by using pairwise tagging method in the Tagger program  as described by de Bakker et al. Pairwise LD analyses were performed between the 89 SNPs identified earlier for each of the ethnic groups. Pairwise LD matrices demonstrated moderate to strong linkage between several SNPs throughout the SLC22A1 tag-SNPs, as well as candidate SNPs from ABCB1 , ABCG2 421C>A (rs2231142), CYP3A5*3 (rs776746) and PXR  was performed in 38 CML patients. The genotypic and allelic frequencies of SLC22A1 tag-SNPs in the patient cohort were found to be similar to that observed in the healthy Chinese subjects as majority of patients were of Chinese descent. Candidate SNPs selected from ABCB1, ABCG2, CYP3A5 and PXR genes were present with high variant allele frequency ranging from 20% to 70% in the patients with CML which was also comparable with healthy subjects from the 3 ethnic groups . The haplowalk score statistic displays a global overview of the association of haplotypes in a particular region with phenotypic traits, and cannot be interpreted as a specific individual haplotypic effect. Therefore, we further employed the haplotype-specific GLM in order to estimate the individual haplotypic effects.The haplotypic effect of −2 L/hr/mg); CAC vs AGT: 4.03 vs 3.16, p = 0.017; CAC vs CGC: 4.03 vs 3.15, p = 0.017]. The SLC22A1 haplotype AGC was found to be present in only 1% of our CML patients, however the IM clearance was observed to be 1.7-fold higher than reference haplotype . Similarly, haplotypes AGT and CGC were modestly associated with a 38% and 30% increase in C0h, respectively, as compared to reference haplotype CAC, although this was not statistically significant . As both of these low clearance-associated haplotypes had similar impact on IM disposition, we examined the combinatorial effect of copy numbers of these two haplotypes (AGT and CGC) on IM pharmacokinetics. Two copies of haplotypes AGT or CGC were observed in 23 patients, 1 copy in 12 patients whereas 3 patients were not carrying any copies of AGT or CGC haplotypes. The patients were divided in two groups according to the number of copies of haplotypes AGT or CGC. The first group (n = 15) comprised of patients carrying 0 or 1 copy (Slow) and the second group (n = 23) comprised of patients carrying 2 copies of AGT or CGC haplotypes (Shigh). The copy numbers of AGT and CGC haplotypes were significantly associated with IM clearance and C0h (high had 33.4% lower clearance and 50% higher C0h than patients with Slow .Haplotype-specific GLM estimates the regression coefficient associated with IM pharmacokinetics . The hig and C0h . PatientABCB1 haplotypes could affect IM clearance and concentrations. Interestingly, although we did not find any direct correlations amongst ABCB1 haplotypes with clearance and C0h, we observed that patients harboring Shigh tended to carry either 0 or 1 copy of ABCB1 haplotypes  TGT or TTT (Alow), rather than 2 copies (Ahigh) (p = 0.030). When stratified according to specific SLC22A1-ABCB1 combination haplotype profiles, a trend towards low clearance and high C0h was observed from Slow-Alow, Slow-Ahigh, Shigh-Alow to Shigh-Ahigh. Notably, the simultaneous possession of Slow with Alow was associated with a 73.2% higher clearance and 41.2% lower C0h than patients carrying other haplotype combinations  (SLC22A1-ABCB1 haplotype profile was not significantly associated with treatment response parameters.We explored if specific  0.0099] . The come.g. hOCT1) and efflux (e.g. ABCB1) transporters Our results demonstrated wide inter-individual variability of trough IM plasma levels in Asian patients with CML, confirming previous reports on Caucasian SLC22A1 gene for polymorphic variation in three healthy Asian ethnic populations and further examined tag-SNPs in Asian CML patients. Our approach revealed a sub-haplotypic region encompassing 1 exonic SNP [1222A>G (rs628031)] surrounded by 2 intronic SNPs [IVS6-878C>A (rs3798168) and IVS7+850C>T] that is significantly associated with IM pharmacokinetics. The SLC22A1 gene was found to be highly polymorphic and displayed significant interethnic variations amongst healthy individuals of Chinese, Malay, and Indian ethnicity  and the other carrying 2 copies of AGT or CGC haplotypes (Shigh). Patients harboring Shigh had 50% higher IM trough level and 33.4% lower clearance than patients with Slow. The haplotype CGC was represented by the presence of the wild type allele of intronic polymorphisms IVS6-878C>A (rs3798168) and IVS7+850C>T, as well as the variant allele of the non-synonymous SNP 1222A>G (rs628031). On the other hand, the haplotype AGT was represented by the presence of the variant allele of all three polymorphisms. To the best of our knowledge, the intronic polymorphisms IVS6-878C>A (rs3798168) and IVS7+850C>T were not studied with phenotypic data previously. Although IM is a substrate of hOCT1 SLC22A1 variants in different ethnic groups. Particularly, strong linkage (|D′| >0.90) was detected amongst these three key variants in the Chinese population, which formed 84.2% of our patient cohort. Hence, the linkage effect of the region may have been the confounding factor in previous studies. The sub-haplotypic region is composed of two intronic and one coding SNP which suggests that the influence of haplotypes on IM pharmacokinetics could be mediated through interactions involving post-transcriptional modification. Taken together, out results highlight the importance of haplotype over single marker analysis to explain the variability in IM disposition and warrant further investigations in other ethnic groups.Amongst our cohort of Asian patients with CML, we were able to identify two groups based on unique haplotype profiles - one composed of those harboring 0 or 1 copy Click here for additional data file.Figure S2Haplowalk Analysis. Haplowalk analysis showing the sub-haplotypic region associated with imatinib clearance. P-values corresponding to polymorphisms are plotted on –log10 base. The sub-haplotypic region, significantly associated with imatinib clearance, is shown in blue dotted box. This region encompasses one exonic SNP (rs628031) surrounded by two intronic SNPs (rs3798168 and IVS7+850C>T).(TIF)Click here for additional data file.Table S1SLC22A1, ABCB1, ABCG2, CYP3A5 and PXR single nucleotide polymorphisms among Asian healthy subjects  and CML patients (n = 38). SLC22A1 24 Tag SNPs and statistically significant interethnic differences in the genotypic distributions among healthy subjects of three ethnic groups are indicated by table footnotes. Table also represents ABCB1, ABCG2, CYP3A5 and PXR gene polymorphism data from our previous publications Genotype and allele frequency of (DOC)Click here for additional data file.Table S2SLC22A1 regions (UCSC RefSeq: NM_003057).Primer Sequences and PCR Conditions for Amplifications of (DOC)Click here for additional data file.


Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB).Protein expression in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies.The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications.We have developed a procedure using  Bovine viral diarrhoea virus (BVDV) infection of cattle is linked to economically important diseases with losses in the USA being estimated to US$10-40 million per million calves . BVDV isEscherichia coli systems. However, the low costs required for veterinary vaccine applications preclude the use of more expensive protein expression systems such as mammalian and insect cell systems [E. coli based expression systems is hindered by the fact that although the recombinant proteins are generally expressed at high yields the resulting proteins are often insoluble and lack post-translational modifications [E. coli has been previously reported, Chia et al. [E. coli expressed Ebola virus antigen recognised the glycosylated antigen expressed in mammalian cells.E2 contains 17 cysteine residues which form both intramolecular disulphide bonds and intermolecular disulphide bonds resulting in dimers of E2-E2 and E2-E1 . Express systems . The useications . Expressa et al.  demonstra et al.  demonstrE. coli often leads to insoluble aggregates known as IB [Expression of recombinant proteins in wn as IB . Though,wn as IB . Furtherwn as IB . IB aggrwn as IB ,19. IB swn as IB .E. coli is contamination with endotoxins. Endotoxins are a major component of gram negative bacterial cell walls and are liberated during extraction of proteins [A potential drawback from the production of recombinant proteins in proteins . Endotoxproteins . Mammaliproteins . Common proteins ,23. Two-proteins . Aida anproteins  demonstrIn this study, we report the expression and purification of a truncated form of the BVDV E2 protein (E2-T1) from IB aggregates and optimised solubilisation conditions for this protein. We also describe a novel process for the removal of endotoxins from IB aggregates using Triton X-114 phase-partitioning to maximise E2-T1 yields. The suitability of this E2-T1 protein for small animal vaccination in mice is also demonstrated.A truncated version of the E2 ORF frame was produced using PCR to remove the 3' region of the ORF which encodes for the membrane binding domain. The resultant 1040 bp PCR product was ligated into pET-SUMO expression vector.Sequence analysis of pET-SUMO-E2-T1 demonstrated the vector encoded start codon, SUMO tag and 6-His region were in frame with E2-T1. The fusion protein encoded by pET-SUMO-E2-T1 has 463 residues; the amino acid composition is shown in Table Pilot expression studies indicated auto-induction of E2-T1 as insoluble protein at 0 hours. Figure . The optPurification of E2-T1 from the soluble protein fraction was attempted using TALON resin. A step gradient of imidazole  was used to determine the optimum elution profile. At 150 mM imidazole, E2-T1 was the major species eluted from the column. This eluted protein was of high purity but of low yield (10 ng/μL).The majority of the E2-T1 protein was present in the insoluble fraction Figure . Using BRefolding of E2-T1 protein was achieved by dialysis in 50 mM Tris (pH 7.0), 0.2% Igepal at room temperature. The resultant protein had high purity Figure , lane 6 Dynamic light scattering (DLS) analysis of purified E2-T1 demonstrated it to be a polydispersed protein with a polydispersity index (PdI) of 0.528 ± 0.041 indicating protein aggregation. The size distribution graph Figure  shows muInitial attempts to refold E2-T1 in 50 mM phosphate buffer (pH 6.0) and PBS (pH 7.2) were unsuccessful, as the protein formed insoluble aggregates, the addition of Igepal CA630 to 0.2% failed to improve protein solubility in these buffers. However, E2-T1 protein remained soluble in 50 mM Tris (pH 8.0), 0.2% Igepal CA630 and 50 mM Tris (pH 7.0), although in the absence of Igepal CA630, E2-T1 showed a much higher level of polydispersity (PdI: 0.931 ± 0.075).Endotoxin assays measured by Limulus Amebocyte Lysate (LAL) assay revealed levels ranging from 5.29 to 199.31 EU/mL . Specific detection of E2 protein was detected at high titre (1:64000) with mouse serum Figure . Non-spein vivo applications.The advantages of IB in terms of biological activity and facilitating protein purification have been highlighted in recent publications . This study describes a procedure for the expression and solubilisation of a highly purified viral antigen derivative, E2-T1 from IB. The methodology includes the efficient removal of endotoxins to allow for potential In vivo refolding studies of purified E2 have shown that intramolecular disulphide bonds form rapidly within 2.5 minutes and intermolecular disulfide bonds result in formation of E2 dimers [E. coli cytosol [E. coli cytosol would lead to the formation of aggregates resulting in IB. In our study consistent and reproducible solubilisation of E2-T1 was only achievable using a highly reducing buffer containing 100 mM DTT. At lower DTT concentrations (20 to 5 mM), the percent solubility of E2-T1 decreased, indicating strong disulphide bond formation. The inclusion of DTT as a reducing agent in the re-suspension buffer would disrupt incorrectly formed disulphide bonds, and then during dialysis into DTT-free buffer, the disulphide bonds could reform in a conformation that is more suitable for soluble protein.2 dimers . Since d cytosol  and natiAlthough refolding of E2-T1 was successful in Tris based buffers, the resultant protein formed soluble aggregates as indicated by a PdI of 0.931. The soluble aggregation could be due in part to the same intermolecular disulphide bond formation reported for native E2. The addition of the detergent Igepal CA630 (0.2%) to Tris based buffers reduced aggregation (PdI 0.528), indicating some aggregation may be due to weaker intermolecular interactions such as ionic and/or hydrogen bonds. Under non-reducing electrophoresis conditions Figure , E2-T1 sE. coli derived proteins have been reported to be safe, causing no adverse reactions in animal trials [E. coli resulted in high endotoxin levels between 5.29 - 199.31 EU/mL. This was considered unsuitable for testing E2-T1 as a component of a veterinary vaccine due to the risk of adverse immunological reactions. Commercially available techniques for endotoxin removal typically employ affinity columns and require buffer changes for washing and elution of the protein. Attempts to use ion exchange chromatography were discontinued due to consistently low recovery of E2-T1.Endotoxin levels lower then 3 EU/mL of l trials ,27. HoweE. coli IB by adding Triton X-114 in the E2-T1 expression and purification protocol prior to solubilisation and recovery of the target protein. Phase separation with Triton X-114 resulted in up to 600 fold reduction in the endotoxin levels of the E2-T1 preparations  observed by Western hybridisation Figure  may be dThe results of this study demonstrate that the bacterially derived E2-T1 protein solubilised from IB can induce an excellent humoral response in small animals and could potentially be used as a subunit vaccine. The next step will be to determine if E2-T1 can elicite similar responses in large animals such as sheep or cattle when evaluated as a potential vaccine antigen.E. coli system and exploiting the formation of IB, we were able to produce a highly pure, stable and endotoxin-free E2-T1 protein. It was highly immunogenic in mice and was recognised by BVDV-E2 specific antibodies raised to native E2 in BVDV. We have developed a procedure for solubilising and refolding E2-T1 as well as the removal of endotoxin by Triton X-114 two-phase extraction which offers an elegant solution for the production of other proteins intended for diagnostic uses and in vivo uses such as subunit vaccines or therapeutics.The development of cost effective veterinary vaccines and diagnostics requires the use of efficient methods that enable the production of large quantities of immunogenic and non-toxic proteins. Using an E. coli strain DH10B (Invitrogen). Positive clones were confirmed by sequencing  and transformed into E. coli strain BL21  cells for protein expression.The E2 gene was amplified from a plasmid containing BVDV isolate MD74 which has been identified as a type-1 isolate . Twenty E. coli BL21 (DE3) containing pET-SUMO-E2-T1 was used to inoculate four 250 mL cultures of LB Miller broth  containing 50 mg/L Kanamycin-sulphate (Amresco). These cultures were grown at 37°C to an OD600 of 0.4 to 0.6, then induced with 1 mM IPTG and grown for a further 2 hours. The bacterial pellet was collected by centrifugation at 3,800 g, at 4°C for 15 minutes in 4 × 250 mL centrifuge tubes. Total protein was extracted by resuspending each bacterial pellet in 50 mL E. coli lysis buffer (50 mM KPO4 phosphate (pH 7.8), 400 mM NaCl, 100 mM KCl, 10% glycerol, 0.5% Triton X-100, 10 mM Imidazole), with the addition of 12.5 mg Lysozyme and 750 units of Benzonase nuclease . The bacterial suspensions were incubated in lysis buffer for 20 minutes with gentle shaking. The samples were frozen in liquid nitrogen, and thawed at 42°C three times. The resultant solution was centrifuged at 37,000 g at 4°C for 15 minutes. The insoluble protein fraction (containing IB aggregates) and the supernatant (containing the soluble protein fraction) were stored at -20°C until required.An overnight culture of E2-T1 protein was purified from the soluble fraction by affinity chromatography using TALON  resin following manufacturer's instruction. Bound protein was eluted from the resin using TALON elution buffer with a step gradient of 50 mM, 80 mM, 100 mM and 150 mM imidazole, collecting ten 500 μL fractions/step. Fractions were analysed by SDS-PAGE.g at 4°C for 15 minutes. Three further washes of 25 mL 1:10 diluted BugBuster™ were performed with vortexing and centrifugation steps as above. Following the final wash step, the pellets were resuspended in 1 mL 1:10 diluted BugBuster™, transferred into 1.5 mL tubes and centrifuged at 16,200 g at 4°C for 15 minutes. The supernatant was removed and the IB pellets were stored at -20°C without any detectable protein degradation.The insoluble protein fraction (including E2-T1) was recovered from the IB using BugBuster™ Master Mix (Novagen-Merck) as follows. IB pellets  were resuspended in 2.5 mL BugBuster™ and vortexed for 2 minutes. Following the addition of 15 mL of 1:10 diluted BugBuster™ and further vortexing for 1 minute, the resuspended IB pellets were centrifuged at 5000 g at room temperature for 7 seconds in a microfuge the 3 phases  were recovered into separate tubes and analysed by SDS-PAGE electrophoresis.All reagents were prepared in endotoxin-free water . Triton X-114 exhibits a cloud point at 22°C, above this temperature micelles aggregate forming a new phase with very low water content. Endotoxin remains in the detergent phase . PurifieTo determine the level of endotoxin in the protein samples, endotoxin assays were performed by using the Limulus Amebocyte Lysate (LAL) assay by AMS Laboratories .Protein pellets from the IB preparations were dissolved in 50 mM Tris (pH 6.8), 100 mM DTT, 1% SDS, 10% Glycerol, vortexed at low speed for 2 minutes and incubated at 37°C for 20 minutes. The resulting solubilised protein was dialysed at room temperature, with 3 buffer changes over 24 hours against 50 mM Tris (pH 7.0), 0.2% Igepal CA630 (Sigma-Aldrich). Following dialysis protein integrity was determined by SDS-PAGE analysis and protein yield determined by colourimetric assay . Other dialysis buffers evaluated in this study were: 50 mM Tris, (pH 7.0); 50 mM Tris (pH 8.0), 0.2% Igepal CA630; PBS (pH 7.2); PBS (pH 7.2), Igepal CA630; 50 mM phosphate buffer (pH 6.0).SureLock® Mini-Cell precast system with NuPAGE 10% BIS-Tris gels according to manufacturer instructions. Size estimations were determined against SeeBlue® Plus2 (Invitrogen) or Precision Plus Protein Kaleidoscope Standards (BioRad) pre-stained standards. The resolved proteins were visualised by staining in 50% methanol, 10% acetic acid, 0.25% Coomassie Blue R250 for 30 minutes, followed by destaining in 30% methanol, 10% acetic acid for 10 minutes three times.SDS-PAGE analysis was performed using Invitrogen's XCell Following SDS-PAGE electrophoresis the resolved polypeptides were transferred to Hybond C nitrocellulose membrane  using Invitrogen XCell II™ Blot Module Kit according to manufacturer's instructions. All antibodies were diluted in BLOTTO (PBS (Amresco), 0.1% Tween 20, 1% skim milk). E2 specific monoclonal antibodies D89 , mAb-157ad libitum. All studies were conducted with 8 week old mice at the time of first injection. All procedures were approved by The University of Queensland Ethics Committee.C57BL/6J mice were purchased from and housed in the Biological Resource Facility, The University of Queensland, Brisbane, Australia under specific pathogen free conditions. Female mice were housed in HEPA-filtered cages with 4 animals per cage in an environmentally controlled area with a cycle of 12 hours of light and 12 hours of darkness. Food and water were given Pre-immunisation blood samples of were collected by retro-orbital bleed using heparin coated hematocrit tubes . Pre-immunisation blood samples collected prior to the first immunisation were referred to as the preimmune samples. Blood samples collected 2 weeks after the third immunisation was referred to as terminal bleed.All doses of E2-T1 were prepared in sterile conditions in a certified biological safety cabinet using sterile reagents, equipment and aseptic technique. The adjuvant QuilA  was resuspended at 2 mg/mL in sterile injectable water .Dose volumes were adjusted to 500 μL using 0.9% saline (Pfizer). 50 μg E2-T1 together with 10 μg QuilA, were administered in a final volume of 100 μL subcutaneously at the tail base using a sterile 27 gauge needle . Three injections were administered at 2 week intervals and mice were sacrificed 14 days after the final immunisation. Animals were closely monitored throughout the study. All the animals remained in good health for the duration of the study with no visible deleterious health effects.ELISA assays for the detection of E2-T1-specific antibodies were performed by coating microtitre plates  with50 μL E2-T1 antigen solution (2 ng/μL in PBS) overnight at room temperature. Antigen solution was removed from the plates which were then washed once with 1× PBS-T  and blocked with 200 μL per well of PBS containing 5% BSA (Sigma-Aldrich), 5% skim milk  for 1 hour with gentle shaking at room temperature. Plates were washed three times in 200 μL 1× PBS-T as described above.Mouse sera samples were diluted from 1:100 to 1:6400 and 50 μL of diluted sera was added to plates then incubated for 2 hours at room temperature. To detect mouse antibodies HRP conjugated polyclonal sheep anti-mouse IgG antibodies (100 μL of 1:10000 dilution in PBS (pH 7.2), Chemicon Australia, Melbourne, Vic, Australia) was added per well and incubated for 1 hour at RT with gentle shaking. Plates were washed three times in 200 μL 1× PBS-T and 100 μL of TMB substrate (Sigma-Aldrich) was added to each well. After 15 minutes at RT 100 μL of 1 N HCl was added to each well to stop the chromogenic reaction. Plates were read at 450 nm within 10 minutes.g, 4°C) until the volume was reduced to the required amount. These columns were also used to perform buffer changes. After concentration of samples, the target buffer was replenished to the original volume.Protein concentration was increased by using 30 kDa Vivaspin 2 centrifugal filters (Millipore). Briefly, up to 2 mL of protein solution was added to the upper chamber and centrifuged (4000 Size and polydispersity data were determined by dynamic light scattering (DLS) using a Malvern Instruments Zetasizer Nano . E2-T1 solution (1 mL) was placed in a clear disposable zeta cell . Analysis was performed using the size standard operating procedure (SOP) for which the following parameters were use: material set to protein, dispersant set to water, taking 5 - 6 independent measurements of 30 runs each. The size and polydispersity index for samples were the average of all measurements. Analysis of the data was carried out by Malvern Zetasizer software.http://au.expasy.org/tools/protparam.html.Physical data was obtained using ProtParam, last accessed 3 March 2011 at The authors declare that they have no competing interests.ASC and DM carried out all experimental work excluding the Western hybridisations. MC performed the western hybridisations. TJM and NM supervised the study and participated in its design and coordination. ASC, DM, TJM and NM were all involved in the experimental design. All authors read and approved the final manuscript.


KRAS-mutant metastatic colorectal cancer patients participating in a phase II multicenter, open-label clinical trial receiving lenalidomide alone or lenalidomide plus cetuximab. The main findings show the T cell immunostimulatory properties of lenalidomide as the drug induced a decrease in the percentage CD45RA+ naïve T cells 3-fold while increasing the percentage HLA-DR+ activated T helper cells and percentage total CD45RO+ CD8+ memory T cytotoxic cells, 2.6- and 2.1-fold respectively (p<0.0001). In addition, lenalidomide decreased the percentage of circulating CD19+ B cells 2.6-fold (p<0.0001). Lenalidomide increased a modest, yet significant, 1.4-fold change in the percentage of circulating natural killer cells. Our findings indicate that lenalidomide significantly activates T cells, suggestive of an immunotherapeutic role for this drug in settings of maintenance therapy and tumor immunity. Furthermore, reported for the first time is the effect of lenalidomide in combination with cetuximab on T cell function, including increases in circulating naïve and central memory T cells. In summary, lenalidomide and cetuximab have significant effects on circulating immune cells in patients with colorectal carcinoma.This study assessed the immunomodulatory effects in previously treated ClinicalTrials.gov NCT01032291 The immunomodulatory drug lenalidomide is an orally active agent with significant activity in a range of hematologic disorders including myelodysplastic syndromes (MDS), multiple myeloma (MM), and non-Hodgkin's lymphoma (NHL).in vitro data indicate lenalidomide has activity in T cells, T regulatory cells (Tregs), B cells, monocytes, natural killer (NK) T cells, and NK cells.Lenalidomide has received US Food and Drug Administration approval for the treatment of MM in patients who have received at least one prior therapy, and for the treatment of transfusion-dependent anemia in patients with International Prognostic Scoring System-defined Low- or Intermediate-1-risk MDS with a del(5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. The molecular target of lenalidomide is the protein cereblon (CRBN). CRBN is a ubiquitously expressed protein and member of the cullin ring ligase 4 (CRL4) E3 ubiquitin ligase complex, and was identified as the primary teratogenic target of thalidomide in vitro+ helper and CD8+ cytotoxic T cells decreased, whereas memory CD4+ helper memory T cells and CD8+ cytotoxic memory cells increased In anti-CD3 stimulated T cells, lenalidomide stimulates T cell proliferation, and the production of interleukin (IL)-2, IL-12, and interferon gamma KRAS -mutant metastatic colorectal cancer patients, we assessed 25 different subpopulations of CD45+ immune cells . This was a phase II multicenter, open-label trial comprising a safety lead-in phase (phase IIa) to determine the maximum tolerated dose, and a randomized proof of concept phase (phase IIb) to determine the response rate of lenalidomide plus cetuximab combination therapy. Phase IIa treatment comprised oral lenalidomide (starting dose 25 mg/day) and intravenous cetuximab (400 mg/m2 followed by weekly 250 mg/m2) in 28-day cycles. In phase IIb patients were randomized to either the phase IIa treatment schedule of lenalidomide plus cetuximab combination therapy or lenalidomide 25 mg/day monotherapy. The combination of lenalidomide and cetuximab appeared to be well tolerated but did not have clinically meaningful activity in KRAS-mutant metastatic colorectal cancer patients (12).In a clinical trial aimed to assess the efficacy and safety of combination treatment with lenalidomide and cetuximab in This phase II, multicenter, open-label trial was conducted in accordance with the ethical principles of the Declaration of Helsinki and Good Clinical Practice, and according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. The study protocol, the proposed informed consent form, and other information to subjects, were approved by the Comitato Etico-Scientifico, Ospedale Niguarda Ca' Granda, Milan, Italy and properly constituted Institutional Review Boards/Independent Ethics Committees of all participating institutions . The study was registered at Clinicaltrials.gov with identifier NCT01032291. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see KRAS-mutant mCRC, and a proof of concept (POC) phase (phase IIb), in which the primary objective was to determine the response rate in these subjects per the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. A total of 48 of 50 subjects who received drug were evaluated for immune flow cytometric analysis.Written informed consent was obtained from all participants involved in the study. The study consisted of a safety lead-in phase (phase IIa), in which the primary objective was to determine the maximum tolerated dose of lenalidomide in combination with cetuximab in subjects with 3 of blood or as a percentage of CD45+ cells.Blood was collected at baseline (cycle 1 day 1 [C1D1]), cycle 2 day 1 (C2D1) and cycle 3 day 1 (C3D1) in either 5 ml Lavender/Black Cyto-Chex® BCT glass tubes  or 2.6 ml yellow top acid citrate dextrose tubes depending upon where the sample was received and maintained at ambient temperature until flow cytometry was performed at ICON Central Laboratories  within 72 hours of sample collection. Stability analyses were conducted during assay validation and supports up to 72 hours processing time from sample collection to analysis. Antibodies  and 100 µl of patient whole blood were transferred to 12×75 mm test tubes  followed by gentle vortexing and 15 min incubation at room temperature. To this, BD Pharm Lyse™ lysing solution (BD Biosciences) was added for 10 min. The tube was centrifuged, washed in PBA (Dulbecco's PBS/0.2%) and the pellet was resuspended in PBA prior to analysis of B, T, and NK cell subsets on a FACSCanto™ II flow cytometer (BD Biosciences). For intracellular protein staining, an additional permeabilization step was added after the first addition of PBA with fixation-permeabilization working solution , followed by two washes in permeabilization buffer , incubation with intracellular antibodies, and another permeabilization buffer wash. Appropriate compensation controls were included during each analysis. Each cell subset population was reported as an absolute value per 1 mmTo assess the changes in different immune cell populations at C2D1 and C3D1 versus baseline, each of the 50 flow cytometry measurements (i.e. one absolute cell count and one percentage measurement for each of the 25 subpopulations of CD45+ immune cells) were first log2 transformed and a linear model was then fitted with cycle number  as an 3-level independent variable and patient as a blocking variable using the R software package LIMMA A total of 48 of 50 subjects who received study drug were evaluable for peripheral blood immune cell analysis. The study schema is shown in The major findings include four immune cell populations with highly significant (p ≤ 0.0001) changes from C1D1 (baseline) to C2D1 and to C3D1 in both the absolute number and the percentage of cells measured in both treatment arms. The changes in these populations are shown in in vitro data showing lenalidomide inhibits Tregs expansion In the lenalidomide only arm (n = 20), 12 T cell populations, total NK cells, total B cells, and total lymphocyte cell populations (either percentage or absolute count) were significantly changed (p≤0.05) in either C2D1 or C3D1 versus C1D1, or both. These T cell populations, starting with the most significant, include total naïve T helper cells, activated T helper cells, activated T cytotoxic cells, T regulatory cells, total naïve T cytotoxic cells, effector T helper cells, effector T cytotoxic cells, total memory T cytotoxic cells, effector memory T cytotoxic cells, T helper cells, T cytotoxic cells, and total T cells. Absolute and percentage B cells decreased 1.78- to 3.41-fold. The percentage of NK cells significantly increased at C2D1 by 1.4-fold across subjects taking lenalidomide only. The absolute lymphocytes significantly decreased 1.36-fold across subjects taking lenalidomide only . Of note+ NK cells significantly increased at C2D1 by 1.15-fold and at C3D1 by 1.25-fold in subjects taking lenalidomide plus cetuximab. The percentage of lymphocytes significantly increased 1.11- to 1.45-fold in subjects taking lenalidomide plus cetuximab , 15 T cell populations, 1 NK cell population, total B cells, and total lymphocyte cell populations (either percentage or absolute count) were significantly changed (p ≤ 0.05) in either C2D1 or C3D1 versus C1D1, or both. These T cell populations, starting with the most significant, include activated T helper cells, total memory T cytotoxic cells, total naïve T helper cells, total naïve T cytotoxic cells, effector memory T cytotoxic cells, activated T cytotoxic cells, effector T cytotoxic cells, central memory T cytotoxic cells, effector T helper cells, effector memory T helper cells, total memory T helper cells, central memory T helper cells, naïve T cytotoxic cells, T cytotoxic cells, and naïve T helper cells. Absolute and percentage B cells decreased 2.01- to 3.6-fold. The percentage of granzyme Betuximab . Of thes+ NK cells significantly increased 1.21-fold at C3D1, whereas the percentage of lymphocytes increased 1.33-fold at C3D1 across all subjects  were significantly modulated (p ≤ 0.05) in either C2D1 or C3D1 versus C1D1, or both. These T cell populations, starting with the most significant, include activated T helper cells, total naïve T helper cells, total memory T cytotoxic cells, total naïve T cytotoxic cells, activated T cytotoxic cells, effector memory T cytotoxic cells, effector T helper cells, effector T cytotoxic cells, central memory T cytotoxic cells, effector memory T helper cells, T regulatory cells, central memory T helper cells, naïve T cytotoxic cells, total memory T helper cells, T cytotoxic cells, and naïve T helper cells. Absolute and percentage B cells decreased between 2.35- and 3.05-fold. The percentage of granzyme Bsubjects .Overall, 19 of the 48 patients were identified as being on either daily or intermittent, concomitant systemic or topical corticosteroids. Of these 19 patients on concomitant corticosteroids for various periods of time, 5 received dexamethasone (ranging from 8 mg daily to 10 mg weekly), 10 received prednisone (ranging from 25 mg twice-daily to 4 mg daily), 5 received betamethasone (ranging from 4 mg daily to 4 mg as needed), and 5 received ultrapotent topical corticosteroids. To rule out any possible effect of these immunosuppressive corticosteroid therapies on the expression of any of the cell populations, a sensitivity analysis was performed excluding the patients on concomitant immunosuppressive medications and the analysis was reperformed. In the sensitivity analyses, the same four cell populations consistently showed significant (p ≤ 0.0001) changes from C1D1 (baseline) to C2D1 or C3D1 in both measurements assessed (absolute number and percentage) signifying these effects are not due to steroid usage. In fact, patients on immunosuppressive concomitant medications had similar magnitude of immune effects (T cell activation and B cell inhibition), and a greater number of immune cell subsets modulated compared with patients not on concomitant therapies. In addition, an analysis of immune changes in concomitant steroid users versus nonusers was performed and the results were comparable (data not shown).As reported by Siena et al. KRAS -mutant metastatic colorectal cancer patients, we assessed 25 different subpopulations of CD45+ immune cells  in this patient population that does not have confounding hematological abnormalities. The major findings are significant (p ≤ 0.0001) changes from baseline to C2D1 and to C3D1 in the following four lymphocyte populations: B cells (decreased), activated T helper cells (increased), total memory T cytotoxic cells (increased), and total naïve T helper cells (decreased). One potential clinical relevance of these findings implicate lenalidomide is an activator of T cells which may play a role in its anti-tumor activity. In addition, the downregulation of B cell counts may be linked to its activity in B cell malignancies. These effects were detected in both treatment arms. As all subjects were treated with lenalidomide, these changes may be ascribed to the pharmacodynamic effects of lenalidomide. The correlative analyses showed that changes in the cell populations (absolute number or percentage) tested did not correlate with treatment arm, response per RECIST version 1.1 criteria, or OS. The clinical responses from this study are reported in detail by Siena et al. Lenalidomide is approved for use in both MM and MDS, and has reported clinical activity in chronic lymphocytic leukemia (CLL) and NHL Furthermore, lenalidomide plus cetuximab had remarkable effects (up to 60-fold increases) in central memory and naïve T cells suggestive that cetuximab itself may play a role in T cell activation and function. Similar effects were reported by Botta et al. + peripheral blood B cells in patients with MM receiving alternate day dosing of 1–10 mg Patients with mCRC administered with the B cell depleting agent rituximab have shown both regression of metastases and achievement of SD + and CD8+ T cells in patients with MM Preclinical and clinical data indicate that lenalidomide activates T cells. Bartlett et al. In vitro, lenalidomide inhibits Tregs; however, in this study an upward trend (p<0.05) in both absolute number and percentage of circulating Tregs in peripheral blood was observed at both C2D1 and C3D1 in subjects taking lenalidomide alone. Similarly, the level of circulating Tregs was increased in patients with MM within 2 weeks of receiving lenalidomide + T cells (both CD4+ and CD8+ subsets). The increase in CD4+FOX-P3+ regulatory T cells was observed after 9 cycles (9 months) of lenalidomide maintenance therapy. Therefore, the increase in regulatory T cells may be a late response to increased effector T cell numbers and activity.in vitro by both increased NK cell cytokine production as well as in functional studies. In a peripheral blood mononuclear cell/tumor cell co-culture model, T cell co-stimulation leads to enhanced NK cell-mediated lysis of various tumor cells, including prostate and ovarian cancer cells in vitro, including NHL and B-CLL + and NKD+) after 28-days of dosing versus baseline. These data do not preclude the possibility that lenalidomide increased NK cells and ADCC at the site of the tumor. Our data previously indicated that dexamethasone antagonizes the stimulatory capacity of lenalidomide on both NK and T cells in vitro+ T cells + cells analyzed in the present study, sensitivity analyses were performed to exclude data for subjects who had used a systemic corticosteroid or a potent topical corticosteroid during the study. The results of the sensitivity analyses were consistent with those of the main analysis, indicating that the inhibition of B cells is a true pharmacodynamic effect of lenalidomide and this effect is not confounded by intermittent concomitantly administered immunosuppressive therapies. The doses and frequency of dexamethasone taken in this study were insufficient to antagonize the T cell effects of lenalidomide, consistent with previous findings that low-dose dexamethasone allows for greater T cell activation by lenalidomide.The activation of NK cells by lenalidomide has been shown KRAS-mutant mCRC to EGFR targeted inhibition by cetuximab. Importantly, these data illustrate the immunomodulatory effects of lenalidomide which helps in understanding the drug's mechanism and potential applicability to other tumor types.In conclusion, despite preclinical evidence, the present clinical data from Siena et al (12) suggest the modulating effect of lenalidomide is unable to overcome primary resistance of Figure S1th percentile whereas the lower edge denotes the 25th percentile. The line inside each box is the median. The lines extend to the maximum and minimum values excluding outliers. The gray lines denote individual subject data. Abbreviation: Abs: absolute.Changes in percentage or absolute number in the remaining 21 immune cell subsets from cycle 1 day 1 (C1D1) to cycle 2 day 1 (C2D1) or cycle 3 day 1 (C3D1) in all subjects. The upper edge of the box denotes the 75(DOC)Click here for additional data file.Table S1a1 patient was enrolled but did not receive study drug. Abbreviation: Len: lenalidomide.Biomarker and correlative analyses performed and number of subjects included in each analysis. (DOC)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(PDF)Click here for additional data file.


The structures were elucidated by spectroscopic means (NMR and MS) and by comparison with the literature. Compound 1 was composed of a glycerol moiety linked to oleic acid (C18:1 Ω9). Compounds 2 and 3 contained a glycerol moiety linked to a galactose unit and eicosapentaenoic acid (C20:5 Ω3) combined with octadecatetraenoic acid (C18:4 Ω3) or linolenic acid (C18:3 Ω3), respectively. The isolated compounds were tested for their cytotoxic and anti-inflammatory activity in RAW 264.7 macrophage cells. All of them inhibited NO production at non-cytotoxic concentrations. The fraction consisting of compounds 2 and 3, in a ratio of 1:1, was slightly more effective than compound 1 . To our knowledge, this is the first report of these compounds from F. spiralis and on their anti-inflammatory capacity.A monoacylglycerol ( Glyceroglycolipids are the most widespread lipids in nature. These amphiphatic compounds constitute a structurally heterogeneous group with functional roles in several biological systems . They arInflammation is a pathological condition in which highly reactive species are produced. Nitric oxide (NO) is a small diffusible molecule responsible for vasodilatation, neurotransmission and inflammation. This molecule is produced by the organism at a basal concentration. Nevertheless, under stimulation by pathogens, NO is generated in higher amounts by the inducible nitric oxide synthase (iNOS) in activated macrophages. The overproduction of NO is involved in the pathogenesis of septic shock, tissue damage, multiple organ dysfunctions and carcinogenesis processes . The antFucus spiralis Linnaeus is a widely distributed species over the Portuguese west coast. This edible seaweed is rich in secondary metabolites, such as phlorotannins, sterols and fatty acids, which are associated with several biological activities, namely antioxidant, anti-allergic, antimicrobial, anti-ageing and anti-diabetic activities + (100), 735 [2M + Na]+ (5), in agreement with data reported in the literature + (10), 797 [M + H]+ (100), 635 [M + H − C6H10O5]+ (24), 617 [M + H − C6H12O6]+ (1), 333 [C17H27CO + 74]+ (3), 285 [C19H29CO]+ (0.5), in agreement with data reported in the literature + (9), 799 [M + H]+ (100), 637 [M + H − C6H10O5]+ (23), 619 [M + H − C6H12O6]+ (1), 335 [C17H29CO + 74]+ (2), 285 [C19H29CO]+ (0.6), in agreement with data reported in the literature  [®). Control values were obtained in the absence of the isolated compounds and after addition of LPS.Macrophage cells release NO into the culture medium, which is converted into different nitrogen derivatives. Of them, only nitrite is stable, and for this reason, easy to measure as an indicator of NO production. After the incubation period with the test compounds  (version 5.2 for Windows). One-way analysis of variance (ANOVA), using the Dunnet Multiple Comparison test was carried out on data obtained from four independent assays performed in duplicate for each sample. Levels of statistical significance at F. spiralis was investigated. Two MGDGs and one monoacylglycerol were isolated and characterized for the first time in this species. The anti-inflammatory capacity of the isolated compounds was also described herein for the first time. The glyceroglycolipids isolated from F. spiralis are mainly composed of polyunsaturated fatty acids. These compounds display anti-inflammatory activity by inhibiting NO release by macrophages.In the present work, the glyceroglycolipids composition of A combination of our study with previous investigations involving similar naturally occurring glyceroglycolipids ,9,11 hig