The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes. in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA.Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes. Persistent infection with human cytomegalovirus (HCMV) is widely prevalent in the human population and mostly asymptomatic in immunocompetent individuals . Howeverin vitro and in vivo, require the genetic manipulation of CMV genomes. However, the large size of CMV genomes (>230 kb) renders genetics challenging. Initially, recombinant CMV was generated by marker rescue . Particularly useful for direct cloning of DNA from a given sample is transformation-associated recombination (TAR) cloning in which linear DNA containing a yeast plasmid flanked by sequences homologous to a target sequence is transformed together with the target sequence . Th. Th11). in vivo , this fiin vivo . Interesin vivo . Thus, tGU937742.2). However, a few single nucleotide polymorphisms (SNPs) were identified, most of them synonymous mutations (see GU937742.2) based on our sequencing results. Individual SNPs were allocated to either one or both Toledo genomes based on the following criteria. If the variation was homogeneous, it was applied to both genomes. If the variation was heterogenous, the major allele was applied to the fibroblast-adapted genome, while the minor allele was applied to the parental genome. Usually, for heterogeneous variations, one of the alleles would match the allele in the reference genome, such that “applying” it would be to take no action to the derivative genome at that locus. Alignment of the full-length sequences of Toledo-F and Toledo-P revealed that, in addition to the inversion of the UL128-UL133 fragment which is flanked by two small deletions, Toledo-F had a 279-bp in-frame deletion in the RL13 gene, as previously reported  and the majority sequence in P7 HCMV Toledo (P7 Toledo-F). (A) Genome alignment of the NGS results obtained by sequencing P7 HCMV Toledo with HCMV Toledo (GenBank accession number GU937742.2). The green bar at the top indicates the percentage of nucleotide identity between both virus sequences with green being 100% identical and yellow being less than 100% identity. The gray bars indicate sections of agreement, the black lines indicate differences, and the black bars indicate deletions and insertions. The green arrows indicate the HCMV ORFs. (B) List of nucleotide differences between the published sequence and P7 Toledo-F. The SNP in the homopolymeric sequences is possibly a false-positive result. Download FIG S1, PDF file, 0.2 MB.Sequence comparison between HCMV Toledo  TAR cloning and analysis of HCMV fragment 4 as an example. (Left) Generation of linear TAR cloning vector. To clone HCMV fragment 4, a linear TAR cloning vector was amplified by PCR using pCC1BAC-his3 as the template and Con04F and Con04R  Depiction of cloning TAR fragments. A linear TAR cloning vector containing YCp and BAC sequences with 40 bp of homology to the desired fragment of HCMV  and I-SceI restriction sites was cotransformed with sheared P7 HCMV DNA into yeast cells. Yeast transformants were screened by PCR, and positive clone DNA was transferred to d Con04R  as primed Con04R  for the d Con04R  for the Copyright © 2017 Vashee et al.2017Vashee et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphereDirect.00331-17.3TABLE S1 TABLE S1, DOCX file, 0.02 MB.Primers used in this study. Download Copyright © 2017 Vashee et al.2017Vashee et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphereDirect.00331-17.4TABLE S2 TABLE S2, DOCX file, 0.02 MB.Design of HCMV fragments. Download Copyright © 2017 Vashee et al.2017Vashee et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To assemble the Toledo-P genome also, we took advantage of the ability to simultaneously change the sequence of each of the 16 fragments either by mutagenesis or by gene synthesis. The major differences between Toledo-P and Toledo-F are located on three fragments: fragment TAR01 contains the RL13 sequence, fragment TAR07 contains open reading frames (ORFs) 59 and 60, and fragment TAR13 contains the UL128-UL133 (UL128-133) region. There were also four SNP differences between Toledo-P and Toledo-F . Howeverin vitro Cas9 digestion followed by assembly with a PCR fragment containing the Toledo-P sequence (E. coli.TAR01 and TAR07 corresponding to the Toledo-P sequence were generated by sequence . TAR13 wE. coli were not as robust as those of Toledo-F. Only 3% of the yeast Toledo-P clones were correct (compared to 15% for Toledo-F), and the efficiency of Toledo-P full-length genome transfer to E. coli was 10% . The reasons for this relative inefficiency for Toledo-P are not yet understood. The resulting full-length YCpBAC containing Toledo-P genome was analyzed by next-generation sequencing (NGS) (The resulting TAR plasmids were sequenced to confirm that the respective Toledo-P fragments had been generated (data not shown). The full-length Toledo-P genome was then generated from two half genomes in which Toledo-P fragments TAR01, TAR07, and TAR13 replaced the corresponding Toledo-F fragments. The assembly of the full-length Toledo-P genome in yeast and transfer to ng (NGS)  and C. Ein vitro and in vivo characterization. At the same time, viral DNA was extracted by the Hirt method and sequenced by NGS. Comparison of the Toledo-P virus with the YCpBAC sequence revealed frameshift mutations in RL13 amino acid residue 193 and in UL128 amino acid residue 154  and AMD3100  was administered intraperitoneally to mobilize human monocytes/macrophages. At 1 week after mobilization, the mice were sacrificed. Total DNA was extracted from the spleens and livers, and HCMV genomes were analyzed using quantitative PCR. When comparing G-CSF-treated to untreated groups, we were able to detect quantitative differences in viral DNA loads in the spleen and liver for both HCMV TR and Toledo-P  in this model, whereas Toledo-F resembles laboratory-adapted strains like AD 169.We previously reported a humanized mouse model in which primary isolates of HCMV are able to establish latency and can be reactivated upon mobilization of myeloid cells from the bone marrow –30. In c–Toledo-P . In contin vitro. Thus, this method potentially enables the direct cloning of CMV from clinical samples, e.g., from viral DNA isolated from congenitally infected infants, without the need for tissue culture. The “TAR hooks” used to generate the 16 genome fragments were designed to represent highly conserved genome sequences so that most HCMV genomes can be cloned by this method. However, in case of sequence mismatches, TAR hooks can be easily modified according to sequencing results obtained from the same DNA sample. This cloning method can also be applied to new DNA virus isolates for which in vitro culture conditions have not been established  strains or species to facilitate mapping of strain- or species-specific traits. The fact that native viral DNA that does not contain residual bacterial sequences is excised from the YCpBAC prior to transfection will additionally facilitate the recovery of live virus without the need of further genome manipulations such as Cre-based excision of the BAC cassette.As a test case for direct cloning, we used the low-passage-number isolate Toledo, a storied virus derived from a congenitally infected boy in Toledo, Ohio. Passage four of HCMV Toledo was used in human challenge studies, and these clinical trials remain the only examples for experimental infection of humans with low-passage-number HCMV . It was in vivo is supported by our experiments in humanized mice. Toledo-F was barely able to establish latency in this model, and we did not observe an increase in the viral genome copy number upon mobilization of monocytes/macrophages, an indicator of reactivation and dissemination. In contrast, similar to other primary isolates, Toledo-P established robust latency and reactivation upon monocyte/macrophage mobilization. This ability correlates to some extent with the broader cell tropism displayed by pentamer-intact HCMV. However, it is also possible that the inversion of the ULb’ fragment affects gene expression within the UL133-138 gene locus which has been implicated in viral latency as well as infection of endothelial cells  were purchased from Lonza (cc-2519) and cultured in endothelial growth medium  containing 2% FBS, human recombinant vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), human epidermal growth factor (hEGF), insulin-like growth factor 1 (IGF-1), hydrocortisone, ascorbic acid, heparin, gentamicin, and amphotericin B (1 μg/ml each) (high-serum medium). HCMV isolate TR was derived originally from a patient with AIDS-related HCMV retinitis  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM etinitis . HCMV Toescribed .Plasmids pCC1BAC-ura3 and pCC1BAC-his3 have been previously described .Taq 5× Master Mix (New England BioLabs [NEB]) using the following primers (indicated in the parentheses) for the regions and diagnostic PCR indicated: UL127 (5′-ATGTGCCAGCTTGATGTCGC-3′) and UL130 (5′-CGCCAAGATTTTTGGAGCGCAC-3′) (PCR-1); UL127 (5′-ATGTGCCAGCTTGATGTCGC-3′) and UL133 (5′-GGTTGTGAACTCACCGTCGG-3′) (PCR-2); UL133 (5′-GGTTGTGAACTCACCGTCGG-3′) and UL148 (5′-CGAGGCAGAACATCTCAACC-3′) (PCR-3); UL128 (5′-GAGGGCCTTACAGCCTATGG-3′) and UL148 (5′-CGAGGCAGAACATCTCAACC-3′) (PCR-4). The thermocycler parameters were 95°C for 2 min, followed by 30 cycles, with 1 cycle consisting of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min.Supernatant from fibroblasts infected with Toledo isolate passage 7 (P7) was used to extract viral DNA using the QIAamp MinElute virus spin kit (Qiagen). HCMV TR DNA was extracted with the same kit as a positive control. Two microliters of viral DNA was amplified with GU937742.2) using CLC Bio’s (Qiagen) clc_ref_assemble_long program, and variations were then extracted and reported using CLC Bio’s find_variations program. Reported variations were manually inspected using CLC Bio’s assembly_viewer program, and variations lacking sufficient evidence  were discarded. In some cases, large structural variations were analyzed and reported manually, for example, a large inversion with indels at the termini that occurred at low frequency had to be determined by bespoke bioinformatic methods to partition sequencing reads based on the presence or absence of junction sequences, and then reassembling the partitions of reads separately and mapping contigs back to the reference sequence.Viral DNA was isolated from the supernatant of fibroblasts infected with P7 Toledo as described above, and 210 μl of isolated DNA was incubated with 30 μl of shrimp DNase to remove host DNA (2 U/μl) (Affymetrix) for 1 h at room temperature. Then, the shrimp DNase was inactivated by incubating at 70°C for 20 min. A 10-μl sample was removed for quantitative PCR (qPCR) analysis. Viral DNA was then purified from the larger remaining sample with the QIAamp MinElute virus spin kit (Qiagen) and eluted in 50 μl of buffer. qPCR was used to determine the removal of host DNA. Viral DNA was then used for sequencing using Illumina HiSeq paired-end sequencing. The sequence reads were sorted by barcode, trimmed, and mapped to HCMV Toledo (GenBank accession number GU937742.2) available from NCBI and to each other for identification of insertions, deletions, and single nucleotide polymorphisms (SNPs).The P7 Toledo-F, P7 Toledo-P, Assembled Toledo-F, Reconstituted Toledo-F, Assembled Toledo-P, and Reconstituted Toledo-P were aligned in Geneious  using the PhyML package to the reference strain  was used for all yeast transformations and grown in YPD  medium supplemented with adenine. S. cerevisiae spheroplasts were prepared using previously described methods with the following modifications (600) of 1.8 to 2.5 and kept overnight at 4°C in 1 M sorbitol. After the cells were harvested, they were resuspended in 10 ml of SPE solution  with 20 μl of β-mercaptoethanol and 20 μl of zymolyase solution. The cells were finally resuspended in 4 ml of STC solution . DNA for transformation-associated recombination (TAR) cloning or assembly was added to 200 μl of spheroplasts, followed by the addition of 900 μl of 20% PEG solution . The suspension was incubated at room temperature for 20 min, the PEG solution was removed, and the cells were then incubated at 30°C for 30 min in SOS medium . Transformed yeast spheroplasts were plated with selective top agar with sorbitol.The  VL6-48N   prior to transformation.Vectors were first PCR amplified using pCC1BAC-his3 as the template with Phusion (NEB) or Q5 (NEB) DNA polymerase. This plasmid contains bacterial artificial chromosome (BAC) and yeast centromeric plasmid (YCp) sequences for replication in Viral DNA, which was isolated from Toledo P7 as described above, was sheared by pipetting. Six hundred nanograms of viral genomic DNA was cotransformed with 20 ng of the appropriate vector into yeast spheroplasts. Transformants were patched on synthetic dropout medium without histidine and supplemented with adenine (SD-HIS) plates and after sufficient growth were selected and transferred into 20 μl of 25 mM NaOH and incubated at 95°C for 30 min to lyse yeast cells. PCR on DNA from lysed yeast using the appropriate detection primers (see E. coli EPI300 (Epicentre) or DH10B (Thermo Fisher) competent cells. DNA was purified from positive transformants for restriction enzyme analysis and sequencing.Positive TAR clone candidates from yeast patches or 3 ml of liquid cultures were resuspended in 500 μl of water containing 5 μl of zymolyase 20T (MP Bio) (10 mg/ml) and 0.5 μl of β-mercaptoethanol (14.2 M) and incubated at 37°C for 1 h. Fifty microliters of 2% SDS was added, and the solution was incubated for 15 min at 70°C, followed by the addition of 50 μl of 5 M potassium acetate and incubation on ice for 5 min. After clarification, the DNA in the supernatant was precipitated with isopropanol and resuspended in 50 μl of Tris-EDTA (TE) buffer. Isolated DNA was then electroporated into E. coli were grown up, and DNA was isolated with PureLink HiPure Plasmid Midiprep kit (Thermo Fisher). Prior to assembly, the clones were digested with I-SceI and heat killed to release the HCMV fragments. Assembly of HCMV genomes was performed by TAR assembly in yeast in two steps. First, half genomes (TAR01 to TAR08 and TAR09 to TAR16) were assembled as follows. Vector DNA was amplified by PCR using pCCIBAC-ura3 as the template and either Con01R (R stands for reverse)and Con08F (F stands for forward) or CMV_1R and CMV_8F  harboring the pJExpress404-FLAG-Cas9-His construct was cultured overnight in 50 ml LB supplemented with 100 μg/ml carbenicillin (Teknova). The starter culture was diluted into 1 liter of antibiotic-supplemented LB broth and shaken at 220 rpm at 37°C to an OD600 of 0.6. The culture was then cooled to 20°C, and isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce protein expression. Cas9 protein was induced overnight in a refrigerated incubator at 20°C under shaking (220 rpm). Cell pellets were washed once with phosphate-buffered saline (PBS), pH 7.4, and then resuspended in 60-ml volumes of xTractor buffer supplemented with EDTA-free protease inhibitor (Clontech), 2 mg lysozyme (Sigma-Aldrich), and 625 U Benzonase (Millipore). Cell suspensions were then gently rotated for 2 h at 4°C prior to mechanical lysis in a Microfluidizer M-110L (Microfluidics). Cell debris was pelleted at 8,000 × g for 1 h, and recombinant protein was purified from the supernatant using a HisTalon column (Clontech) according to the manufacturer’s instructions. Purified protein was stored at −80°C in Cas9 buffer  at a concentration of 4.5 mg/ml. The Cas9-targeting chimeric synthetic guide RNAs (sgRNAs) were designed, in vitro synthesized, and purified as described previously (A single colony of eviously .E. coli-yeast shuttle pCC1BAC_HIS3 were purified using the QIAprep Spin Miniprep kit (Qiagen). Plasmid-borne genomic fragments were then independently digested with Cas9 in conjunction with two sgRNAs , liberating DNA fragments of roughly 350 bp and 70 bp, respectively. One-hour digestions were performed at 37°C in 50-μl volume and contained 500 ng of plasmid DNA, 1 μg each gRNA, 10 mM spermidine (Sigma-Aldrich), 10 mM MgCl2 (Ambion), 20 mM HEPES, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA, and 3.6 μg of Cas9 protein. Following digestion, Cas9 was heat inactivated at 80°C for 10 min, and digested DNA was extracted with UltraPure phenol-chloroform-isoamyl alcohol , precipitated with ethanol, and resuspended in sterile water. DNA fragments used to generate Toledo-P insert fragments 1 and 7 were obtained from de novo-synthesized gblocks (IDT). The Toledo-P fragment 1 insert encoded a 279-bp insertion not found in the Toledo-F sequence. Conversely, the Toledo-P fragment 7 insert removed a 52-bp insert found only in Toledo-F. Both DNA inserts contained 20- to 100-bp overlap sequences homologous to regions upstream and downstream of the Cas9 digestion sites to facilitate in vitro assembly. Synthesized insert fragments were mixed with corresponding digested products at a 3:1 molar ratio (insert-vector) and covalently joined via standard Gibson Assembly reaction (SGI-DNA). Following assembly, constructs were transformed into E. coli TransforMax EPI300 electrocompetent cells (Epicentre) and plated on selective media. Transformants were verified to contain new inserts using PCR and Sanger sequencing prior to final confirmation of the full Toledo-P genome sequence by next-generation sequencing (NGS).Previously assembled HCMV Toledo-F fragments TAR01 and TAR07 contained within the E. coli-yeast shuttle pCC1BAC_HIS3 vector. In a second PCR, the 14.3-kb inverted sequence was amplified with Ultramer oligonucleotides 5′-CTCTCCAGGTACTGATCCAGGCCCACGATCCGGGTTATCTTGTCGTATTCCAGGTTGATCCATCGATAGGGAACGCTGCCAGCGGCGCCCAGCAGGTACTGCGCCTTGTCGTTCACTTTGCCGCAGCGTATTCGCCCGTCAGCTTCGAGGTATAACCTACAACACGGAGGGGAAGGGGGGTACAAAACGTGAAATTAGAC-3′ and 5′-GAGACGACGCCGCTGGTAGAGGATGCCGAACCGCCGGCCGAGCTGGAGATGGTGGAAGTGTCGGACGAGTGTTACTAGGAGATCGCCGCGGCCGATGGGCGCCGGCGGACGTGACTCGGCAGCCGCTGTAGGGATAAATAGTGCGATGGCGTTTGTGG-3′, generating an inverted sequence with the indicated terminal inserts flanked by 121-bp overlap sequences homologous to regions at the ends of the first amplicon. Both PCRs were performed with the KOD Xtreme Hot Start DNA polymerase (Millipore) using the Toledo-F genomic fragment 13 as the template and according to the manufacturer’s specifications. The amplicons were digested with DpnI (NEB) and purified using the QIAquick PCR purification kit (Qiagen). Following purification, amplicons were mixed at an equal molar ratio and covalently joined via standard Gibson Assembly reaction (SGI-DNA). Following assembly, constructs were transformed into E. coli TransforMax EPI300 electrocompetent cells (Epicentre) and plated on selective media. Transformants were verified to contain new inserts using PCR and Sanger sequencing prior to final confirmation of the full Toledo-P genome sequence by NGS.NGS sequencing data indicated that the Toledo-P genome contained a large 14.3-kb genomic inversion flanked by two small insertions (14 and 23 bp). This large inversion was entirely contained within the previously generated Toledo-F genomic fragment 13. Utilizing primers 5′-GCAAAGTGAACGACAAGGCGCAGTACCTGCTG-3′ and 5′-CCTAGTAACACTCGTCCGACACTTCCACCATCTCCAGC-3′ (IDT), the ends of genomic fragment 13 backbone were PCR amplified together with the scid IL2Rγcnull (NSG) mice were maintained at a pathogen-free facility at Oregon Health & Science University in accordance with procedures approved by the Institutional Animal Care and Use Committee. Both sexes of animals were used. Humanized mice were generated as previously described   using a subcutaneous micro-osmotic pump  and 125 μg AMD3100 bis-1,4,8,11-tetra-azacyclotetradecane octahydrochloride or plerixafor] administered intraperitoneally to mobilize hematopoietic progenitor cells (HPCs). The remaining mice serve as a direct comparison for the effects of virus reactivation and dissemination that follow HPC mobilization. One week after HPC mobilization, the mice were sacrificed, their organs were harvested, and samples for PCR were frozen in RNAlater solution and stored at −80°C for subsequent analysis.All animal studies were carried out in strict accordance with the recommendations of the American Association for Accreditation of Laboratory Animal Care (AAALAC). The protocol was approved by the Institutional Animal Care and Use Committee (number IS00003498) at the Oregon Health & Science University. NOD-escribed . The ani2 sections of mouse spleen or liver using the DNAzol kit (Life Technologies). HCMV genomes were analyzed using quantitative PCR (TaqMan) performed on 1 µg of total DNA and using TaqMan FastAdvance PCR master mix (Applied Biosystems) according to the manufacturer’s instructions. Primers and a probe recognizing HCMV UL141 were used to quantify HCMV genomes . The probe contains a 5′ FAM (6-carboxyfluorescein) reporter molecule and a 3′ quencher molecule (Applied Biosystems). The reaction was initiated using TaqMan Fast Advanced master mix (Applied Biosystems) activated at 95°C for 10 min followed by 40 cycles (1 cycle consists of 15 s at 95°C and 1 min at 60°C) using a StepOnePlus TaqMan thermocycler. Results were analyzed using ABI StepOne software. Data were analyzed using the statistical program GraphPad Prism 5. Statistical significance was determined using two-way analysis of variance, followed by Bonferroni’s posttest correction. A P value of <0.05 or lower was considered significant.Total DNA was extracted from approximately 1-mmMF783090 to MF783093.The genome sequences of P7 Toledo-F, P7 Toledo-P as well as assembled Toledo-F and Toledo-P have been submitted to GenBank under accession numbers


Spib, Ccl9 [chemokine (C-C motif) ligand 9], Tnfaip2 (TNF-α-induced protein 2), Anxa5 (annexin A5), and Marcksl1  in 1 day. The mature M cell marker glycoprotein 2 (Gp2) was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-κB pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the mitogen-activated protein kinase kinase kinase 14 (Map3k14) gene encoding NF-κB-inducing kinase failed to induce M cell-associated genes. While the cytokine TNF-α alone had little, if any, effect on expression of M cell-associated genes, addition of TNF-α to RANKL consistently resulted in three- to sixfold higher levels of multiple M cell-associated genes than RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2 (NF-κB subunit 2), genes encoding the two subunits of the noncanonical NF-κB heterodimer. We conclude that endogenous activators of canonical NF-κB signaling present in the gut-associated lymphoid tissue microenvironment, including TNF-α, can play a supportive role in the RANKL-dependent differentiation of M cells in the follicle-associated epithelium.Microfold (M) cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium of Peyer's patches that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine receptor activator of NF-κB ligand (RANKL) and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. Addition of RANKL to enteroids induced expression of multiple M cell-associated genes, including  M cells are responsible for the highly efficient uptake of particulate antigens into gut-associated lymphoid tissue structures, such as PPs and isolated lymphoid follicles, that serve as inductive sites for mucosal immunity in the intestine  and Anxa5 (annexin A5) by M cells is independent of Spi-B (Tnfrsf11a (TNF-α receptor superfamily member 11A) gene encoding RANK in the intestinal epithelium have a phenotype characterized by the absence of intestinal M cells, reduced formation of germinal centers in PPs, and substantial impairment in development of a secretory IgA response after weaning  mice and aly/+ controls were bred in our mouse colony at Emory University starting with aly/+ mice backcrossed onto the C57BL/6 background . Mice were genotyped for the wild-type and aly mutant alleles of Map3k14 (mitogen-activated protein kinase kinase kinase 14) gene by running two separate PCRs with allele-specific forward primers [5′-CACATCCCGAGCTACTTCAACA-3′ for aly and 5′-CACATCCCGAGCTACTTCAACG-3′ for wild-type NF-κB-inducing kinase (NIK)] and a common reverse primer (5′-CCTTCGGGGACTCTACAGGC-3′ for NIK)  and RANKF/F littermate controls were bred at Emory University and genotyped as previously described  were used for wild-type enteroid cultures. Alymphoplasia (for NIK) . The mutescribed . The anid-sorbitol (Thermo Fisher Scientific) in Dulbecco's PBS . After filtration through 70-μm mesh and a 4-min 200-g spin, the sedimented crypts were resuspended in 50 μl of Matrigel (Corning Life Sciences) and placed in the center of the wells in a 24-well plate. The plates were incubated at 37°C for 30 min to allow for polymerization of Matrigel before addition of culture medium (500 μl/well) as previously described  and 54.9 mM escribed . The ENRescribed .Ultra-LEAF-grade purified anti-mouse TNF-α antibody  was added to some enteroid cultures at a final concentration of 5 μg/ml to neutralize TNF-α. Unconjugated monoclonal rat anti-mouse GP2  was used to stain frozen sections of enteroids. Alexa Fluor 546-conjugated goat anti-rat secondary antibody  was used to detect the anti-GP2 primary. FITC-conjugated monoclonal anti-E-cadherin antibody  was used to stain cell junctions of enteroids on frozen sections.t) results were normalized by comparison with the housekeeping genes Gapdh and Rpl13a . The ΔΔCt method was used to determine fold induction of a gene of interest in a comparison of two samples, with normalization of each experimental Ct result to the geometric mean of the Ct values of Gapdh and Rpl13a .Enteroids in Matrigel were incubated with Cell Recovery Solution (Corning Life Sciences) with shaking for 1 h at 4°C to dissolve Matrigel prior to RNA extraction. The contents of three separate replicate wells were pooled for each experimental condition. After two PBS washes, RNA was extracted using the RNeasy Mini Kit . The iScript cDNA synthesis kit  was used to make DNA from 0.5–1.0 μg of RNA, with the same amount of RNA used from all samples in each experiment. iTaq Universal SYBR Green supermix (Bio-Rad) was used for PCRs run on a CFX Connect thermal cycler (Bio-Rad). All PCR primers are listed in d Rpl13a . ExpressSpib-1- or Spib-2-specific primers was ligated into pJET1.2 using the CloneJET PCR cloning kit (Thermo Fisher Scientific). Plasmids with inserts of the correct size were sequenced by automated dideoxy sequencing. The sequences of the Spib-1 and Spib-2 amplicons confirmed use of the expected promoters and splice sites.cDNA amplified from RANKL-treated enteroids using Enteroids were removed from Matrigel by treatment with 500 μl of Cell Recovery Solution (Corning Life Sciences) with shaking for 1 h at 4°C followed by washes. The recovered enteroids were embedded in optimum cutting temperature compound  and snap-frozen in isopentane on dry ice. Blocks containing the enteroids were sectioned on a cryostat to yield 5-μm-thick frozen sections. Slides were fixed with 4% paraformaldehyde for 20 min at room temperature. Sections were stained overnight with rat anti-mouse GP2 monoclonal antibody and then incubated for 2 h with goat anti-rat IgG-Alexa Fluor 546 secondary antibody and monoclonal FITC-anti-E-cadherin antibody. 4′,6-Diamidino-2-phenylindole  was used to stain nuclei. Fluorescence staining images were acquired with a Nikon 50i microscope using an ×40 oil-immersion objective.t-test with Prism . P < 0.05 was considered significant.Mean values of relative expression in quantitative PCR experiments were compared by a two-tailed Student's Spib, Ccl9, Tnfaip2 (TNF-α-induced protein 2), Marcksl1, and Ccl20, was strongly upregulated  and Saa1 (serum amyloid A1), but did not induce Spib (A–C). No induction of Reg3g and Saa1 was observed after RANKL stimulation under conditions that resulted in strong induction of Spib. TNF-α is a cytokine in the TNF superfamily that rapidly activates the canonical, but not the slower noncanonical, NF-κB pathway (Ccl20 expression at 4 h and 1 day but failed to induce Spib expression at 1 day (D–F). RANKL also induced Ccl20 expression, but not until the 1-day time point (E and F). While the enteroid cultures responded to all three cytokines tested, only RANKL induced an increase in Spib expression.IL-22 has potent effects on intestinal epithelial cells in vivo and in enteroid cultures, signaling through a heterodimeric receptor consisting of IL-22R1 and IL-10R2 to activate STAT3 and induce epithelial proliferation and expression of antimicrobial proteins , 34. To uce Spib , A–C. No pathway , 18, 36.at 1 day , D–F. RAme point , E and Faly/aly mice with a nonfunctional NIK allele and aly/+ control mice were stimulated with RANKL, IL-22, or TNF-α  149 bp upstream of a consensus TATA box sequence (TATATATA) located just 5′ to the transcriptional start site. The Spib-2 promoter includes a recognition site for octamer transcription factors (ATTTGCAT) but does not include a TATA box . The isoform-specific quantitative PCR amplifications revealed that RANKL exclusively induced the Spib-1 transcript. A low constitutive level of Spib-2 mRNA was detected in enteroids, but no additional Spib-2 mRNA was induced by addition of RANKL . Thus, RANKL-induced Spib expression in enterocytes depends on noncanonical NF-κB induction of the Spib-1 transcript.In B lymphocyte cell lines, transcription of the mouse d Spib-2 , 7. The t intron A. The SpTATA box , 7. Becaerocytes , B and Cof RANKL C. The pr+ cells in enteroids (to 14% compared with 11% with RANKL only) that was not statistically significant  and capable of enhanced phagocytic activity  was an important technical advance in stem cell and epithelial cell biology , 44, 45.activity , 40, 41.Spib and Gp2 is strongly induced by RANKL in the mouse enteroids, we investigated which other known M cell-associated genes were activated during the course of in vitro M cell differentiation. We found that Spi-B-dependent (Ccl9 and Tnfaip2) and -independent (Marcksl1 and Anxa5) M cell-specific genes were efficiently induced by RANKL. Because M cells found in RANKL-supplemented enteroids faithfully replicate the pattern of gene expression of natural M cells, the enteroid system is a significant improvement over the Caco-2/Raji coculture system for gene discovery applications and functional studies of bona fide M cells  and a Senior Research Award from the Crohn's & Colitis Foundation of America (to I. R. Williams).This work was supported by No conflicts of interest, financial or otherwise, are declared by the authors.M.B.W. and I.R.W. developed the concept and designed the research; M.B.W. and D.R. performed the experiments; M.B.W., D.R., and I.R.W. analyzed the data; M.B.W., D.R., and I.R.W. interpreted the results of the experiments; M.B.W. prepared the figures; M.B.W. drafted the manuscript; M.B.W. and I.R.W. edited and revised the manuscript; M.B.W. and I.R.W. approved the final version of the manuscript.


Adenovirus (Ad) vectors deleted of the early region 1 (E1) are widely used for transgene delivery in preclinical and clinical gene therapy studies. Although proteins encoded within the E1 region are required for efficient virus replication, previous studies have suggested that certain viral or cellular proteins can functionally compensate for E1, leading to expression of the early region 2 (E2)-encoded replicative proteins and subsequent virus replication. We have generated a series of E1-encoding and E1-deficient Ad vectors containing a FLAG-epitope tag on each of the E2-encoded proteins: DNA-binding protein (DBP), terminal protein (TP) and DNA polymerase (Pol). Using these constructs, we show that for the replication-competent virus, the expression level of each E2-encoded protein declines with increasing distance from the E2 promoter, with E2A-encoded DBP expression being ~800-fold higher than E2B-encoded TP. Pol was expressed at extremely low levels in infected cells, and immunoprecipitation from cell lysates was required prior to its detection by immunoblot. We further show that DBP was expressed 200- to 400-fold less efficiently from an E1-deficient virus compared to a replication-competent virus in A549 and HepG2 cells, which was accompanied by a very small increase in genome copy number. For the E1-deficient virus, late gene expression (a marker of virus replication) was only observed at very high multiplicities of infection. These data show that E1-deleted Ad gives rise to limited expression of the E2-encoded genes and replication in infected cells, but highlight the importance of considering viral dose-dependent effects in gene therapy studies. Human adenovirus (Ad) mainly causes self-limiting respiratory illnesses and can rapidly spread through confined populations such as day care centers, hospitals, retirement homes and military training venues . Since tThe E2 transcription unit consists of two regions, E2A and E2B, which have separate polyadenylation sites . The E2AIn general, the late transcription units, L1-L5, are produced from alternative splicing of a common major late transcript, and code for structural and capsid proteins  required for the production of progeny virions. In addition to these major early and late proteins, four other small products are produced by Ad: protein IX (pIX), virus-associated (VA) RNA I, VA RNA II and the U exon protein (UXP). pIX is a structural protein expressed at an intermediate time during infection (after initiation of early gene expression and before late gene expression), and it plays a role in virion stability ,16. VA Rin vitro [in vitro or in vivo [in vivo is the transient expression of the therapeutic transgenes due to rapid clearance of the transduced cells, caused in part by induction of immune responses to Ad proteins produced from the viral protein coding regions retained in the vector [i.e. cells that do not complement the E1A/E1B deletion in these vectors) at multiplicities of infection (MOI) as low as 10 [Ad vectors are one of the most commonly used platforms for gene delivery in human gene therapy clinical studies, as well as in biochemical studies conducted in vitro . First g in vivo . One maje vector ,25. Furtow as 10 . In suchow as 10 ,28. For ow as 10 ,30,31. Cow as 10 ,27.Although antibodies against the E2-encoded proteins have been generated by some research laboratories, they are not commercially available or readily accessible ,33,34. TAll Ad constructs were cloned using a combination of conventional and bacterial RecA-mediated cloning . Clones 3016F1-GCAACGCGTCGCAGATAATGGCGAC and 3116R-CTTATCGTCGTCGTCCTTGTAATCGGGAGGCGGCGGCGACGGGGAC; 3116F-GATTACAAGGACGACGACGATAAGCGGGCGCCCCCAAAAAGC and 3016R2-CCAACGCGTTTAGCAGGTCGGGC. The two resulting DNA products were combined into a single PCR reaction and re-amplified with 3016F1 and 3016R2. The DNA product from this second amplification was digested with MluI and cloned into MluI-digested pGEM7, generating pRP3116. A 5520 bp BamHI/SpeI fragment from pRP2014 was also cloned into BamHI/SpeI-digested pBluescript, generating pRP3037. An 1821 bp MluI fragment from pRP3116 was then used to replace the MluI fragment in pRP3037, generating pRP3118. A 5544 bp BamHI/SpeI fragment from pRP3118 was cloned into pRP2014, generating pRP3120 which is an Ad5 genomic plasmid lacking E1 and E3, but containing a FLAG-tag on the DBP. pRP3120 was recombined with pRP2483 [+/E3- infectious plasmid containing the FLAG-tagged DBP. pRP3121 and pRP3122 were digested with PacI to liberate the inverted terminal repeats, transfected into 293 cells and recovered as viruses, which are designated AdE1-DBP-F and AdE1+DBP-F, respectively, in this study. In these viruses, the coding sequence for the FLAG-tag is located immediately before 23916 bp of the conventional human Ad5 genome (GenBank AC_000008). Please note that the E2 coding sequences runs right to left, so before 23916 bp actually places the FLAG-tag downstream of this position in the E2 open reading frame. The specific amino acid sequence of this region of DBP is 36-PPPPDYKDDDDKRAPP, where 36 refers to the position in the DBP of the first listed amino acid , the FLAG-tag is shown in italics and bold, and the native DBP sequence is in normal type.The coding sequence for the FLAG-tag was added to DBP using overlap PCR. pRP2014  was subj pRP2483  to produ pRP2483  to gener+/E3- infectious plasmid containing the FLAG-tagged pTP. pRP2997 and pRP3000 were digested with PacI to liberate the inverted terminal repeats, transfected into 293 cells and recovered as viruses, which are designated AdE1-TP-F and AdE1+TP-F, respectively. In these viruses, the coding sequence for the FLAG-tag is located immediately before 9201 bp of the Ad genome. The specific amino acid sequence of this region of TP is 463-EALGDYKDDDDKINES. Of note, the 467th amino acid (D) is native to the TP protein and also constitutes the first residue of the FLAG-tag.A 1615 bp MfeI fragment (8016 bp to 9631 bp of the Ad5 genome) containing a portion of the pTP coding sequence from pRP2014 was cloned into MfeI-digested pNEB193, generating pRP2982. The synthetic oligonucleotide CTTATCGTCGTCATCCTTGTA and its complement were ligated into EcoRV-digested pRP2982, generating pRP2988. A 1069 bp EagI/XbaI fragment from pRP2110a  was clonBRS100F-GCGAAGCTTCCGCCTGACCTGACGCCGC and BRS100R-GCGAAGCTTATCGTCGTCATCCTTGTAATCCTCCTGCAGGTTTACCTCGCATAGACG, which prime in opposite directions around the circular plasmid. The resulting DNA product was digested with HindIII and re-circularized, generating pBRS100. A 602 bp ApaI/XhoI fragment from pRP2110a was cloned into XhoI/ApaI digested pBRS100, generating pBRS101 and adding an extra region of Ad genome homology and convenient restriction sites for the next step of cloning. A 309 bp BsrGI/Sse8387I fragment from pBRS101 was cloned into pRP2110a, generating pBRS103. Lastly, a 7127 bp XbaI fragment from pBRS103 was cloned into XbaI digested pRP2014ΔXbaI, generating pBRS104, an Ad5 genomic plasmid lacking E1 and E3 but containing a FLAG-tag on the Pol. pBRS104 was recombined with pRP2483, generating pBRS105, an E1/E3-deleted Ad5 genomic plasmid containing a CMV-RFP-BpA expression cassette replacing the E1 region and the FLAG-tagged Pol. pBRS104 was also recombined with the pXC1, generating pBRS106, an E1+/E3- infectious plasmid containing the FLAG-tagged Pol. pBRS105 and pBRS106 were digested with PacI to liberate the inverted terminal repeats, transfected into 293 cells and recovered as viruses, which are designated AdE1-Pol-F and AdE1+Pol-F, respectively. In these viruses, the coding sequence for the FLAG-tag is located immediately before nucleotide 8404 bp of the Ad genome. The specific amino acid sequence of this region of Pol is 127-NLQEDYKDDDDKLPPD.A 327 bp XhoI/NsiI fragment (XhoI at 8255 bp to NsiI at 8582 bp of the Ad5 genome) from pRP2014 was cloned into XhoI/NsiI-digested pGEM7, generating pRP2183. pRP2183 was amplified by PCR with synthetic oligonucleotides 293   and A549+DBP-F, AdE1-DBP-F, AdE1+TP-F or AdE1+Pol-F diluted in phosphate-buffered saline  for 1 hour at 37°C. Medium was then added to the cells and incubated in a humidified CO2 incubator at 37°C until the indicated time point. For the quantitative PCR (qPCR) experiments, the viral inoculum was removed after the 1-hour incubation and the monolayers were washed once with PBS before adding medium.Viruses were propagated in 293 cells and purified by cesium chloride buoyant density centrifugation, using standard procedures . Viral tTo prepare protein samples, medium was removed from infected cells at the indicated time points, 2x protein sample buffer  was added, and the resulting crude protein extract was collected. The protein samples were boiled prior to separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore) and the membrane was probed with monoclonal antibodies to Ad5 fiber , FLAG , Ad5 E1A  or a monoclonal antibody to human vinculin . The monoclonal antibody against Ad5 DBP  was a ki10 virus particles (VP) were diluted in 10 mM Tris pH 7.5 to a final volume of 25 μl and denatured for 10 min at 60°C. The samples were cooled to room temperature, prior to adding reaction buffer  and 2 units of DNaseI . After a 20-min incubation at 37°C, an equal volume of 2x protein sample buffer was added, the samples were boiled for 5 minutes, and analyzed by immunoblot as described above.For detection of the viral DNA-associated 55-kDa TP present within the virion, 3x105’-CTT ACC CCC AAC GAG TTT GA and 5’-GGA GTA CAT GCG GTC CTT GT.Media from infected cells was removed at the indicated time points and the cells were incubated overnight at 37°C in SDS-proteinase K buffer , 1 mg/ml proteinase K) . Followi+Pol-F or wildtype Ad5 at an MOI of 100, and lysed 24 hours later in modified radio immunoprecipitation assay (RIPA) extraction buffer . Immunoprecipitation was conducted as previously described [Ten centimeter dishes of A549 cells were infected with AdE1escribed , using 3th of the confluency density in 12-well tissue-culture plates, and infected as described above with FLAG-tagged Ad constructs at MOIs varying from 10 to 50. Eighteen hours post infection (hpi), media was removed and the coverslips were washed once with cold PBS. The cells were fixed with 4% paraformaldehyde (pH 7.0) in PBS for 15 mins, rinsed with cold PBS, permeabilized with PBS containing 0.25% Triton X-100 (Thermo Fisher Scientific) for 10 min, and then washed three times with PBS for 5 min each. PBST supplemented with 1% BSA was added for 30 min to block nonspecific antibody binding, prior to overnight incubation at 4°C with the primary antibody diluted in the blocking solution. The next day, the primary antibody was decanted and the cover slips were washed three times as before. The coverslips were incubated for 1 hour at room temperature with secondary antibody, also diluted in the blocking solution. After secondary antibody removal and three washes with PBS in the dark, the cells were incubated with Hoechst 3342  for 1 min. Lastly, the coverslips were rinsed with PBS and placed on glass slides using Dako Fluorescent Mounting Medium (Agilent Technologies). The following antibodies were used: mouse monoclonal anti-FLAG antibody  and Alexa Fluor 594 anti-mouse IgG . Fluorescent images were obtained with a Plan-APOCHROMAT 63x/1.4 oil-immersion Ph3 objective on a Zeiss Axio Observer.Z1 microscope equipped with an Axiocam MRC camera and ZEN 2 software for image processing.A549 cells were seeded on 1 cm rounded coverslips at 1/10et al. used insertion mutagenesis studies to identify regions of the DBP that were tolerant for insertion of small peptides [et al. identified the region between amino acids 39 and 41 of DBP as tolerant for insertion of small peptides with no loss of virus replicative ability [+DBP-F, was easily rescued and showed similar fitness to wildtype Ad5  of the Ad5 DBP is relatively weakly conserved, and is dispensable for ssDNA binding activity . In contpeptides . In this ability . Thus, ttype Ad5 . An E1-det al. [et al. identified two insertion sites, ins318 and ins467, that retained full activity during DNA replication [+TP-F, was rescued and grew similar to wildtype Ad5 , suggesting that presence of the FLAG-tags does not impact virus infectivity or growth.To determine whether insertion of the FLAG-tag affected DNA replication or late gene expression of the viral constructs, monolayers of A549 cells were infected at an MOI of 10 with wildtype Ad5, AdE1d to Ad5 . Viral Dd to Ad5 . All of +DBP-F at an MOI of 50, and crude protein extracts were prepared at varying times post-infection. As shown in +DBP-F with wildtype Ad5 to determine whether presence of the FLAG tag altered expression. A549 cells were infected with the two viruses at an MOI of 50, protein samples were collected 8 and 24 hpi, and DBP expression was evaluated by immunoblot using an antibody to native DBP. As shown in We next determined whether the presence of the FLAG-tag on the various E2-encoded proteins could allow them to be detected by immunoblot and immunofluorescence. To characterize expression of the DBP, A549 cells were infected with AdE1Unlike the abundantly expressed DBP, biochemical studies on Ad Pol and pTP have been difficult to conduct due to low expression levels of these two proteins, and existence of these proteins as a pTP-Pol complex in infected cells ,34. Cons+DBP-F and undiluted lysate from cells infected with AdE1+TP-F. Comparison of the signal obtained for pTP with the dilution series of DBP indicates that DBP is expressed at an approximately 800-fold higher level than TP under the conditions examined   .Repeated attempts to detect the FLAG-tagged Ad DNA Pol in crude protein lysates by standard immunoblot, even at as high an MOI as 100, were unsuccessful (data not shown), suggesting that Pol is expressed at very low levels in infected cells. However, using a standard immunoprecipitation assay, we could detect a 140-kDa species representing the full-length Pol . As migh+ virus at 72 hpi [Previous studies have suggested that E1-deleted Ad vectors can replicate in many cell types, particularly transformed cells . For exat 72 hpi . Varyingt 72 hpi , it has t 72 hpi ,56. In tt 72 hpi ,27,56.+DBP-F and the AdE1-DBP-F constructs to take advantage of the higher DBP expression levels, and thus greater sensitivity. A549 cells were infected at an MOI of 10 with AdE1+DBP-F or AdE1-DBP-F and 24 hr later, crude protein extracts were prepared and analyzed for DBP and fiber expression by immunoblot. As shown in +DBP-F, but the level of DBP expression from AdE1-DBP-F was very low and only visible on overexposure of the blot. Similarly, although expression of the late fiber protein, which is only expressed following DNA replication, was easily detected in cells infected with AdE1+DBP-F, no fiber expression was observed in cells infected with AdE1-DBP-F. To better gauge the relative level of DBP expression between the two viruses, we analyzed a dilution series of lysate from cells infected with AdE1+DBP-F relative to undiluted AdE1-DBP-F cell lysate. AdE1-DBP-F expressed the E2A-encoded DBP at a level approximately 1/200th that of the E1+ virus . Although cells infected with both viruses showed comparable levels of viral DNA at 4 hpi (prior to initiation of DNA replication), there was a ~4-log increase in viral genome copy number at 24 hpi for AdE1+DBP-F, but no change for AdE1-DBP-F . 293 cells were infected at low MOIs with AdE1+DBP-F or AdE1-DBP-F, crude proteins samples were isolated 24 later, and evaluated for the level of fiber protein . As shown in in trans. We next examined replication for the E1-deleted virus in A549 cells as a function of MOI. We saw an approximately linear increase in expression of DBP from AdE1-DBP-F between an MOI of 50 to 1000 . If each virus contributes a small amount of E2 proteins, then with increasing MOI, the cumulative expression could reach the threshold required for efficient replication. Consequently, with increasing MOI, we should observe a non-linear increase in replication and late gene expression. We first examined whether the E1-deleted and the E1 to 1000 ; howeverof fiber . Similarigh MOIs . Comparaigh MOIs . Taken tIn this study, we have created Ad constructs containing a FLAG-tag in each of the three E2 protein coding regions . Determi+ virus at an MOI of 1 (e.g. SP1) to directly activate expression from the MLP [Using the FLAG-tagged Ad constructs, we showed that during low MOI infections, the level of DBP expressed from E1-deleted Ad in A549 and HepG2 cells is very low and does not support efficient replication . These oMOI of 1 . NeverthMOI of 1 . It is p the MLP , E1A del the MLP ,59. E1A  the MLP . This wiTaken together, our work underscores the crucial role of E1A in orchestrating and modulating various cellular and viral processes to achieve efficient Ad replication. However, the results also clearly show that MOI-dependent effects on replication of E1-deleted Ad is an important consideration when using Ad vectors for gene delivery. Attempts to increase transgene expression through use of high MOI infections may have the undesired effect of enabling Ad replication, which could ultimately compromise the integrity of the study.


There was a significant inverse relationship between the extent to which BCF samples inhibited hydrolysis in MCF-7 cells and stimulated it in MDA-MB-231 cells. BCF stimulated E1S formation in MCF-7 cells while inhibiting formation in MDA-MB-231 cells. No difference in the ability of BCF from British or Hungarian women to inhibit or stimulate E1S hydrolysis was detected in ER+ or ER– breast cancer cells. In contrast, BCF from British women stimulated E1S formation in ER+ cells (median 82%) to a significantly greater extent (P< 0.01) than BCF from Hungarian women (median 33%). The role that E1S has in breast cancer development remains unclear. The greater stimulation of E1S formation by BCF from British women, who have a higher risk of breast cancer than Hungarian women, suggests that it may act as a storage form of oestrogen within cells that can be activated by oestrone sulphatase. © 2000 Cancer Research CampaignWomen with gross cystic breast disease may have an increased risk of breast cancer. In this study the ability of breast cyst fluid (BCF), obtained from British or Hungarian women, to modulate oestrone sulphate (E1S) formation or hydrolysis, has been examined. For this, oestrogen receptor-positive (ER+) MCF-7 or MDA-MB-231 (ER–) breast cancer cells were employed. The formation and hydrolysis of E1S was measured using radiometric techniques. BCF from British and Hungarian women mainly inhibited E1S hydrolysis in MCF-7 cells while stimulating hydrolysis in MDA-MB-231 cells. The extent of inhibition or stimulation of E1S hydrolysis in these cells was related to the Na


Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology . Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the  mqsR, the gene most highly upregulated in persisters, together with mqsA, function: they are the founding members of a new family of toxin:antitoxin (TA) systems. Unexpectedly, the structure of MqsR reveals that it is a ribonuclease, a protein that controls the production of other essential proteins. Moreover, we identified multiple features of this TA system that are so unique that each is a starting point for drug development. Unlike other antitoxins, MqsA is structured throughout its entire sequence, its structure is unchanged between the free and toxin-bound states and it binds zinc. It also binds DNA via its C- and not N-terminal domain. Finally, MqsA binds both its own promoter and additional genes important for E. coli physiology. Taken together, our data provide fundamental new insights into the role of MqsR and MqsA in bacterial persistence and biofilms.Most bacteria live in biofilms, microbial communities that cause more than 80% of human infections. Biofilms have a genetically identical sub-population of dormant cells, named persister cells, which are the well-recognized source of antibiotic resistance. Recently, it was demonstrated that toxins are highly upregulated in persisters and have therefore been postulated to play a role in the persister state. Using an inter-disciplinary approach, we reveal how  E. coli, the frequency of persisters in planktonic cultures is only about one in a million The emergence of increasing numbers of bacteria that are resistant to antibiotics portends a major public health crisis. One well-recognized but poorly understood mechanism used by bacteria to survive environmental stress is through the formation of persisters, a subpopulation of cells that survive prolonged exposure to antibiotics Recent studies have demonstrated that the persister state is correlated with the increased expression of chromosomal toxins from toxin:antitoxin (TA) genes E. colirelBEmazEFdinJ-yafQhipBAhicAByefM-yoeBrelE, mazF and yoeBmqsR (ygiU/b3022), a gene originally identified as one that encodes a regulator of motility, curli and quorum sensing and that influences biofilm development by mediating the response of the cell to autoinducer-2 To date, more than ten TA loci have been identified in mqsA (ygiT/b3021), the second gene in the two-gene mqsRA operon, is lethal mqsRA constitutes a novel TA module mqsRA has many characteristics that differ from canonical TA systems. First, in the mqsRA operon, mqsR precedes, instead of follows, mqsA. This unusual genetic organization has only been observed in two other recently characterized TA systems, that of higBAhicABhicABDeletion of bona fide TA pair that, because of the unique features of MqsA, define a novel family of TA modules. We show that MqsR is toxic and forms a tight complex with its antitoxin, MqsA, an interaction that mitigates MqsR toxicity. MqsA and the MqsR:MqsA complex also bind the promoters of the mqsRA operon and, unexpectedly, genes critical for E. coli physiology, including mcbR and spy. To the best of our knowledge, this is the first time a TA pair has been shown to bind and regulate promoters other than its own. The structure of MqsR reveals that it is a member of the RelE/YoeB family of bacterial RNase toxins. Based on its similarity with RelE, MqsR likely functions as a ribosome-dependent RNase. This suggests that MqsR is important for bacterial persistence via its ability to inhibit translation and, in turn, cell growth. MqsA itself is a two-domain protein with a novel fold that, unlike every other antitoxin, is well-ordered throughout its entire sequence and whose structure does not change upon toxin binding. It is also the first antitoxin known that binds metal, in this case zinc. These studies reveal the molecular mechanisms by which MqsR and MqsA mediate the cessation of cell growth and provide novel targets for the development of a new class of antibiotics that target TA pairs.In this paper, we employed a combination of biochemical and structural studies to show that MqsR, along with MqsA, are a 2:MqsR), as determined using size exclusion chromatography  and cell viability. Expression of MqsR alone leads to cell growth arrest in multiple bacterial strains (BW25113 and MG1655), while co-expression of MqsR with full-length MqsA (referred to hereafter as MqsA-F) rescues the cell growth arrest phenotype . In additography  and confr PmqsR; . It was  classes . Taken t and spy .1, with two molecules in the asymmetric unit. The two monomers are identical in terms of the overall fold with well-ordered electron density throughout both chains. The final model contains 262 residues, and includes all 131 residues for each MqsA-F molecule.The structure of full-length MqsA  of the 76 residues for MqsA-N and 97 residues (1–97) of the 98 residues for MqsR in the first complex and 59 residues  of MqsA-N and 90 residues  for MqsR in the second complex. X-ray diffraction data quality and refinement statistics are reported in The structure of the MqsR:MqsA-N complex  was solvThe structure of the MqsA-F dimer is shown in MqsA-N binds zinc and adopts a novel, elongated fold . It is cThe interaction between the two, long twisted β-strands and the five turn α-helix of the MqsA zinc binding domain is stabilized by an extended hydrophobic core composed of 11 residues: Ile18, Tyr20, Phe22, Leu29, Ile32, Tyr36, Met45, Phe53, Val57, Phe60 and Val64 , they arMqsA-C, the helix-turn-helix (HTH) domain, is composed of five tightly packed α-helices , which b2 of solvent accessible surface area  binds MqsR, we carried out two co-expression toxicity experiments using the same protocol as that used to produce the proteins for structural studies: 1) MqsR co-expressed with MqsA-N and 2) MqsR co-expressed with MqsA-C. When MqsR and MqsA-C are co-expressed, bacterial cell growth is arrested , and conmqsR promoter (PmqsR), as incubation of PmqsR with MqsA-C results in a shift in the electrophoretic mobility of the DNA  and three α-helices, with α-helix 2 adjacent to and α-helices 1 and 3 abutting the backside of the β-sheet , both ofThe RelE-like fold is characterized by a central, antiparallel β-sheet and adjacent α-helix, which is conserved among the MqsR, YoeB and RelE bacterial toxins  were K56A, Q68A, Y81A and K96A , S7B. Th2 of SASA while the secondary interface buries 479 Å2 of SASA. A total of 2016 Å2 of SASA is buried upon complex formation, or 17.7% of the total SASA, well within the range expected for biological interfaces. The primary interface is centered on MqsA β-strand 3 (Ser43-Met45), which interacts with MqsR β-strand 2  to form a single continuous β-sheet . Since the structure of the MqsA-N is invariant among all structures determined , we used the MqsA-F and MqsR:MqsA-N structures to generate an accurate model of the full MqsR:MqsA2:MqsR complex. As can be seen in 2:MqsR complex forms a highly extended structure with the MqsA-C domains located at the center of the complex  and the MqsR toxins (magenta) bound at the periphery. In overall shape, the MqsR:MqsA2:MqsR complex is most similar to the MazF:MazE complex The MqsR:MqsA2:MqsR complex bind directly to the mqsR promoter , bind DNA via the same helix, the HTH-XRE DNA binding helix. In MqsA, the HTH-XRE DNA binding helix corresponds to α-helix 4  2:MqsR complex , and thus may be able to adopt a slightly different conformation when bound to DNA, it is highly unlikely that it could shift sufficiently to permit binding to unbent DNA.We generated a model of the MqsR:MqsA complex . As can promoter . Notably another  may helpE. coli persister cells gamma- delta- and epsilon proteobacterial classes. This work has provided the first steps for understanding this novel, unique TA system at a molecular level and provides a basis for developing novel antibacterial therapies that target TA pairs.Taken together, the crystal structures of full-length MqsA and the MqsR:MqsA-N complex reveal that the MqsR:MqsA TA complex is the founding member of a novel family of TA pairs. It forms a dimer of dimers, in which DNA binding and MqsR recognition by MqsA are mediated via distinct, structured domains. Because MqsR is the gene most highly upregulated in E. coli strain BW25113 were conducted in LB medium at 37°C. Growth experiments were monitored using pBS(Kan)-based plasmids lac promoter, the fragments from genomic DNA were amplified by PCR . For the MqsR:MqsA-N complex, MqsR was subcloned into the pET28a vector (Novagen) which contains an N-terminal his6-tag with a thrombin cleavage site. MqsA-N was subcloned into the untagged pCA21a vector . The complex was co-expressed and purified by sequential his6-tag, thrombin cleavage, second subtraction his6-tag and SEC.Full-length MqsA , the MqsA N-terminal domain  and the MqsA C-terminal domain  were subcloned into a modified pET28a vector which contained an N-terminal his2, and 19% (w/v) PEG3350). Crystals of MqsR:MqsA-N were obtained using the sitting drop vapor diffusion method at 4°C. Crystals were grown by mixing 0.2 µL MqsR:MqsA-N protein complex  with 0.4 µL of precipitant (0.1 M Bis-Tris pH 5.5 and 25% (w/v) PEG3350). MqsA-F and MqsR:MqsA-N crystals were cryoprotected in precipitant containing 20% glycerol and frozen in liquid nitrogen for data collection.Crystals of MqsA-F were obtained using the sitting drop vapor diffusion method at 4°C. Crystals were grown by mixing 0.2 µL of MqsA-F protein  ethanol) with 0.2 µL of precipitant  and sequence verified. Growth experiments were conducted in LB at 18°C and all mutants were tested in parallel. Briefly, 10 ml of an overnight culture was used to inoculate 1 L of LB and then incubated at 37°C with rigorous shaking. Once a mutant culture reached an ODThe P22 C2 repressor protein (PDBID 2R1J) The genes/proteins mentioned in the text include (UniProtKB/Swiss-prot ID unless stated otherwise): MqsR/YgiU (Q46865), MqsA/YgiT (Q46864), McbR (P76114), Spy (P77754), RelE (P0C077), RelB (P0C079), MazE (P0AE72), MazF (P0AE70), DinJ (Q8X7Q6), YaFQ (Q47149), HipA (P23874), HipB (P23873), HicA (P76106), HicB (P67697), YefM (P69346), YoeB (P69348), HigA (Q9KMG4/Q9KMA5), HigB (Q9KMG5/Q9KMA6). The PDB files mentioned in the text are RelE (PDBID 2KC8), YoeB (PDBID 2A6Q) and RNase Sa (PDBID 1RSN).The structure factors and coordinates for MqsA-F and the MqsR:MqsA-N complex have been deposited with the Protein Databank with accession numbers 3GN5 and 3HI2, respectively.Figure S1E. coli strain MG1655 (A–C) and BW25113  containing pBS(Kan) , pBS(Kan)-mqsA-F (open circle), pBS(Kan)-mqsR (black triangle) and pBS(Kan)-mqsR-mqsA-F (open triangle) at 37°C in LB with 1 mM IPTG induction upon inoculation (incubation time  = 0 min). Electrophoretic mobility shift assay controls that show both MqsR:MqsA-F (F) and MqsA-F (G) do not bind the PtomB DNA probe, which was used as a non-specific competitive control for the EMSA assays shown in MqsR and MqsA are a Toxin:Antitoxin Pair. The effect of MqsR, MqsA-F and the MqsR:MqsA-F complex on cell growth , cell viability (CFU/ml)  and colony formation (C) for (1.25 MB TIF)Click here for additional data file.Figure S21–131 (14.9 kDa), while the proteolytic fragments are denoted with *. The trypsin digested samples analyzed by MALDI-TOF MS and LC-MS/MS are boxed. Proteolytic digestion of a stable, folded domain (SPAR PDZ domain) and an intrinsically unstructured protein (DARPP-32) are shown on the right for comparison.MqsA is Susceptible to Proteolytic Degradation. SDS-PAGE analysis of MqsA-F incubated with chymotrypsin, trypsin or proteinase K for 5, 15, 30 and 60 minutes at 30°C. The upper band indicated with an arrow corresponds to undigested MqsA(0.82 MB TIF)Click here for additional data file.Figure S3MqsR and MqsA are conserved among multiple bacterial species. Sequence alignment of MqsR (A) and MqsA (B). Secondary structural elements for MqsR and MqsA are represented as cylinders (α-helices) or arrows (β-strands) and shaded according to the protein/domain to which they belong . Identical residues are highlighted in cyan whereas similar residues are shaded in gray; symbols below the sequence: ‘*’ identical residues; ‘:’ highly similar residues; ‘.’ similar residues.(1.59 MB TIF)Click here for additional data file.Figure S4Constructs used in this study. MqsA residues 62–76 represent the ‘linker’ between the two domains of MqsA. Subsequent structure determination demonstrates that the physical linker between the MqsA N- and C-terminal domains is short, centered on residue T68.(0.20 MB TIF)Click here for additional data file.Figure S5Crystal Structures of the Individual MqsA Domains and their Independent Rotation in the MqsA dimer.(A) Ribbon model of MqsA-N. Bound zinc is illustrated as a teal sphere. (B) Ribbon model of MqsA-C. α-helix 4, which is predicted to mediate DNA binding, is shaded in green. (C) The N- and C-terminal domains of MqsA rotate independently via the short flexible linker. Both monomers of MqsA were superimposed on the MqsA-C domains  to illustrate the relative rotation of the MqsA-N domains , resulting in a shift of the bound zinc ions by 18 Å.(1.46 MB TIF)Click here for additional data file.Figure S6oF-dcF map at 1.5 σ. MqsA in green, zinc ion in teal. (B) Stick representation of a portion of the MqsR:MqsA secondary interface (MqsR residues T59-Q68 in blue and MqsA residues K2-H7 in green) with σA-weighted 2moF-dcF map at 1.5 σ.Stereoimages of MqsA and MqsR. (A) Stick representation of the MqsA zinc binding pocket (residues K2-M11 and C37-E42) with σA-weighted 2m(2.08 MB TIF)Click here for additional data file.Figure S7Semi-log plots of MqsR toxicity assays illustrated in (0.39 MB TIF)Click here for additional data file.Protocol S1Supporting Protocols and References.(0.07 MB PDF)Click here for additional data file.Table S1Bacterial strains and plasmids used in this study.(0.05 MB PDF)Click here for additional data file.Table S2Oligonucleotides used for this study.(0.02 MB PDF)Click here for additional data file.Table S3Data collection and refinement statistics for MqsA-N.(0.05 MB PDF)Click here for additional data file.Table S4Data collection and refinement statistics for MqsA-C.(0.05 MB PDF)Click here for additional data file.


Disturbances in peripheral blood memory B cell subpopulations have been observed in various autoimmune diseases, but have not been fully delineated in rheumatoid arthritis (RA). Additionally, the possible role of tumour necrosis factor (TNF) in regulating changes in specific peripheral blood memory B cell subsets in RA is still unclear.The frequency and distribution of B cell subsets in the peripheral blood and synovial membrane of active RA patients with long-standing disease have been analysed. Additionally, the possible role of TNF in causing disturbances in memory B cell subsets in RA patients was assessed in a clinical trial with the specific TNF-neutralising antibody, infliximab.+CD27+ memory B cells than healthy individuals, whereas post-switch IgD-CD27+ accumulate with increased disease duration. Notably, both pre-switch IgD+CD27+ and post-switch IgD-CD27+ memory B cells accumulate in the synovial membrane of RA patients. Finally, anti-TNF therapy increased the frequency of pre-switch IgD+CD27 memory B cells in the peripheral blood.RA patients, independent of disease duration, have a significantly lower frequency of peripheral blood pre-switch IgD+CD27+ pre-switch memory B cells in RA reflect their accumulation in the synovial tissue. Moreover, the significant increase in the peripheral blood pre-switch memory B cells in patients who underwent specific TNF-blockade with infliximab indicates that trafficking of memory B cells into inflamed tissue in RA patients is regulated by TNF and can be corrected by neutralising TNF.The data suggest that decreases in peripheral blood IgD Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterised by inflammatory polyarthritis and joint damage resulting in progressive disability . The infThe importance of B cells in RA has been emphasised by the success of therapeutic approaches using anti-CD20 monoclonal antibodies (mAbs) . It is c+CD27-; pre-switch memory IgD+CD27+; and post-switch memory IgD-CD27+ [- population of memory B cells with mutated Ig genes has been described [Functionally distinct B cell subsets can be defined by the surface expression of immunoglobulin (Ig) D and CD27. These include naïve IgDgD-CD27+ -12. ImpogD-CD27+ , althougescribed -16, whicescribed . Abnormaescribed , and Sjöescribed . Howeverescribed -21.+memory B cells in the blood [Treatment with TNF blockers ameliorates the signs and symptoms of RA and disease progression -25. Recehe blood . Howeverhe blood ) and alshe blood , suggest+CD27+ memory B cells than healthy individuals, whereas post-switch IgD-CD27+ memory B cells accumulate with increased disease duration. Additionally, we present evidence that pre-switch IgD+CD27+ memory B cells accumulate in the synovial membrane of RA patients, and that this accumulation might be related to the influence of TNF, because anti-TNF therapy increased the frequency of pre-switch IgD+CD27 memory B cells in the peripheral blood. These results document disease-related and TNF-dependent abnormalities in memory B cell subsets in RA and suggest that part of the success of TNF neutralising therapy could relate to normalisation of memory B cell abnormalities.In order to study the changes in peripheral memory B cell subpopulations in RA patients, and to understand the possible role of TNF in regulating changes in specific memory B cells, we analysed the frequency and distribution of B cell subsets in the peripheral blood and synovial membrane of active RA patients with long-standing disease. Subsequently, we assessed whether treatment with the specific TNF-blocker, infliximab, normalised the distribution of these peripheral B cell subsets. Our results show, for the first time, that RA patients, independent of disease duration, have a much lower frequency of peripheral blood pre-switch IgDPeripheral blood samples from 40 healthy donors  were obtained from the National Institutes of Health blood bank, and from 33 patients  with long-standing RA  enrolled in a natural history protocol (00-AR-0222) at the Warren G. Magnuson Clinical Center .In addition, blood samples were obtained from 23 patients  with active RA  greater than 20 mm/hour or C-reactive protein (CRP) greater than 0.8 mg/dl) who failed treatment with methotrexate  and were entering a clinical trial of infliximab therapy (00-AR-0220). For this trial, patients on prednisone had to be on 7.5 mg or less per day to be eligible to participate. Patients were randomised to receive either monthly infliximab infusions (3 mg/kg infliximab with MTX 15 mg/week), or monthly control infusions and weekly MTX alone (<25 mg/week). All patients fulfilled the revised American College of Rheumatology criteria for RA . MB, carThe group of patients enrolled in the natural history protocol, with a median disease length of 13 years, were considered as the long-standing disease group. The group of patients enrolled in the clinical trial for infliximab, with a median disease duration of 4.4 years, were considered as the group with shorter disease duration.Synovial specimens and peripheral blood samples were collected at the Department of Rheumatology, Tokyo Medical and Dental University from 10 RA subjects with long-standing disease (median disease length of 13.5 years).The characteristics of all patients studied are shown in Table The local institutional review board or the ethics committees  approved the studies and all patients signed an informed consent before participating in this study. Patient's management was performed in accordance with the local standard practice and the study was conducted in accordance with the regulations governing clinical trials, such as the Declaration of Helsinki as amended in Edinburgh (2000).6 cells/100 μL). Isolated PBMCs were stained by standard methods with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein Cy5.5 (PerCpCy5.5) or allophycocyanin (APC) conjugated mAb specific for the following human cell surface markers: anti-CD19 PerCpCy5.5, anti-CD27 PE, anti-IgD FITC and anti-IgM FITC . Data were acquired on a FACSCalibur .Peripheral blood samples from the controls and natural history patients were obtained during a single scheduled outpatient visit. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation and re-suspended in 1.5 ml PBS and 1% BSA  and anti-IgD FITC . T cells were identified by anti-CD3 APC or PE, anti-CD4 PE, anti-CD8 FITC or APC, anti-CD45RA FITC and anti-CD45R0 APC .To calculate absolute numbers of each lymphocyte subset, the percentage of cells staining positively was multiplied by the absolute peripheral blood lymphocyte count, which was determined by cell counting with a Celldyne 3500  blood cell counting machine. With all experiments, peripheral blood from healthy adult patients was stained and analysed as controls.To determine the chemokine receptor expression by B cells and their subsets, the following APC-conjugated anti-human mAbs were used: anti-CXCR1, anti CXCR2 and anti-CCR2 ; and anti-CXCR4 .+ or CD19+) were gated and the percentages of CD27+ , IgD+CD27- (naïve), IgD+CD27+ (pre-switch memory) and IgD-CD27+ (post-switch memory) populations in the gated B cells were calculated. Although anti-CD20 mAb do not identify all plasmablasts, most of which are CD19+CD27++IgD-, the results from both staining protocols were pooled together, because no significant differences in total or post-switch memory B cells were observed when analysing the results separately.Irrelevant, directly conjugated, murine IgG1  was used to ascertain background staining. Samples were run on a FACScan or a FACSCalibur . Data were analysed using the WinList software, version 5.0, and FloJo software . B cells (CD20+) were gated, and the precentages of CD4+ , CD8+ , CD4+CD45RA+  and CD4+CD45R0+  populations within the T cell population were calculated.T cells .Synovial tissues were obtained during joint replacement surgery from 10 RA patients. Specimens were minced and incubated with 0.3 mg/ml of collagenase  for one hour at 37°C in Dulbecco's Modified Eagle's Medium . Partially digested pieces of the tissue were pressed through a metal screen to obtain single cell suspensions. Cells were stained with anti-CD19 PECy5 , anti-CD27 FITC, anti-IgM PE and anti-IgD PE , anti-CXCR1 PE, anti-CXCR2 PE, anti-CXCR4 PE and anti-CCR2 PE . Synovial tissue cells were adjusted to 1 × 10+IgD+CD27- naïve B cells and CD19+IgD+CD27+ memory B cells from four patients with RA were sorted using a Beckton Dickinson FACS DIVA  or a Dako Cytomation MoFlo  and 1 to 1.5 cells/5 μL PBS, and then plated into 96-well PCR plates containing 10 μL lysis buffer ), subjected to primer extension pre-amplification and then VH3 and VH4 genes were amplified by nested PCR, as previously described [® 96-Well Standard Plate kit  and sequenced on a model 3100 capillary sequencer  using the Big Dye® Terminator v1.1 Cycle Sequencing Kit . Ig variable heavy chain rearrangements were analysed for somatic mutations using the web-based algorithm JOINSOLVER®  [CD19escribed . PCR prond, USA) .The level of soluble CD27 was determined in serum samples from RA patients in the natural history protocol and healthy controls using the PeliKline Compact human soluble CD27 ELISA kit  according to the manufacturer's instructions.Data were checked for a normal distribution in order to decide whether to use parametric or non-parametric tests. Median group values (with standard error of the mean) for percentage and absolute numbers of the different B cell populations were compared in patients and healthy controls using the nonparametric unpaired Mann-Whitney test.+ memory B cell population were compared between the synovium and peripheral blood of 10 RA patients undergoing synovectomy using a paired Student's t-test.Mean values (with standard deviation) of the CD27P < 0.05 was considered statistically significant.Median group values (with standard error of the mean) of the different B cell populations compared pre- and post-treatment in the 23 RA patients who were treated with infliximab plus MTX or MTX monotherapy using the nonparametric paired Wilcoxon Signed Rank test. A The demographic and clinical characteristics of the RA patient groups evaluated in this study are shown in Table The 23 RA patients enrolled in a clinical trial comparing MTX plus infliximab with MTX alone had disease of shorter duration .P = 0.0031) lower frequency of IgD+CD27+ pre-switch memory B cells than the healthy donors (median RA 10.4 ± 1.3% vs control 15.1 ± 1.1%). This significant difference (P = 0.0036) was maintained when analysing the absolute number of pre-switch memory B cells (median RA: 13.8 ± 4.7 cells/μl vs control: 21.3 ± 3.9 cells/μl). On the other hand, the frequency – but not the absolute numbers – of the IgD-CD27+ post-switch memory population was significantly (P = 0.0101) increased in subjects with long-standing RA when compared with the control individuals (median RA 19.6 ± 2.9% vs control 13.2 ± 1.0%). Interestingly, no significant difference could be seen between RA patients and controls in the frequency or absolute number of the total CD27+ memory B cell pool . Finally, the frequency of IgD+CD27- naïve B cell population in the peripheral blood of subjects with long-standing RA was comparable with the healthy donors (median RA 57.3 ± 4.1% vs control 65.6 ± 1.7%). However, the absolute number of naïve B cells was significantly (P = 0.0231) lower in the long-standing RA patients when compared with the control individuals (median RA: 61.4 ± 28.6 cells/μl vs control: 100.5 ± 10.7 cells/μl). These differences could not be the result of B cell lymphopenia, because the absolute number of the total B cell pool in the long-standing RA patients was comparable to the healthy donors (median RA 151.0 ± 35.8 cells/μl vs control: 148.5 ± 20.0 cells/μl).The frequencies of B cell subsets defined by the expression of IgD and CD27 in the peripheral blood of patients with long-standing RA were compared with healthy donors Figures . One strP = 0.8156; median shorter disease 14.4 ± 2.9 cells/μl vs long-standing disease: 13.8 ± 4.7 cells/μl, P = 0.4003), each of which was significantly (P < 0.03) lower than that found in the healthy controls. Notably, however, the frequency of post-switch memory B cells was significantly (P = 0.0025) lower in the patient group with shorter disease duration when compared with the long-standing group (median shorter disease: 9.7 ± 2.9% vs longer standing disease: 19.6 ± 2.9%). Furthermore, the frequency of the total CD27+ memory B cell population was significantly (P = 0.0184) lower in the patient group with shorter disease duration when compared with the long-standing group (median shorter disease: 19.1 ± 3.2% vs longer standing disease: 31.3 ± 3.8%).In order to assess whether disease duration had an influence on the B cell subset disparities between RA patients and healthy individuals, the frequencies of the different B cell subpopulations in the RA patients with long-standing disease  were compared with the baseline values of another group of patients with shorter disease duration (median disease duration 4.4 years) who had enrolled in a clinical trial examining the impact of MTX versus that of the combination of MTX and infliximab  genes from sorted peripheral blood, IgD+CD27+ and IgD+CD27- RA B cells, was carried out to determine the frequency of somatic mutations in those subsets. As expected, most of the IgD+CD27- B cells expressed unmutated IgVH genes (76%) and those that were mutated contained few mutations. Both the frequency of mutated Ig sequences and the mutational frequency in the IgD+CD27- subset was significantly (P < 0.05) lower when compared with the IgD+CD27+ subset. Moreover, the mean number of somatic mutations per IgVH gene was significantly (P < 0.05) lower in the IgD+CD27- subset .It is known that the rheumatoid synovial membrane is infiltrated by B and plasma cells ,6. In or+ memory B cells could contribute to the skewed distribution of the different B cell subsets in the RA synovial membrane, the frequencies of CD27+ memory B cells expressing specific chemokine receptors was also determined. Notably, expression of CXCR1, CXCR2, CXCR4 and CCR2 was significantly elevated in synovial compared with blood memory B cells (P < 0.00001) of either controls or RA patients (Table P < 0.02).Chemokine receptor expression by RA B cells can be indicative of their preferential capability for homing into the inflamed tissues. To assess whether the expression of chemokine receptors by RA synovial CD27ts Table . MoreoveP < 0.05) improvement of several laboratory and clinical parameters, including disease activity score, rheumatoid factor titres, ESR and the number of swollen and tender joints. By contrast, after the sixth treatment, the group receiving MTX monotherapy manifested significant (P < 0.05) improvement only in disease activity score and in the number of tender joints.As shown in Table +CD27- naïve B cells, IgD+CD27+ pre-switch memory B cells and IgD-CD27+ post-switch B cells. Before any of the therapies had been initiated all RA patients and healthy controls had a comparable frequency of naïve B cells . Similarly, the frequency of the post-switch memory B cell population in all RA patients before treatment was comparable to healthy donors , whereas the pre-switch memory B cell subset was significantly (P < 0.03) lower in RA patients than in the control individuals .In order to determine whether anti-TNF therapy had an effect on the distribution of the different peripheral blood B cell subsets in RA, the frequencies of those subsets were calculated after six administrations of study drug Figure . At the P < 0.01) increased the frequency of total and pre-switch peripheral memory B cells after the sixth round of treatment (median baseline 11.2 ± 1.5% vs after treatment 14.7 ± 1.8%) However, no changes could be observed in the patients receiving MTX alone (median baseline 5.4 ± 3.1% vs after treatment 5.9 ± 2.4%).When compared with the baseline values from the first visit, the RA patients receiving infliximab plus MTX significantly .+ memory B cells, have been reported in several autoimmune diseases including RA [+CD27+ memory B cells when compared with healthy individuals. Importantly, memory B cells accumulate in the synovial membrane of subjects with RA, suggesting that accumulation of pre-switch memory B cells within inflamed tissue may contribute to a decrease in this B cell subset in the blood. It should be pointed out that post-switch IgD-CD27+ memory B cells are also enriched in the rheumatoid synovium, although these cells are not decreased in the blood in early RA. This discrepancy may be explained by the more complex homeostasis of post-switch memory B cells. Although these cells accumulate in the synovium, they are also generated in increased numbers in patients with RA [Abnormal distributions of peripheral B cell subsets, particularly of CD27uding RA -18,20,21uding RA -21. In t with RA . As a re+CD27+ and post-switch IgD-CD27+ B cells [-CD27+ memory B cells are post-germinal centre highly mutated memory B cells [+CD27+ subpopulation is still a matter of controversy. Despite some characterisation [+CD27+ memory population only participates in T cell-independent immune responses, because this population expresses heavily mutated Ig genes [+ memory cells in the peripheral blood [-CD27+ memory subset, whereas the IgD+CD27+ subset was reported to be comparable to healthy donors [+CD27+ subset. At baseline the group of patients engaged in the clinical trial and those with shorter disease duration had similarly lower frequencies and absolute numbers of peripheral blood pre-switch IgD+CD27+ memory B cells compared with the group of patients with long-standing disease, so this abnormality would seem to be an integral feature of RA, independent of disease duration. Preliminary assessment of a group of patients with very early RA, who had disease duration of less than six weeks and had received no DMARD therapy, also indicated a decrease in IgD+CD27+ pre-switch memory B cells and is consistent with the conclusion that this abnormality in memory B cell homeostasis is characteristic of RA independent of disease duration and DMARD therapy . Notably, in none of the analysed RA patient groups did we observe an increase in the total CD27+ memory B cells when compared with control subjects. Nevertheless, with long-standing disease in both the National Institutes of Health and Japanese cohorts, the post-switch IgD-CD27+ population was increased. This is likely to be related to the increased production of post-switch memory B cells owing to persistent immunological stimulation that is sufficient to overcompensate for the enhanced sequestration of these cells in the synovium.The human memory B cell population is heterogeneous, comprising mutated pre-switched IgD B cells ,32, that B cells . It is w B cells ,13,32,34risation , it has Ig genes ,36. Howeal blood ,21, and y donors . We were+ memory B cells were analysed in patients receiving only MTX therapy, a remarkably broad range of distributions was noted, with some patients with very high and others with low frequencies [The disparities between our data and the results previously reported -21 may bquencies . Finallyquencies .+CD27+ B cells in RA patients could be the result of proteolytic cleavage of CD27  from th+CD27+ B cells, we have also observed significant shifts in the expression of several chemokine receptors in the RA peripheral blood B cell subsets and in the synovial memory B cells: the frequency of CD27+ memory B cells expressing the pro-inflammatory CXCR1, CXCR2 and CCR2 chemokine receptors [+ memory B cells in the RA synovium. CXCR4 is the receptor for the homeostatic and pro-inflammatory chemokine CXCL12 and is expressed by mature naïve B cells when they recirculate through germinal centres of secondary lymphoid organs [+ memory B cells increase CXCR4 expression upon differentiation into plasma cells [+ memory B cells may be recruited into the synovial membrane where they accumulate, and might be involved in seeding follicular-like structures and/or differentiating into autoantibody-secreting plasma cells, thus perpetuating the chronic synovitis.Chemokine receptor imbalances have been reported in several autoimmune diseases ,40-43. Aeceptors ,50 was eeceptors ), stressd organs . CXCR4 ed organs . Moreoved organs , whereasma cells . The impAnti-TNF therapy in RA has been linked to a reduction of B cells expressing the early activation marker CD23 in the peripheral blood . NeutralIn summary, the present findings indicate that the reduction of circulating memory B cells in RA patients, particularly the pre-switch memory subset, might be linked to their accumulation in the inflamed rheumatoid synovium under the influence of TNF.H: immunoglobulin heavy chain variable region; IL: interleukin; LT: lymphotoxin; MAb: monoclonal antibodies; MTX: methotrexate; PBMC: peripheral blood mononuclear cells; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PE: phycoerythrin; PerCpCy5.5: peridinin-chlorophyll-protein Cy5.5; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; SS: Sjögren's syndrome; TNF: tumour necrosis factor; VCAM: vascular cell adhesion molecule; VEGF: vascular endothelial growth factor.APC: allophycocyanin; BSA: bovine serum albumin; CRP: C-reactive protein; DMARD: disease-modifying anti-rheumatic drugs; DMEM: Dulbecco's Modified Eagle's Medium; ELISA: enzyme-linked immunosorbent assay; ESR: erythrocyte sedimentation rate; FCS: fetal calf serum; FDC: follicular dendritic cell; FITC: fluorescein isothiocyanate; ICAM: intercellular adhesion molecule; IgVThe authors declare that they have no competing interests.MMS-C collected data, designed experiments, carried out statistical analysis and wrote the manuscript. VM carried out the clinical study and collected data. RG-M designed and carried out the clinical study. RF, KT, TN, MW, MB and TAF collected patient data. PEL designed and coordinated the study and wrote the manuscript.