Whole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC  using this approach.RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband's daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband's husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years).We sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.The results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of  RET) protooncogene containing 21 exons is mapped on chromosome 10q11.2 and encodes a tyrosine kinase receptor that plays a crucial role in regulating cell proliferation, migration, differentiation, and survival through embryogenesis RET are found in multiple endocrine neoplasia type 2 (MEN 2) and Hirschsprung disease (HSCR) in an autosomal dominant pattern RET mutations are dispersed throughout the gene with poor genotype-phenotype correlation RET mutation typically occurring at one of the five cysteine residues . Biochemical and biological analyses revealed that these cysteine mutations induce disulfide-linked dimerization of the RET protein, leading to constitutive activation of its intrinsic tyrosine kinase activity RET mutations of codons 805, 806, and 904 in cis configuration with the p.V804M mutation have also been reported in individuals with MEN 2B et al found a p.V292M mutation within exon 5 in an Italian man with MTC and PHEO and suggested that the mutant had low-grade transforming potential RET mutations appear to show modulation of the degree of aggressiveness and the development of unusual features of MTC The REarranged during Transfection  were randomly fragmented by Covaris to provide DNA fragments with a base pair peak at 150 to 200 bp, then adapters were ligated to both ends of the resulting fragments. The adapter-ligated templates were purified by Agencourt AMPure SPRI beads, and fragments with an insert size of ∼200 bp were excised. Extracted DNA was amplified by ligation-mediated PCR (LM-PCR), purified, and hybridized to the SureSelect Biotinylated RNA Library for enrichment; hybridized fragments were bound to strepavidin beads whereas non-hybridized fragments were washed out. Captured LM-PCR products were subjected to an Agilent 2100 Bioanalyzer to estimate the magnitude of enrichment. Each captured library was then loaded onto the Hiseq2000 platform, and we performed GA sequencing for each captured library independently to ensure each sample had at least 50-fold coverage. Raw image files were processed by Illumina Pipeline v1.6 for base-calling with default parameters and the sequences of each individual were generated as 90 bp paired-end reads.http://soap.genomics.org.cn/.SOAPaligner (soap2.21) was used to align the clean reads to the human reference genome. Based on the SOAP alignment results, the software SOAPsnp was used to assemble the consensus sequence and call genotypes in target regions http://www.ncbi.nlm.nih.gov/Ftp/). Target regions were from the captured regions of the Agilent Exome Array (http://www.nimblegen.com/downloads/annotation/seqcap_exome/index.html). The exome variation set of eight HapMap samples was obtained from “Supplementary data 2” of 12 exomes in Ng et alData from the dbSNP129 and 1000 Genome Project (20100208 release) were downloaded from the NCBI FTP site  respectively, and performed polymerase chain reaction (PCR) amplification of all RET exons followed by Sanger sequencing, restriction endonuclease digestion, reverse transcription PCR (RT-PCR)/sequencing, and haplotype analysis of four RET-flanking microsatellite markers .To further confirm the All new data have been deposited in GenBank.2 pneumoperitoneum; bilateral PHEO was confirmed by histopathological examination. In 2006, the proband's 52-year-old female cousin (III-20) underwent a total thyroidectomy with modified bilateral neck dissection based on the evidence of elevated Ct and CEA and multi-centric nodules with calcifications in both thyroid lobes but without adrenal abnormalities. In 2010, some previously hesitant members of the family  agreed to participate in biochemical testing, image examinations and RET screening (2 pneumoperitoneum (III-12) were performed. Histopathological examination revealed bilateral PHEO in III-12 and bilateral multi-focal MTC with lymph node metastases in III-15, III-20 and III-23, while IV-21 showed no evidence of lymph node metastases. All six patients  were followed up. Biochemical and imaging examination did not reveal any evidence of recurrence of MTC or PHEO, and showed no HPT during this period except for III-12. Biochemical examination of III-12 showed Ct was 1360 ng/L and CEA was 13.2 ng/mL. US-guided fine needle aspiration and cytology was suggestive of MTC with lymph node metastases.The proband III-12) was a 43-year-old woman who had been diagnosed as having MTC in 1995 with a 4-year history of a palpable neck mass in the right thyroid. Biochemical examination disclosed increased CEA  and Ct , while US scanning disclosed multi-centric hypoechoic nodules with calcifications in both thyroid lobes. Then a total bilateral thyroidectomy with modified neck dissection was performed and histopathological examination showed bilateral multi-centric MTC with lymph node metastases T2N1aM0, . Since s was a 43creening  and 2. ARET were found by WES in him and his daughter (IV-10) respectively in 2010, neck US screening was performed on all 6 carriers and the results disclosed multi-focal hypoechoic nodules (range 0.4–0.8 cm) in the upper third of left/right or both thyroid lobes in II-5, III-3, III-8 and III-11 but not in IV-3 and IV-7. Ct was normal while the pentagastrin-stimulated Ct was slightly elevated in II-5 and III-11. In 2010, a total thyroidectomy with modified bilateral level VI lymph node dissection was performed in II-5, then histopathological examination revealed bilateral MTC without lymph node metastases  and his family members have not felt any special abnormality to date. As p.V292M/R67H/R982C and p.C634Y/V292M/R67H/R982C mutations of  T1N0M0, . III-11  T1N0M0, . None ofWe excluded the possibility of HSCR in this family based on the fact that none of the affected individuals presented evidence of HCSR involving severe constipation, vomiting, abdominal distension, or enterocolitis. Meanwhile, cutaneous lichen amyloidosis was also excluded because there was no evidence of pruritic lichenoid skin lesion located on the upper back.RET gene and found four recurrent missense substitutions: c.G200A (p.R67H), c.G874A (p.V292M), c.G1901A (p.C634Y) and c.C2944T (p.R982C) (Sequence Read Archive accession number: SRA029363.2). Analysis to explain or eliminate the pathogenicity of the other 29 genes (including 90 NS/SS) in MEN2A is ongoing and further data will be reported elsewhere.We generated an average of approximately 4.0 billion bases of sequence per affected individual as paired-end, 88-bp reads, and the fraction of effective bases on target was about 64% with a minimum 53-fold of average sequencing depth on target. At this depth of coverage, more than 99% of the targeted bases were sufficiently covered to pass our thresholds for variant calling . We focuRET gene was completely consistent with the whole exome sequencing. The proband (III-12) carried the p.C634Y mutation within exon 11, and so did her family members III-15, III-20, III-23 and IV-21. Unfortunately and interestingly, the proband's husband (III-11) and his family members II-5, III-3, III-8, IV-3 and IV-7 carried the p.R67H mutation within exon 2, the p.V292M mutation within exon 5 and the p.R982C polymorphism within exon 18 . In comparison, the all-inclusive cost of WES per sample is 25000 Yuan (∼$3788), about twice as much as Sanger sequencing. However, WES would be a more cost-effective and robust method for a causative gene embracing more than 42 exons  such as NIPBL. Additionally, with rapid commercialization, the cost of WES for gene testing will be considerably reduced in the coming years We used WES here and successfully confirmed several pathogenic variants in a Chinese family with MEN 2A/FMTC. WES can serve as a genome-wide scan for the causative gene RET mutations responsible for MEN 2A are p.C634Y/R/W/F/S/G/L, which target to one of five cysteine residues in the extracellular domain of RETet alThe most frequent RET and is a gain-of-function alteration with weaker pathogenicity et al reported another mutation, p.G321R, within exon 5 in a three-generation Czech kindred with FMTC RET p.V292M/R67H/R982C mutation. In contrast to the previous literature, our patients presented with a later onset of MTC or CCH and showed no evidence of PHEO even in the 70-year-old male (II-5), while the previously reported case with p.V292M had PHEO before MTC RET double mutations associated with MEN 2 are summarized in RET polymorphisms RET or components of the RET signaling pathway have also been reported to modify the risk of MTC et al demonstrated that the transforming activity of RET with the cis p.V804M/Y806C mutation is dramatically higher than that of RET with a single p.V804M or p.Y806C mutation et al indicated that p.V778I might potentially affect the phenotype through an additive effect in the presence of p.V804M in a Spanish family cis double mutation carries a codon 634-like pattern of MTC development and is associated with increased susceptibility to unusually large bilateral PHEO et al concluded that the RET p.V804M/R844L cis double mutation predisposes the individual to the development of FMTC with a mild phenotype RET screening showed that cis p.R67H/R982C combined with p.V292M in cis co-segregated with the FMTC phenotype. Since we did not find PHEO, in contrast to the phenotype of p.V292M reported previously, we speculated that the cis p.R67H/R982C variant has a modifying effect on the disease and may inhibit the genesis of PHEO, although p.R982C seems to have normal biological and biochemical behavior in in vitro expression experiments Previous studies provided evidence that some variants play roles in modifying other variants in the genesis of MEN 2A or FMTC. The p.V292M mutation maps to the third cadherin-like domain of RET mutations may be associated with unusual MEN 2 phenotypes (et al believed that the RET trans p.C634R/V648I double mutation may have modified and contributed to the rare MEN 2A phenotype in a patient with bilateral adrenocorticotropin-producing PHEO and MTC in silico are moderately deleterious in isolation but severely deleterious in cis combination due to their distinctly synergistic effect trans. A “second hit” mechanism advocates that allelic imbalance between mutant and wild-type RET is a possible mechanism of tumorigenesis in some patients with MEN 2A-related MTC RET might play a neutralizing role and thereby partly compensate for the activating effects of the mutant allele, but this protective effect of wild-type RET may disappear in the trans double mutant, which, from another perspective, could explain the more aggressive pathogenicity of the p.C634Y/V292M/R67H/R982C compound mutation Previous studies have shown that double enotypes . The casenotypes , in contRET sequencing studies to the gene's entire coding region, even though initial testing of a patient with clinical signs of MEN-2 reveals RET hot-spot mutations, or when genotype-phenotype discrepancies emerge. Meanwhile, in the present study, the p.R67H/R982C polymorphism had a modifying effect on the disease and may inhibit the genesis of PHEO, and p.C634Y/V292M/R67H/R982C may lead to higher MTC pathogenicity due to the additive effect of p.V292M/R67H/R982C and p.C634Y in trans combination.Briefly, WES provides an instructive, comprehensive, fast and accurate method for genetic diagnosis of a disease whose pathogenic genes embrace a large cohort of exons. Our case suggests the importance of extending


Toll like receptors (TLRs) are pattern-recognition molecules that initiate the innate immune response to pathogens. Pulmonary surfactant protein (SP)-A is an endogenously produced ligand for TLR2 and TLR4. SP-A has been proposed as a fetally produced signal for the onset of parturition in the mouse. We examined the effect of interactions between SP-A and the pathogenic TLR agonists lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C))  on the expression of inflammatory mediators and preterm delivery. Three types of mouse macrophages  were treated for up to 7 hours with pathogenic TLR agonists with or without SP-A. SP-A alone had no effect upon inflammatory mediators in mouse macrophages and did not independently induce preterm labor. SP-A significantly suppressed TLR ligand-induced expression of inflammatory mediators (interleukin (IL)-1β, tumor necrosis factor (TNF)-α and the chemokine CCL5) via a TLR2 dependent mechanism. In a mouse inflammation-induced preterm delivery model, intrauterine administration of SP-A significantly inhibited preterm delivery, suppressed the expression of proinflammatory mediators and enhanced the expression of the CXCL1 and anti-inflammatory mediator IL-10. We conclude that SP-A acts via TLR2 to suppress TLR ligand-induced preterm delivery and inflammatory responses. Preterm birth is the most important cause of neonatal morbidity and mortality not due to congenital anomalies in the developed world Toll like receptors (TLRs) are a family of membrane bound proteins that recognize pathogen-associated molecular patterns and mediate innate immune responses th week of human pregnancy, reaching fully functional levels at about the 34th week ex utero survival of the fetus. It has been suggested that in mice, the appearance of fetal SP-A toward the end of gestation acts as an intrauterine signal for the onset of parturition via an inflammatory mechanism In addition to exogenous  ligands, TLRs can bind substances produced by the host (‘endogenous ligands’). Surfactant protein (SP)-A, an endogenous ligand for TLR2 SP-A is a member of the collectin family of proteins. Collectins have a C-terminal globular region with a carbohydrate recognition domain , a hydrophobic alpha-helical neck region, a collagen-like region with Gly-X-Y repeats and an N-terminal disulfide bond-forming region Using a well-validated mouse model of infection-induced preterm delivery, we demonstrated previously that combined activation of TLR2 and TLR3 using peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C)) yields dramatic synergy in the labor response and uterine expression of inflammatory mediators All procedures involving animals were approved by the NorthShore University HealthSystem Animal Care and Use Committee and conform to the Guide for Care and Use of Laboratory Animals .Three sources of macrophages were used for cell culture experiments: 1) the mouse macrophage cell line RAW 264.7 (American Type Culture Collection TIB-71); 2) thioglycolate-stimulated peritoneal macrophages from male mice of the following mouse strains : B6.B10ScN-Tlr4lps-del/JthJ (TLR4-KO), B6.129-Tlr2tm1Kir/J (TLR2-KO), C57BL/6J (wild-type (WT) controls), C3H/HeJ  and C3HeB/FeJ (WT control); and 3) amniotic fluid macrophages from the CD-1 mice on day 14.5 and 16.5 of pregnancy.2/95% air and were passaged every 2 or 3 days to maintain logarithmic growth. Prior to each experiment, cells (4×105 cells per well) were plated in triplicate in 6-well plates and cultured overnight.RAW 264.7 cells were cultured in DMEM High Glucose (GIBCO 11965-092) supplemented with 10% fetal bovine serum, 1% streptomycin and 1% penicillin in tissue culture flasks at 37°C in 5% CO2 inhalation and peritoneal exudate cells were isolated by flushing the peritoneal cavity with 15 ml ice-cold PBS. These cells were centrifuged, washed once, plated at a density of 1.5×106 cells per well and incubated for 2 hours in 6-well plates under similar conditions as described above, supplemented with 10 mM HEPES. Adherent cells were confirmed as macrophages using the F4/80 macrophage cell marker.Peritoneal macrophages were freshly harvested as previously described Amniotic fluid macrophages were harvested from pregnant CD-1 mice on days 14.5 or 16.5 of pregnancy. Amniotic fluid was aspirated from amniotic sacs using an 18-G needle, mixed in RPMI 1640 medium (ATCC 30-2001) in 12-well plates and incubated for 1 hour. Medium was changed and adherent cells were used for experiments. In separate incubations, these adherent cells were confirmed as macrophages using the F4/80 antibody macrophage cell marker.in-vitro experiments were conducted in triplicate and repeated twice (i.e. three triplicate experiments).Cells were cultured for up to 7 hours with either LPS , PGN , poly(I:C) , PGN plus poly(I:C), or medium. The effect of either simultaneous or sequential incubation with SP-A (20 µg/ml) was studied . All Viability of cultured cells was assessed using trypan blue dye exclusion. For RAW 264.7 cells, viability prior to plating was 95% and 5 hours after plating was as follows for each treatment group: 93.5% for control (medium) treatment; 90% for LPS - 91.3% for PGN; 92.5% for poly(I:C); 90.5% for PGN+poly(I:C); 90% for SP-A; 90.5% for SP-A+LPS; 88% for SP-A+ PGN+poly(I:C). For peritoneal macrophages, the corresponding viability values were: pre-plating: 92%; after plating: control - 90%; LPS −88.4%; PGN −87%; poly(I:C) –88.1%; PGN plus poly(I:C) −85%; SP-A −89.5%; SP-A+LPS –87.3%; SP-A+ PGN+poly(I:C) –83.21%. For amniotic fluid macrophages, the corresponding viability values were: after plating: control-87.69; LPS −84.8%. The differences between post-plating values were not statistically significant from each other.SP-A purification was done from bronchial lavage fluid collected from patients with alveolar proteinosis. The lavage fluid was donated by and was processed according to method reported by Condon At the end of cell culture experiments, medium was aspirated and cells were washed twice with PBS and then lysed in the wells with TRIzol reagent  to extract total RNA according to the manufacturer’s protocol. For tissue specimens, total RNA was extracted after homogenization in TRIzol reagent.The quantity and quality of the RNA were verified by spectrophotometry and formaldehyde gel electrophoresis, respectively. Two µg of total RNA were used as a template for cDNA synthesis. cDNA was prepared using random primers and the Moloney Murine Leukemia Virus (MMLV) reverse transcriptase system (Invitrogen). All PCR primers and probes were purchased from Applied Biosystems   Mm01302428; TNF Mm00443258; CXCL1 Mm00433859; IL-10 Mm00439614; Mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (20X) 4452339E). Use of TaqMan PCR Reagent Kits was in accordance with the manufacturer's manual. Reactions were performed in a 10 µL mixture containing 0.5 µL cDNA. Duplex RT-PCR was performed with one primer pair amplifying the gene of interest and the other an internal reference (GAPDH) in the same tube. Thermocycler parameters were 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Semi-quantitative analysis of gene expression was done using the comparative CT (ΔΔCT) method, normalizing expression of the gene of interest to GAPDH. PGN/poly(I:C) values were set to 100. PCR assays were performed in duplicate for each of the triplicate cell culture samples.Primary antibodies were as follows: phosphorylated ERK  and total ERK , IκBα , actin , F4/80  and IL-1β . Secondary antibodies were: goat anti-mouse IgG-HRP , goat anti-rabbit IgG-HRP , rabbit anti-rat IgG-FITC  and goat anti-rabbit IgG-FITC .g for 10 minutes at 4°C. Supernatants were collected and used as whole cell lysates for western blotting. Protein concentration was measured spectrophotometrically  at A280. Equal amounts of protein (50 µg) from cell lysates were separated by SDS-PAGE and blotted onto PVDF transfer membranes. The membranes were blocked either at room temperature for 1 hour or at 4°C overnight. 5% BSA or 5% nonfat dry milk in TBS-T buffer  was used as blocking buffer for the detection of phosphorylated and non-phosphorylated proteins, respectively. Blots were incubated with the appropriate primary and secondary antibodies either for 1–2 hour at room temperature or overnight at 4°C with slight agitation. Proteins were detected using the ECL western blot detection system  in a Storm imager .For western blotting from cell culture, cells were washed twice with cold PBS and lysed in ice-cold 1X radioimmune precipitation assay (RIPA) buffer  and phosphatase inhibitors I and II (Santa Cruz)). Lysates were collected and incubated on ice for 1 hour and then centrifuged at 10,000 After detection of signal for pERK, membranes were stripped by incubating in stripping buffer  at 50°C for 30–40 minutes. Following stripping, blots were washed five times for 10 minutes each with TBST buffer, redeveloped to check for the removal of pERK signal, and re-probed with primary antibody for total ERK. IκBα was detected on a separate membrane with actin as a loading control. Quantification of bands was performed using ImageQuant software (Molecular Dynamics). Background intensity was subtracted from each sample and then fold change was determined . Control values were set to 100. Each experiment was done twice in duplicate.5 cells per well) were plated in triplicate in 12-well plates, cultured overnight and stimulated with either LPS (5 ng/ml) or PGN plus poly(I:C)  for 4 hours in the presence or absence of SP-A (20 µg/ml). Supernatants were incubated with a detection substrate  at 37°C for 30 minutes, and relative SEAP activity was determined using a spectrophotometer at 620–655 nm.RAW-Blue™  is a mouse macrophage reporter cell line that stably expresses a secreted embryonic alkaline phosphatase (SEAP) gene inducible by the transcription factor NF-κB. RAW-Blue cells were cultured under similar conditions as mentioned above, in medium supplemented with Zeocin, a selection marker (200 µg/ml). Prior to each experiment, cells . Fluorescent antigen distribution was examined with a Leica DMLB florescence microscope , and images were captured using a Retina 1350B camera  and IPlab image acquisition/analysis software .For pregnancy outcome experiments, CD-1 female mice in estrus were selected by the gross appearance of the vaginal epithelium and were impregnated naturally. Mating was confirmed by the presence of a vaginal plug. Intrauterine (IU) injections were performed on day 14.5 of a 19–20 day gestation, as previously described in utero and the survival status of these retained fetuses  were recorded.Animals recovered in individual, clean cages in the animal facility. Observations were made twice-daily for both preterm delivery and maternal health status. Preterm delivery was defined as the finding of at least one fetus in the cage or in the lower vagina within 48 hours of surgery. Necropsies were performed either after delivery or at the latest by 48 hours after surgery. The number of fetuses delivered or remaining For experiments requiring tissue harvests, animals were euthanized 8 hours after surgery. The inoculated horn was incised longitudinally along the anti-mesenteric border. Gestational tissues (uteri (full thickness biopsies from the middle region), fetal membranes , fetuses and placentas) were harvested, washed in ice-cold PBS, flash-frozen in liquid nitrogen and stored at −85°C for later RNA extraction.P<0.05 was considered a statistically significant difference.Continuous variables (e.g. relative mRNA levels) were assessed with Student’s t-test or ANOVA or, when data were not normally distributed and two groups were compared, the Mann-Whitney U test. Categorical variables (e.g. preterm delivery) were analyzed using Fisher’s Exact Test. in vitro macrophage culture system , we performed sequential incubations in RAW 264.7 cells, with SP-A incubations occurring either before or after incubation with TLR ligands. In all cases, an intermediate washing step ) was performed between the first and second exposure . SP-A suTo test whether the anti-inflammatory effect of SP-A is TLR-mediated, we used peritoneal macrophages isolated from TLR2-deficient (KO), TLR4-KO and WT control mice . AdherenTo identify intracellular mechanisms underlying suppression of the inflammatory response by SP-A, we focused on two signaling events downstream of TLR activation. The first is nuclear translocation of the transcription factor NF-κB, which occurs upon phosphorylation and subsequent degradation of its inhibitor IκBα . Thus, degradation of IκBα can be used as an indirect measure of NF-κB activation. A second signaling event downstream of TLR activation is phosphorylation of extracellular signal-regulated kinases (ERK) -1 and ERK-2, mitogen-activated protein kinases that play important roles in cell proliferation, apoptosis, differentiation, cell migration, and gene expression.RAW 264.7 cells were cultured in the presence or absence of TLR ligands with or without supplemental SP-A. Degradation of IκBα resulting from treatment with LPS, PGN alone or PGN+poly(I:C) was reversed by SP-A . SP-A alTo address the possibility that the effects of SP-A might be compartment-specific , we isolated amniotic fluid macrophages from CD-1 mice on day 14.5 and 16.5 of pregnancy. Adherent cells were confirmed as macrophages using the specific F4/80 marker . IL-1β pin vitro anti-inflammatory effect of SP-A has an in vivo correlate, a well-validated mouse model of infection-induced preterm labor using local administration of bacterial products was employed. The right uterine horns of pregnant mice on day 14.5 of a 19–20 day gestation were injected with TLR ligands (either LPS or PGN+poly(I:C)) with or without SP-A. These injections were performed in the space between the uterine lumen and the fetal sacs. Delivery of one or more pups within 48 hours was considered preterm. This model been shown to reproduce faithfully many aspects of infection-induced preterm labor in women, including the expression of cytokines, prostaglandins and other mediators; the lack of dependence on a drop in circulating progesterone; neonatal brain injury; and other characteristics In order to test whether the p<0.02) or PGN+poly(I:C)  and led to higher rates of retention of fetuses in utero 48 hours after surgery , or SP-A plus PGN+poly(I:C) and washed. Total RNA was extracted and real-time PCR was performed to determine the relative quantitative expression of CXCL1, the pro-inflammatory genes IL-1β, CCL5 and TNF-α and the anti-inflammatory gene IL-10 . IL-10 win vivo. In macrophages (a key effector cell for infection-induced preterm labor), suppression of inflammation by SP-A is not due to sequestration of stimulating ligand by SP-A, is accompanied by decreased degradation of IκBα, and is TLR2 dependent.A substantial body of evidence has accumulated showing that TLR signaling plays an important role in infection- and inflammation-induced preterm labor. In the present study we report that SP-A suppresses TLR ligand-induced preterm delivery and the expression of inflammatory mediators while increasing the expression of anti-inflammatory mediators Other studies also report that the mechanism for inhibition of PGN-induced cellular responses by SP-A involves interaction of SP-A with TLR2 in vitro and in vivo studies An inhibitory effect of SP-A on the production of different inflammatory mediators has been reported in various ex utero survival, is an inflammatory signal for the onset of parturition. A potential role for SP-A in the extra-membranous space within the pregnant uterine lumen is unknown, as is the degree to which amniotic fluid SP-A crosses the fetal membranes or is otherwise transported to other compartments. The discrepant observations between the present in vivo study (in which SP-A was administered into the extra-amniotic space within the uterus) and that of Condon and colleagues (in which SP-A was injected into amniotic sacs) might be due to factors specific for these different compartments. Such factors might include soluble elements or differentially functioning resident inflammatory cells. However, in the present study we show that in amniotic fluid macrophages (the cells proposed by Condon to mediate the parturition-inducing effect of SP-A), the effect of SP-A is to suppress the expression of molecules that promote labor. It is also notable that in contrast to the findings of Condon in mice, in human pregnancies fetal macrophages are not observed to cross into maternal tissues Our results stand in contradistinction to those of Condon In summary, SP-A suppresses preterm labor and reduces inflammation in response to TLR ligands. This suppression is mediated, at least in part, by TLR2. These observations may hold a key toward preventing preterm birth, which remains the most important preventable cause of neonatal morbidity and mortality in developed countries. Understanding these molecular mechanisms may lead to interventions using SP-A or like molecules to prevent preterm birth.Figure S1Immunoidentification using the F4/80 macrophage cell marker. Adherent cells from peritoneal exudates were exposed to F4/80 or an isotype control antibody, each labeled with the fluorescent dye FITC. Top row: Fluorescent microscopy for FITC stain (green). Bottom row: DAPI nuclear stain (blue), merged with FITC. Original magnification: 200X. Bars: 10 µm.(TIF)Click here for additional data file.Figure S2The anti-inflammatory effect of SP-A is not mediated through TLR4. Expression by RT-PCR of IL-1β, CCL5 and TNF-α in WT and TLR4-defective peritoneal macrophages (C3H/HeJ mice) after treatment with PBS, LPS, or PGN+poly(I:C), with or without SP-A. Concentrations of reagents are provided in the text (Methods). P values were calculated by ANOVA and compare four treatment groups (KO or WT with or without SP-A) for each TLR ligand. n = 3 replicates per condition per experiment. Depicted is a representative figure from three repeat experiments. Error bars = standard deviation. Values for the PGN+Poly(I:C) group were set to 100.(TIF)Click here for additional data file.Figure S3Effect of SP-A on phosphorylation of ERK-1 and ERK-2 in macrophages stimulated with TLR ligands. (A) Western blots showing phosphorylated and total ERK1 and ERK2. Representative blots of 2 independent experiments, each with duplicate sets, are shown. (B) and (C) Densitometric analysis of the ratios of band intensities of phosphorylated to total ERK-1 (B) and ERK-2 (C). P values were calculated by t-test and compare exposures with and without SP-A. Error bars = standard deviation.(TIF)Click here for additional data file.Figure S4Immunoidentification using the F4/80 macrophage cell marker in amniotic fluid macrophages. Adherent cells from amniotic fluid were exposed to F4/80 or an isotype control antibody, each labeled with the fluorescent dye FITC. Top row: Fluorescent microscopy for FITC (green). Bottom row: DAPI nuclear stain (blue), merged with FITC. Original magnification: 200X. Bars: 10 µm.(TIF)Click here for additional data file.Figure S5Effect of SP-A on the TLR ligand-induced expression of inflammation markers in the fetal membranes. RT-PCR analysis of mRNA for IL-1β (A), CCL5 (B), TNF-α (C) and CXCL1 (D) normalized to GAPDH in fetal membrane collected from CD-1 mice on day 14.5 of a 19- to 20-day gestation 8 hours after intrauterine administration of either PBS or PGN+poly(I:C) with or without SP-A. No transcripts were detected for IL-10. Data depicted are expression levels of cytokine for individual mice. N = 7 for control, n = 11 for PGN/poly(I:C), n = 7 for SP-A and n = 12 for SP-A/PGN/poly(I:C). P values were calculated by Mann-Whitney U test and compare PGN+poly(I:C) with and without SP-A.(TIF)Click here for additional data file.


Moreover, we explored the possible influence of the 3′ untranslated region (3′UTR) of HLA-G, which has been associated with HLA-G expression, on AML susceptibility. Results showed that HLA-G-expressing DC-10 and CD4+ T cells are highly represented in AML patients with HLA-G positive blasts. None of the HLA-G variation sites evaluated was associated with AML susceptibility. This is the first report describing HLA-G-expressing DC-10 and CD4+ T cells in AML patients, suggesting that they may represent a strategy by which leukemic cells escape the host's immune system. Further studies on larger populations are required to verify our findings.Human Leukocyte Antigen-G (HLA-G) contributes to cancer cell immune escape from host antitumor responses. The clinical relevance of HLA-G in several malignancies has been reported. However, the role of HLA-G expression and functions in Acute Myeloid Leukemia (AML) is still controversial. Our group identified a subset of tolerogenic dendritic cells, DC-10 that express HLA-G and secrete IL-10. DC-10 are present in the peripheral blood and are essential in promoting and maintaining tolerance via the induction of adaptive T regulatory (Treg) cells. We investigated HLA-G expression on blasts and the presence of HLA-G-expressing DC-10 and CD4 It i T cells  and prol T cells , 8, and  T cells . APCs ov T cells –12. Our  T cells , 14. DC- T cells , 14. We  in vivo .Studies on solid tumors revealed that HLA-G could be potentially expressed by all tumors, either as membrane-bound or as soluble isoform , 17. In  HLA-G, several polymorphisms are present in the noncoding region of the gene both at 5′ upstream regulatory region (URR) and 3′ untranslated region (UTR), which may influence the HLA-G expression [ HLA-G mRNA stability [ HLA-G mRNA degradation [Despite low degree of genetic variability in the coding region ofpression . The mostability –30. In atability , 32. Theradation . Beside radation , 35.The 14 bp Ins/Del has been associated with tolerance in different clinical conditions including autoimmunity –38, path+ T cells, in the peripheral blood of AML patients at diagnosis. We also determined whether polymorphisms at 3′ UTR of HLA-G locus correlate with AML susceptibility.We investigated the expression of HLA-G on leukemic blasts and tolerogenic immune cells, DC-10 and CD4All protocols were approved by the institutional review board and samples were collected under written informed consent according to the Declaration of Helsinki. 22 patients affected by AML were included in this retrospective study and analyzed for biological and clinical characteristics. AML diagnosis was based on standard cytological criteria according to the French-American-British (FAB) classification. Patients' diagnosis was subclassified by morphological and immune phenotyping. None of the patients received medical interventions before the study. Patients' characteristics are listed in Tables Peripheral blood mononuclear cells (PBMCs) from AML patients were isolated by Ficoll density gradient centrifugation and cryopreserved in gas phase of liquid nitrogen to the time of analysis. Serum was obtained from the blood samples of AML patients by centrifugation and cryopreserved in gas phase of liquid nitrogen for ELISA test.Cytological analysis was performed using standard G-band karyotyping technique. Results were described according to the International System for Human Cytogenetic Nomenclature .dim according to Lacombe et al. [Frozen PBMCs were thawed in X-VIVO 15 medium , supplemented with 5% pooled AB human serum  and 100 U/mL penicillin/streptomycin , and washed twice in Phosphate Buffered Saline (PBS)  with 2% Fetal Bovine Serum (FBS) . PBMCs were initially incubated for 10 min at room temperature with FcR blocking reagent  and stained for additional 20 min at room temperature in the dark with monoclonal antibodies (mAbs) specific for the following human antigens: CD45 , CD16 , CD4 , CD14 , and HLA-G . Cells were identified using a multiparametric approach based on the combination of mAbs. Samples were acquired using a FACS Canto II flow cytometer , and data were analyzed with FCS express . Quadrant markers were set accordingly to unstained controls. Leukemic blasts were identified as CD45e et al. .β2-microglobulin or W6/32 mAbs , respectively. Supernatants from HLA-G transfected LCL721.221 [Levels of shed HLA-G1 and soluble HLA-G5 were determined by enzyme-linked immunosorbent assay (ELISA), as previously described , 56. To L721.221  and HeLaμL reaction containing 1X polymerase chain reaction (PCR) buffer , 0.2 mM dNTP mix , 1.5 mM MgCl2 , 0.8 U Taq Polymerase , and 1 μM of each primer . The initial denaturation step was carried out at 95°C for 3 min, followed by 30 cycles at 93°C for 60 s, 58°C for 60 s, and 72°C for 60 s and by a final extension step at 72°C for 10 min. The amplification product was evaluated using a 2.5% agarose gel, purified using a commercial kit  according to the manufacturer's instructions, and subjected to direct sequencing on both strands. All polymorphic sites observed at the 3′UTR were individually annotated and named according to previous reports [Genomic DNA was extracted from PBMCs using a commercial kit  according to the manufacturer's instructions. Briefly, 100 ng of genomic DNA was amplified in a 25  reports . U test for continuous variable and Fisher's exact test for categorical data. Differences were regarded as significant at P < 0.05. The results were analyzed using GraphPad Prism 3.0 .All results are presented as mean values ± SD. Comparison of parameters between subgroups of patients was performed using the nonparametric Mann-Whitneyn = 22; range 13.3–96.4%). Patients' PBMCs were analyzed by flow cytometry and the expression of HLA-G was determined on blasts identified as CD45dim cells with MEM-G/9 mAb, which detects the full-length HLA-G1 isoform. HLA-G expression on AML blasts less than 1% was considered negative. Results showed that HLA-G was expressed in 15 out of 22 (68.2%) AML patients, and percentage of HLA-G+ blasts varied from 1.5% to 59% (+ blasts (We investigated the expression of HLA-G on leukemic blasts from 22 patients affected by Acute Myeloid Leukemia (AML), referring to the San Raffaele Hematology and Bone Marrow Transplantation Program . The coh% to 59% . Althoug+ blasts . Neverth+ blasts and only in 1 patient with HLA-G− blasts . Previous reports indicated that HLA-G itself promotes the expression of HLA-G on T and myeloid cells [+ or HLA-G− blasts. Results showed a significantly higher frequency of HLA-G+ DC-10 in patients with HLA-G+ blasts as compared to those with HLA-G− blasts , which have been identified in the peripheral blood of healthy donors and patients [+ T cells in the peripheral blood of leukemic patients and of healthy donors was similar . In parallel, we detected HLA-G5 in 10 out of 18 patients with a mean value of positive samples of 2.33 ± 1.84 ng/mL (mean ± SD), and similar to sHLA-G, no differences were found between sera from patients with HLA-G+ and HLA-G− blasts , +3003 C/T (rs1707), +3010 C/G (rs1710), +3027 A/C (rs17179101), +3035 C/T (rs17179108), +3142 C/G (rs1063320), +3187 A/G (rs9380142), and +3196 C/G (rs1610696), previously described [ HLA-G locus, and particularly the 14 bp Ins/Del, have been associated with different clinical conditions including autoimmunity and pathological pregnancy. So far, limited information has been published on the association of HLA-G polymorphisms in tumor cells with the levels of HLA-G expression and/or clinical outcome of patients [To define whether variations in the 3′UTR of HLA-G are associated with HLA-G expression, we analyzed 8 polymorphic sites at theescribed , 35. Thens/Del rs04, +3003patients , 63, 64.+ CD4+ Tregs can be used to evaluate the effectiveness of anti-tumor therapies. Moreover, the analysis of HLA-G polymorphisms will allow the identification of specific HLA-G genotypes that could be associated with AML susceptibility.Results from this study further improve the knowledge on the role of HLA-G in promoting tolerance. Moreover, they opem new clinical perspectives: HLA-G expression can be used as prognostic tumor biomarker to monitor disease state or as therapeutic target for improving immune responses against leukemia. The expression of HLA-G on blasts and the analysis of DC-10 and HLA-G


Theileria is the only eukaryote known to transform its mammalian host cells. We investigated the host mechanisms involved in parasite-induced transformation phenotypes. Tumour progression is a multistep process, yet ‘oncogene addiction’ implies that cancer cell growth and survival can be impaired by inactivating a single gene, offering a rationale for targeted molecular therapies. Furthermore, feedback loops often act as key regulatory hubs in tumorigenesis. We searched for microRNAs involved in addiction to regulatory loops in leukocytes infected with Theileria parasites. We show that Theileria transformation involves induction of the host bovine oncomiR miR-155, via the c-Jun transcription factor and AP-1 activity. We identified a novel miR-155 target, DET1, an evolutionarily-conserved factor involved in c-Jun ubiquitination. We show that miR-155 expression led to repression of DET1 protein, causing stabilization of c-Jun and driving the promoter activity of the BIC transcript containing miR-155. This positive feedback loop is critical to maintain the growth and survival of Theileria-infected leukocytes; transformation is reversed by inhibiting AP-1 activity or miR-155 expression. This is the first demonstration that Theileria parasites induce the expression of host non-coding RNAs and highlights the importance of a novel feedback loop in maintaining the proliferative phenotypes induced upon parasite infection. Hence, parasite infection drives epigenetic rewiring of the regulatory circuitry of host leukocytes, placing miR-155 at the crossroads between infection, regulatory circuits and transformation.The intracellular parasite  Theileria is the only intracellular eukaryotic parasite known to transform its host cell into a cancer-like state. Infection by the T. annulata parasite causes tropical theileriosis, killing large numbers of cattle in North Africa and Asia, and the related T. parva parasite causes East Coast Fever. We investigated whether transformation of host bovine leukocytes was associated with deregulation of small, non-coding RNAs. We discovered that transformation by Theileria leads to upregulation of an oncogenic small RNA called miR-155 which is contained within the BIC gene. Parasite induction of the microRNA involves activation of the transcription factor c-Jun which controls the BIC gene promoter. We identified a new target for the miR-155; the DET1 protein which is responsible for degradation of the c-Jun factor. This leads to a regulatory feedback loop that is critical for the transformed phenotype of the infected cells. We show that miR-155 expression inhibits DET1 protein translation, leading to accumulation of c-Jun protein and activation of the BIC gene containing miR-155. This is the first study to report regulation of oncogenic non-coding RNAs by Theileria and the novel feedback loop underlying the parasite-induced transformation. Both infection and cancer have been extensively linked to the induction of microRNAs (miRs) which can exert diverse effects on cellular phenotypes by targeting many genes Theileria constitutes a powerful model system to explore the signaling and epigenetic mechanisms underlying transformed phenotypes T. annulata and T. parva infect bovine leukocytes leading to proliferative and invasive phenotypes which partially mirror lymphoma pathologies when injected into immunocompromised mice Theileria-infection causes hyperproliferation, invasiveness and escape from apoptosis, presumably through the manipulation of host cellular pathways Theileria-infected cells are curable by treatment with the theilericidal drug Buparvaquone (BW720c), which kills the parasite without any apparent toxicity towards host cells in vitro infection with T. annulata of BL3 cells, an immortalized, bovine B lymphocyte cell line. Specifically, we investigated whether the transformed phenotype of the Theilieria-infected cells is associated with deregulation of miRNA pathways. miRNA networks are affected by several parasites of the apicomplexa phylum  have been identified in humans and mice and contain the precursor hairpin encoding miR-155. BIC and miR-155 are overexpressed in lymphomas, including Hodgkin's lymphoma, and acute myeloid leukemia patients, as well as several solid tumours BIC gene contains a highly conserved recognition motif for the transcription factor AP-1 formed by heterodimers of Jun and Fos proteins The oncomir miR-155 is one of the best studied oncogenic miRNAs and it has been extensively linked to inflammation, induced by a range of bacterial pathogens and viruses miR-155 and BIC upregulation are features of cells infected by the parasite Theileria. We identified AP-1/Jun as a transcriptional regulator of BIC in these cells. We also identified a new miR-155 target, transcripts encoding the DET1 protein which is involved in targeting c-Jun for degradation by ubiquitination. Thus, miR-155 expression leads to DET1 down-regulation, accumulation of the c-Jun protein and activation of the BIC promoter. This feedback loop is essential for miR-155 oncogenic function and, thus, Theileria infection of the host leukocytes creates a transformed state involving addiction to both parasite and oncomiR.Here we show that Theileria-infected cells, we studied TBL3 cells which were derived by in vitro infection with T. annulata of BL3 cells, an immortalized, bovine B lymphocyte cell line. Treatment with the theilericidal drug Buparvaquone caused reduced proliferation in TBL3 cells, while it had no effect on the growth of the parental BL3 cells . The activity of the BIC promoter was significantly higher in TBL3 cells compared to non-infected BL3 cells . We found that Theileria-infected leukocytes.These results suggest that DET1 protein translation is directly targeted by miR-155 binding to the 3′UTR sequence. To confirm this at the protein level, we performed Western blot analysis; DET1 levels were reduced in TBL3 cells compared to BL3 cells . In TBL3BIC promoter. We tested this hypothesis in BL3 cells using the BIC-Luc reporter that we showed above was Theileria-regulated via AP-1   . Finallyng siRNA , middle.To test the functional significance of this miR-155-DET1-Jun loop, we investigated the effect of blocking the hubs in the loop on the ability of transformed TBL3 cells to form colonies in the soft agar assay. Transfection of parasitized TBL3 cells with either the miR-155 Sponge or DN c-Jun caused a dramatic decrease in the number of colonies . NotablyTheileria-induced transformation offers an attractive experimental model, as it appears that infection of host leukocytes is accompanied by a rewiring of the cellular circuitry Theileria-infected cells may highlight examples of oncogene addiction for future studies. We have extended this hypothesis to investigate microRNA pathways and identified molecular targets that create an addictive regulatory loop. This is the first study to show that Theileria manipulates host gene expression via microRNAs. This observation underlines the increasing importance being given to non-coding RNAs in the regulation of gene expression, inflammatory response and tumour cell phenotypes ToxoplasmaCryptosporidiumEimeriaC. parvum infection of epithelial cells was also shown to induce a range of host miRNAs which are regulated by NFκB-dependent transcription Theileria. Similarly, T. gondii was shown to induce transcriptional regulation of a distinct set of host miRNAs, whereas the related Neospora caninum parasite did not Helicobacter pylori infection, which is associated with gastric adenocarcinoma, also induces miR-155 expression in T cells, but via Foxp3 BIC gene, leading to increased miR-155 expression in both artificially infected bovine B cells and in naturally-infected bovine Thei macrophages. We provide evidence that miR-155 targets the DET1 protein, which leads to accumulation of the c-Jun protein and increased transcription of the miR-155-encoding BIC gene  is also driven by AP-1 activity Host cell infection by Theileria-infected cells. Human DET1 (de-etiolated 1) is a component of the Cul4A-DDB1 ubiquitin ligase complex and was shown to promote the ubiquitination and degradation of the proto-oncogenic transcription factor c-Jun Theileria infection. It is also possible that miRNA targeting of ubiquitination machinery may contribute to other aspects of Theileria-induced host signaling, such as effects on p53 and NFκB pathways We report here that miR-155 represses DET1 in Overexpression of miR-155 has been functionally linked to tumorigenesis and inflammation in animal models in vitro infection of the spontaneous bovine-B lymphosarcoma cell line, BL3, with Hissar stock of T. annulata. The culture conditions and B cell characteristics of these two cell lines have been described previously T. annulata naturally infected cow. Cell were provided by G Langsley . All cell lines were cultured in RPMI 1640 , supplemented with 10% heat-inactivated Fetal calf serum, 4 mM L-Glutamine, 25 mM HEPES, 10 µM β-mercaptoethanol and 100 µg/ml penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37°C. For all experiments, cells were seeded at a density of 3×105 cells/ml and exponentially growing cells harvested. The number of cells and viability, as judged by the trypan blue dye exclusion test, were determined by counting the cells in a Mallassen chamber. The anti-parasite drug Buparvaquone (BW720c) The TBL3 cell line was derived from 6 cells, which had been cultured in the absence or presence of the indicated agents, were harvested and RNAs were prepared with the Nucleospin miRNA kit  according to the manufacturer's instructions. The quality and quantity of the resulting RNAs were determined using a Nanodrop spectrophotometer. Oligonucleotides were designed (Supplementary For isolation of long (>200 nt) and small (<200 nt) cellular RNA, 5×10Cells were sonicated in 2× Laemmli buffer : 15 secs ON/30 secs OFF for 5 mins. Proteins extracts were resolved on 10.5% acrylamide/bis acrylamide SDS-PAGE gels and transferred to nitrocellulose membranes  in transfer buffer. Protein transfer was assessed by Ponceau-red staining. Membranes were blocked in Tris-buffered saline pH 7.4 containing 0.05% Tween-20 and 5% milk for 1 hour at room temperature. Incubations with primary antibodies were carried out at 4°C overnight using antibody dilutions as recommended by the manufacturer in Tris-buffered saline pH 7.4, 0.05% Tween-20 and 5% milk. Following 1 hour of incubation with goat-anti-rabbit peroxidase-conjugated antibody  at room temperature, proteins were detected by the chemiluminescence method  according to the manufacturer's instructions. Antibodies used in immunoblotting were as follows: Rabbit anti-DET1 , Rabbit Anti-c-Jun , Mouse Anti-αTubulin , Rabbit Anti-Active Caspase 3 , Mouse Anti-Ubiquitin (P4D1) . c-Jun or DET1/Tubulin ratios were calculated after western blot signal quantification with the Plot Lanes Analysis tool of Image J software (NIH).BIC promoter extends from −1556 to +166 and was cloned into pGL3basic . AP1 and NFκB point mutations were generated using the QuikChange II site-directed mutagenesis kit (Stratagene) as previously described 5′- ACTAGTACCCCTATCAGTCTAGCATTAAGGGTTTACCCCTATCAATGTAGCATTAACACAGAACCCCTATCAGAGTAGCATTAAGAGCAGACCCCTATCATTGTAGCATTAAGTGGAAACCCCTATCAACTTAGCATTAACCTTGAACCCCTATCAAGGTAGCATTAAGGACCAACCCCTATCATACTAGCATTAACGAGATACCCCTATCATCTTAGCATTAACCAGGTACCCCTATCAGGATAGCATTAAGGTGCTACCCCTATCAGCCTAGCATTAATCTAGA-3′) were cloned downstream from the GFP gene in the vector, pMSCV-puro-GFP-miRcntl between the NotI and EcoRI sites, as previously described www.flemingtonlab.com. Unique/different short spacer regions are included between inverted miR-155 sequences to prevent the formation of exact repeats (to prevent recombination events). A Flag-tagged c-Jun dominant negative (DN) mutant Δ169 cDNA was cloned into pCDNA1, and c-Jun cDNA into pHT108. These two plasmids were kindly provided by G Langsley . The DET1 gene was targeted using siRNA oligonucleotides against the bovine DET1 sequence (AAAACCACCTGTTTATCAAGT) and results were compared to transfection with a non-relevant ‘scrambled’ control siRNA.Most Luciferase reporters were constructs previously described Thei cells were transfected using Nucleofector kit solution L according to the manufacturer's instructions using Amaxa Nucleofector II device (program T-20) . BL3 and TBL3 cells were transfected using Neon Transfection kit . Cells were seeded in 24-well plates for 24 h, and then transfected or co-transfected with 1 µg of the indicated constructs for 36 h.Renilla reporter plasmid (Promega Ref: E6241). Luciferase assay was performed 36 h after transfection using the Dual-Luciferase Reporter Assay System  in a microplate luminometer. Relative luminescence was represented as the ratio firefly/renilla luminescence and then compared with the corresponding empty vector as a control.The cells are transfected with luciferase constructs with or without miR-155, Sponge, c-Jun, DN c-Jun and with or without Buparvaquone treatment. Efficiencies of transfections were normalized to Renilla activity by co-transfection of a pRL-TK Cells were collected and fixed in 3.7% Formaldehyde for 40 min on ice and then cold Ethanol 70% for 15 min at 4°C. The cells were stained with propidium iodide (50 µg/ml) and RNase A (1 µg/ml) for 15 min at room temperature. Flow cytometric analysis was done using a FACScan instrument  and CellQuest software.Total RNAs was prepared using QIAGEN RNeasy mini kit  according to the manufacturer's protocol from two separate Buparvaquone-treated samples of TBL3 cells. The quality and quantity of the RNA samples were assessed using the Experion machine . The microRNA expression profiling service from Dharmacon (Thermo Fisher Scientific) performed the miRNA microarray analysis.SPSS 19.0 program  was used for statistics. The results presented in all the figures represent the mean ± SEM of at least three independent experiments. Statistical analysis was performed using the paired-samples t-test to analyze the significant difference between the control and treatment groups. p values of <0.05 were considered statistically significant and are indicated with asterisks.2 in air, and control plates were monitored for growth using a microscope. At the time of maximum colony formation (10 days in culture), final colony numbers were counted manually after fixation with 0.005% Cristal Violet .A two-layer soft agar culture system was used. Cell counts were performed on a Malassen chamber. A total of 20,000 cells were plated in a volume of 1.5 ml +2× DMEM 20% Fetal calf Serum) over 1.5-ml base layer  in 6-well plates. Cultures were incubated in humidified 37°C incubators with an atmosphere of 5% COCells were treated for 3 h at 37°C with 20 uM MG132 and lysed 10 min on ice in the following buffer: 150 mM NaCl, 1% Nonidet P-40, 0,5% Deoxycholate, 0,1% SDS, 50 mM Tris-HCl pH 7,5, 20 mM NEM, 5 mM Iodoacetamide, 100 uM MG132, 2 mg/mL Pefabloc SC (Roche) and 5 ug/mL each Aprotinin, Leupeptin, Pepstatin. Equal amounts of total cellular proteins were immunoprecipitated with Rabbit Anti-c-Jun (E254)  coupled to protein G sepharose beads  for 90 min at 4°C. After three washes, immunoprecipitated proteins were eluted in Laemmli sample buffer at 95°C for 5 min, resolved by SDS-PAGE and analyzed by western blot using the indicated antibodies. Immunoprecipitation was repeated for three independent biological replicates.Transient transfected cells with indicated constructs were treated 30, 60 or 120 min with 100 µg/mL Cycloheximide, 36 h post transfection. Cells were lysed in Laemmli sample buffer, resolved by SDS-PAGE and analyzed by western blot using the indicated antibodies. Relative quantification indicates the c-Jun/Tubulin ratios calculated with Image J software (NIH) and c-Jun levels at time 0 was set as 1. Cycloheximide chase experiments were repeated for three independent biological replicates.Transient transfected TBL3 cells with miR-155 Sponge were treated 3 h with 20 µM MG132. Cells were lysed in Laemmli sample buffer, resolved by SDS-PAGE and analyzed by Western blot using the indicated antibodies. Relative quantification indicates the c-Jun/Tubulin ratios calculated with Image J software (NIH). MG132 treatment was repeated for three independent biological replicates.Cells were plated on Fibronectin coated slides and then fixed in PBS 3.7% Formaldehyde for 15 min at room temperature. Slides were rinsed in PBS and permeabilized with PBS 0.2% Triton X-100 for 5 min and then blocked for 30 min with PBS 1% SVF and 1% BSA to prevent non-specific staining. The slides were incubated with Mouse monoclonal anti-Ki67  in PBS 1% SVF and 1% BSA at room temperature for 40 min. After washing in PBS 0.2% Tween, the slides were incubated with Cy2 AffinyPure anti-mouse IgG  for 30 min. Slides were subsequently washed in PBS 0.2% Tween, mounted on slides and coverslipped with ProLong Gold Antifade Reagent with Dapi . Images of immunofluorescence staining were photographed with a camera attached to a fluorescent microscope (Leica Inverted 6000) and percentage of Ki67 positive cells was calculated. This staining was repeated for three independent biological replicates.Figure S1The effect of Buparvaquone treatment on the growth and survival of infected cell lines.(A) The parasite-infected THEI cells were grown in the presence or absence of Buparvaquone (+Bup) and cell numbers were monitored by counting live cells followed by trypan blue exclusion (left panel). Representation of flow cytometry analysis indicating the induction of apoptosis (sub-G1 population) and growth arrest (G1 population) of THEI cells following treatment with Buparvaquone (open histograms - right panel) . *p<0.05, **p<0.01 (B) BL3 and TBL3 were treated with Buparvaquone for 64 h and cycling cells were measured by immunofluorescence using an anti-Ki67 antibody. Quantification of Ki67-positive cells was monitored by fluorescence microscopy . *p<0.05, **p<0.01(TIF)Click here for additional data file.Figure S2Analysis of putative miR155 oncomiR target genes.(A) Conservation of the seed sequences of miR155 in the 3′UTR of predicted target genes, DET1, JARID2 and TP53INP1, identified by computational analysis, in human, cow, mouse and chicken sequences. (B) Luciferase reporters containing the 3′UTR of DET1, JARID2 and TP53INP1 were compared with a control Luciferease reporter (pMIR-REP-dCMV), demonstrating that Buparvaquone (+Bup) induced Luciferase activity in parasitized THEI cells . *p<0.05, **p<0.01 (C) Buparvaquone treatment had no effect on the mRNA levels of DET1 or TP53INP1 in the three different cell lines, as assessed by qPCR analysis for the bovine genes. Transcript levels in untreated cells are shown relative to the control and normalized against β-actin and β2M mRNA .(TIF)Click here for additional data file.Figure S3miR155 does not affect c-Jun transcription.(A) The overexpression of miR155 or siDET1 or the miR-155 Sponge had no effect on c-Jun mRNA levels in BL3 cells (left) or TBL3 cells (right), as assessed by qPCR analysis. Transcript levels are shown relative to the control plasmids or scrambled siControls (gray bars) and normalized against β-actin and β2M mRNA . (B) The miR155 Sponge had no significant effect on c-Jun mRNA levels in TBL3 cells treated or not with MG132, as assessed by qPCR analysis. Transcript levels are shown relative to the control plasmid and normalized against β-actin and B2M mRNA . (C) miR155 inhibition in TBL3 cells increased c-Jun ubiquitination. Transfected TBL3 cells were treated with MG132 for 3 h, followed by immunoprecipitation of endogenous c-Jun protein and immunoblot analysis with antibodies against Ubiquitin or c-Jun .(TIF)Click here for additional data file.Table S1Summary of additional microRNAs downregulated more than two-fold (Log2) upon Buparvaquone treatment in TBL3 or Thei cells. The table shows the known functions and known target genes and references.(PPT)Click here for additional data file.Table S2Oligonucleotide primer sequences used to analyze the expression of genes. List of oligonucleotide sequences (sense and antisense) used for PCR analysis.(PPT)Click here for additional data file.


Human enteroviruses (HEV) have been linked to hand, foot, and mouth disease (HFMD) in the Pacific and Southeast Asia for decades. Many cases of HFMD have been attributed to coxsackievirus A16 , based on only partial viral genome determination. Viral phenotypes are also poorly defined. Herein, we have genetically and phenotypically characterized multiple circulating CV-A16 viruses from HFMD patients and determined multiple full-length sequences of these circulating viruses. We discovered that the circulating CV-A16 viruses from HFMD patients are genetically distinct from the proto-type CV-A16 G10. We have also isolated circulating CV-A16 viruses from hospitalized HFMD patients and compared their virological differences. Interestingly, circulating CV-A16 viruses are more pathogenic in a neonatal mouse model than is CV-A16 G10. Thus, we have found circulating recombinant forms of CV-A16 (CRF CV-A16) that are related to, but different from, the prototype CV-A16 G10 that have distinct biological phenotypes. Coxsackievirus A16 (CV-A16) and enterovirus 71 (EV71) have been isolated and identified as the main causes of the large outbreaks of this disease.Hand, foot, and mouth disease (HFMD) is a common infectious disease that mainly affects children ≤5 years of age Human enteroviruses (HEV) belong to the picornaviridae family, which can be further divided into 12 subgroups on the basis of molecular typing: Enterovirus A, Enterovirus B, Enterovirus C,Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, Enterovirus H, Enterovirus J,Rhinovirus A, Rhinovirus B and Rhinovirus C. CV-A16, along with enterovirus 71 (EV71), CV-A2 and CV-A4, are members of the Enterovirus A subgroup. The first and prototype CV-A16 strain, CV-A16 G10, was isolated in South Africa almost 60 years ago Based on partial viral sequencing and/or serologic characterization, a significant proportion of the viruses from HFMD patients have been found to be related to the prototype CV-A16 G10 and have thus been classified as CV-A16 strains. Most reports of CV-A16 genetic characterization are based only on the VP1 or VP4 region In the present study, we have sequenced the full-length genome of eight new CV-A16 viruses from HFMD patients. Surprisingly, we have found that prevalent CV-A16 viruses are recombinants with circulating HEV-A viruses in which only one-third of the viral genome is related to CV-A16 G10. Furthermore, significant differences in pathogenicity were observed between the newly identified circulating recombinant CV-A16 viruses and the prototype CV-A16 G10 in a neonatal mouse model.This study has been approved by the Ethics Committee of the First Hospital of Jilin University. Written informed consent was obtained from the parents of all the children involved in our study. Eight throat swabs were collected from pediatric patients with clinical diagnoses of CV-A16-related HFMD at the First Hospital of Jilin University in 2010. All specimens were positive for CV-A16 VP1-based characterization assays 2. The cycling conditions consisted of 4 min at 95°C, followed by 35 cycles of 94°C for 30 s to 45 s, 50°C to 55°C for 30 s to 45 s, and 72°C for 1 to 2 min, and finally 72°C for 8 min. The PCR products were examined by agarose gel electrophoresis. The primers used for CV-A16 detection are listed in The enterovirus sequencing strategy used has been previously described The full-length enteroviral sequences were retrieved from Genbank (Genbank number in parentheses): shzh00-1 (AY790926), shzh05-1 (EU262658), GZ08 (FJ198212), coxsackievirus A2 (AY421760), coxsackievirus A3 (AY421761), coxsackievirusA4 (AY421762), coxsackievirus A5 (AY421763), coxsackievirus A6 (AY421764), coxsackievirus A7(AY421765), coxsackievirus A8 (AY421766), coxsackievirus A10 (AY421767), coxsackievirus A12 (AY421768), coxsackievirus A14 (AY421769), coxsackievirus A16 (U05876), enterovirus 71A (U22521), enterovirus 71B (AM396587), enterovirus 71C (DQ341359), enterovirus 76 (AY697458), enterovirus 89 (AY697459), enterovirus 90 (AY697460), enterovirus 91(AY697461), enterovirus 92 (EF667344), enterovirus 68 (AY426531), and poliovirus 1 (V01150).Bootscanning and nucleotide similarity tests were carried out with the Simplot 3.5.1 program Vero cells were used to isolate the Changchun024, Changchun045, and Changchun090 strains of the CV-A16 virus from the throat swabs of patients with HFMD in 2010. The viral samples were diluted in DMEM medium and filter-sterilized using 0.22-µm filters ; 300 µl of each filtered sample was inoculated into T25 flasks containing approximately 50% confluent Vero cells. Cultures were monitored daily for evidence of cytopathic effect. Culture supernatants from infected Vero cells showing cytopathic effect were collected, aliquoted, viral-titered, and stored at −80°C. Viral titer was determined according to the Reed–Muench method One-day-old specific pathogen-free (SPF)-level imprinting control region (ICR) neonatal mice  were randomly divided into several groups of three litters per group and 8–10 neonatal mice per litter. The neonatal mice were injected intracerebrally with different viral strains or DMEM medium. Body weight, activity, limb paralysis, morbidity, and death were noted for 21 days post-injection For immunohistochemical analysis, tissue samples were embedded in optimal cutting temperature (OCT) compound and frozen in liquid nitrogen. The frozen tissue samples were then cut into 4-µm sections, placed on poly-L-lysine-coated glass slides, and fixed in 3.7% paraformaldehyde. The endogenous peroxidase activity of the tissues was inhibited by treatment with hydrogen peroxide (2.5%). CV-A16 antigen was captured by rabbit anti-CV-A16 polyclonal antibody (made in our laboratory) and detected by a Streptavidin-Peroxidase anti-rabbit IgG Kit , followed by color development with diaminobenzidine for detection of the antigen-antibody reactions.To detect the viral loads of challenged mice, all tissues  were individually weighed and homogenized in sterile phosphate-buffered saline, disrupted by freeze-thawing, and centrifuged. RNA was extracted from the samples using TRIzol (Invitrogen) and viral load was determined by real-time PCR as previously described The sequences of the eight CV-A16 stains have been entered into the GenBank database under accession numbers KF055238-KF055245.Samples from all eight patients with HFMD were collected from the First Clinical Hospital of Jilin University in 2010. Their clinical features are listed in To characterize circulating CV-A16 related to HFMD, full-length viral sequences were obtained from these eight HFMD patients. Phylogenetic analysis indicated that these full-length CV-A16 sequences from Changchun are not closely related to the prototype CV-A16 G10. A neighbor-joining phylogenetic tree was constructed for the eight sequences, along with 19 reference sequences for HEV-A, using EV68 and poliovirus 1 as outliers . This anSince the new HFMD viral sequences contained the CV-A16 VP1 region (data not shown) but clustered within the HEV-A group but not with CV-A16-G10, we examined the viral sequences for evidence of recombination. Bootscanning was performed with the Simplot program to investigate the possibility of recombination within the changchun024 sequence. Various HEV-A sequences were used as reference sequences. The results indicated that the 5′UTR of the changchun024 sequence was closely related to CA4 . The P1 We therefore carefully examined the 5′-UTR. Phylogenetic analysis of the fragment 24–718bp in the 5′UTR showed a small cluster of circulating CV-A16 viruses with CA4 and CA14, with a bootstrap value of 74% . On the The bootscanning results indicated that the P2 and P3 regions of circulating CV-A16 viruses showed no significant similarity to CV-A16 G10 . To helpIn order to reconfirm our conclusion, we used a series of algorithms in the RDP program, including RDP,GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD, and 3Seq, to detect the recombination events. The results confirmed those obtained with Bootscan in the Simplot program: when the sequence of changchun024 was analyzed in the absence of reference sequences CV-A2, CV-A3, CV-A6, CV-A10, and CV-A12, seven algorithms detected significant recombination with EV71A at the P2-P3 region; when the sequence of changchun024 was analyzed in the absence of reference sequences EV71A, CV-A3, CV-A6, CV-A10, and CV-A12, recombination was detected in two regions: five algorithms detected recombination with CV-A2 in the P2 region and four algorithms detected recombination with CV-A2 in the P3 region. This was consistent with the bootscan results. Of particular interest was the recombination with CV-A2 detected in the 3D region, which has not been previously reported. In a similar fashion, when the sequence of changchun024 was analyzed in the absence of reference sequences EV71A, CV-A2, CV-A3, CV-A6 and CV-A10, recombination with CV-A12 was detected using six algorithms. All of the recombination events had high P-values .Since circulating recombinant CV-A16 viruses differ from CV-A16 G10 genetically, we were interested in whether these viruses also differed in their biological properties. We evaluated viral replication in multiple cell lines. Like CV-A16 G10, CV-A16 changchun045 and CV-A16 changchun090 replicated and caused a cytopathic effect in African green monkey kidney Vero cells, a cell line sensitive to most enterovirus infections . The CPE5 copies/mg tissue) than the loads in these muscles from CV-A16 G10-challenged mice, which contained no significant detection of CV-A16 G10. These findings are consistent with Changchun024 being much more virulent in this model system.As described above, the Changchun circulating CV-A16 strains are recombinant and differ from the prototype CV-A16 G10 in most regions of the viral genome. Although viral replication in certain cell lines appeared to be unaltered by the recombination, whether these recombinants have different pathogenetic potential still needed to be evaluated. To address this issue, we established a mouse model of lethal CV-A16 infection in order to compare the pathogenesis of the prototype CV-A16 G10 and the Changchun-circulating CV-A16 strains. We found that CV-A16 changchun024, which was isolated from a HFMD patient with viral meningitis, was highly pathogenic in newborn mice. To evaluate any possible differences in viral pathogenesis, we established a grading system for evaluating the clinical condition of the mice: no abnormalities (grade 0), lethargy and inactivity (grade 1), wasting (grade 2), limb shaking and weakness (grade 3), hind-limb paralysis (grade 4), and moribund or dead (grade 5) Coxsackievirus A16, like other enteroviruses, shows a high mutation rate during viral replication due to the deficiency of proofreading activity. In the current study, we have determined eight full-length sequences of CV-A16 viruses from recent HFMD patients. Our study revealed significant differences in the genomic structures between the prototype CV-A16 G10 and the currently circulating CV-A16 viruses. Further analysis of several CRF CV-A16 viruses isolated from hospitalized patients revealed similar viral phenotypes, which were distinct from that of CV-A16 G10.We first demonstrated that circulating CV-A16 viruses from recent HFMD patients in China are actually complex recombinant viruses involving multiple type A HEV, including CA4, CV-A16, and possibly EV71, CV-A2, CV-A3, CV-A6,CV-A10 or CV-A12. The 5′UTR region of these viruses had the highest similarity to CV-A4. Most of the structural protein (P1) region resembled that of the prototype CV-A16 G10 strain. However, the non-structural protein domains (P2 and P3) showed similarity to multiple type A HEV viruses but not CV-A16 G10.The detailed characterization of eight new CV-A16-related full-length viral sequences as well as three earlier CV-A16-related sequences from China confirm the previous report et al. and Cyril C.Y.Yip et al. claimed that the P2 and P3 regions of circulating CV-A16 strains in China were mainly from EV71A et al. found that only a 1.5-kb fragment  of these nonstructural domains was similar to EV71A The non-structural protein domains (P2 and P3) of circulating CV-A16 viruses are most divergent from the same regions in CV-A16 G10. The origin of these P2 and P3 regions is also not clear. Recent reports have suggested that circulating CV-A16 strains are recombinant viruses that have recombined with different HEV-A type viruses. However, these reports are not in total agreement concerning the possible origin of the P2 and P3 regions in these circulating recombinant CV-A16 viruses. Chan Yoke-Fun Despite the fact that only a third of the viral genome of the circulating CV-A16 viruses shares similarity with CV-A16 G10, these viruses had a similar replication capacity in multiple mammalian cell lines. All the viruses replicated well in human neuroblastoma SK-N-SH cells and African green monkey kidney Vero cells, and none could efficiently replicate in the mouse fibroblast cell line L929. Thus far, two cellular receptors have been identified for CV-A16 and EV71 viruses: human scavenger receptor B2 (SCARB2) and P-selectin glycoprotein ligand-1(PSGL-1) Surprisingly, circulating CV-A16 viruses were more pathogenic than the prototype CV-A16 G10 in neonatal mice despite similar replication patterns in tissue culture. Circulating CV-A16 viruses induced more disease-related symptoms in neonatal mice than did CV-A16 G10. Furthermore, whereas circulating CV-A16 viruses produced a 100% death rate in infected mice, the majority of the CV-A16-G10-infected mice survived. To our knowledge, this is the first example of a positive or negative effect of a CV-A16 recombinant on viral pathogenesis in an animal model. It is well known that many enterovirus recombinants have gained activity related to viral replication or spread in animal models, and, while the implications of these findings for human disease have yet to be determined, our results raise the possibility of evolution to increased virulence. The 5′UTR and the nonstructural P2/P3 regions showed the most difference between CV-A16 G10 and the circulating recombinant CV-A16 viruses. The contribution of these regions to viral pathogenesis should be explored in future experiments. In addition, it is still not clear if the significantly higher viral loads in the spine muscle and hind limb muscle of CRF CV-A16-infected newborn mice are due to specific receptor expression or more suitable viral replication environments; similar results were also detected in our CV-A16 vaccine candidate studies To date, there is still no effective vaccine or drug against CV-A16 infection. The significant differences among CV-A16 viruses in cellular infection systems and animal models may be important factors for vaccine development and evaluation. The establishment of animal models of lethal CV-A16 infection that could distinguish between different CV-A16 viral strains would also be critical for subsequent studies of the pathogenesis of CV-A16. Further elucidation of the mechanisms that govern the differences in these different model systems may also shed light on our understanding of CV-A16 enterovirus recombination and viral pathogenesis.Figure S1Phylogenetic analysis of full-length genomic sequences of CV-A16 viruses.(TIFF)Click here for additional data file.Figure S2Characterization of circulating Changchun CV-A16 viruses with a similar origin as other enteroviruses in the P2 and P3 regions. (A) Bootscanning analysis of Changchun024 was performed with all HEVA sequences except EV71,CV-A2,CV-A6,CV-A10 and CV-A12. (B) Bootscanning analysis of Changchun024 was performed with all HEVA sequences except EV71,CV-A2,CV-A3,CV-A10 and CV-A12. (C) Bootscanning analysis of Changchun024 was performed with all HEVA sequences except EV71,CV-A2,CV-A3,CV-A6 and CV-A12.(TIFF)Click here for additional data file.Table S1A list of primers used for CA16 sequence PCR amplifications.(DOC)Click here for additional data file.


Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the β-chain has been shown to affect localization of the complex. We generated mice in which the endogenous β-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-β-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-β-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-β-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-β-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the β-chain is not essential for the function of class II MHC products in antigen presentation or antibody production. Class II major histocompatibility complex (MHC) molecules are expressed by professional antigen presenting cells (APCs), and are necessary for the presentation of antigenic peptides to CD4 T cells and the subsequent initiation of an adaptive immune response. The timing of class II MHC-dependent antigen presentation is carefully controlled in order to achieve specificity of immune responses; the regulated trafficking of class II MHC molecules is one aspect of such control Class II MHC molecules can be ubiquitinated on a single lysine residue that is present in the cytoplasmic tail of the β-chain (I-Aβ-K225) In dendritic cells, internalization of class II MHC from the cell surface is decreased when the cytoplasmic lysine of the β-chain is mutated It is clear from these previous studies that the ubiquitination of class II MHC dramatically affects its trafficking. These previously published reports have not, however, addressed the physiological role of this modification in vivo in the context of the immune system. In order to address this issue we generated a targeting construct with a mutant class II MHC β-chain tagged with EGFP in which the single cytosolic lysine was replaced by an arginine (K225R). This construct was knocked-in to the endogenous I-Aβ locus, creating the I-Aβ-K225R-EGFP mouse. We show that I-Aβ-K225R-EGFP mice retain normal populations of CD4 T cells and B cells, display unaltered efficiency of antigen presentation in vitro and are capable of in vivo generation of antibody responses.Ubiquitination of class II MHC regulates the localization of class II MHC molecules in both B cells and dendritic cells (DCs) bβ locus was previously targeted to generate an I-Aβ-EGFP expressing mouse bβ locus, followed by fused exons 5 and 6, in frame to the coding region for EGFP. The K225R mutation in the cytoplasmic tail of class II MHC was introduced into exon 4 of this construct. The long arm of homology and a short arm of homology corresponding to the 3′ untranslated region (UTR) of I-Aβ were cloned into the pACN-TV targeting vector. pACN-TV contains a self-excisable ACN cassette that consists of a neomycin resistance gene, and a gene for Cre recombinase under the control of a sperm-specific promoter flanked by loxP sites The I-ACorrectly targeted ES cell lines, as confirmed by PCR and Southern blotting, were injected into blastocysts to generate chimeric mice, which were then bred to obtain germline transmission of the targeted I-Aβ locus. The generation of the I-Aβ-K225R-EGFP mice was confirmed by sequencing of exon 4 of I-Aβ, which showed that the I-Aβ locus in these mice does in fact harbor the K225R mutation.The mice that received the I-Aβ-K225R-EGFP targeted allele were analyzed by flow cytometry to assess the cell specific distribution of GFP fluorescence. In splenocytes from both control I-Aβ-EGFP mice and I-Aβ-K225R-EGFP mice, all of the B220 positive B cells and CD11c positive DCs in the spleens of I-Aβ-K225R-EGFP mice were positive for GFP, while none of the CD3 positive T cells expressed GFP  indicatiIn order to confirm that the K225R mutation abolished ubiquitination of class II MHC molecules we crossed I-Aβ-EGFP and I-Aβ-K225R-EGFP mice with mice that express HA-tagged ubiquitin Altered ubiquitination of class II MHC molecules has been linked to changes in the levels of class II MHC that are present in the cell To confirm that the overall levels of class II MHC were increased in the I-Aβ-K225R-EGFP mice, we analyzed protein lysates from splenocytes by SDS-PAGE and immunoblotting . LysatesThe increase in class II MHC levels in I-Aβ-K225R-EGFP mice could be caused by a change in the synthesis, maturation or degradation of class II MHC. We analyzed cells from these mice by metabolic labeling in a pulse chase experiment, followed byimmunoprecipitation . Rates oSince ubiquitination has been implicated in the degradation of class II MHC molecules In order to determine the effects of ubiquitination on the localization of class II MHC molecules, we performed confocal microscopy on live bone marrow-derived dendritic cells (BMDCs). We took advantage of the fact that our targeted mice expressed EGFP tagged I-Aβ to allow us to visualize both wild type and K225R mutant I-Aβ in the same cell simultaneously. Dendritic cells that had been cultured for 5 days from either I-Aβ-EGFP or I-Aβ-K225R-EGFP mice were transduced with lentiviral vectors that express either I-Aβ-K225R-Cherry or I-Aβ-Cherry, respectively, to enable simultaneous imaging . Since tDisrupting the ubiquitination of class II MHC molecules alters both the total levels of class II MHC and their distribution. Does this change in class II MHC dynamics affect the function of class II MHC? We started to address this question by surveying lymphocyte populations in both I-Aβ-EGFP and I-Aβ-K225R-EGFP by flow cytometry. Total numbers and percentages of splenic B cells (B220), CD4 T cells, CD8 T cells and dendritic cells (CD11c) were comparable between the I-Aβ-EGFP and I-Aβ-K225R-EGFP mice . In addiWe next investigated antigen presentation by bone marrow-derived DCs from I-Aβ-K225R-EGFP mice. At day 5 the majority of BMDCs were immature as judged by low levels of cell surface CD80, and no difference was observed between the WT and KR dendritic cells (data not shown). These Day 5 BMDCs were incubated with ovalbumin (Ova) and were subsequently fixed. Fixed BMDCs were then incubated overnight with T cells isolated from OTII mice, whose T cell receptor recognizes the Ova 323–339 peptide-class II MHC complex. Activation of CD4 T cells was measured by upregulation of the cell surface marker CD69 . AntigenClass II MHC molecules play an essential role in the T cell-dependent antibody response. If correct class II MHC homeostasis is important for these responses then the altered class II MHC dynamics in I-Aβ-K225R-EGFP mice could affect the ability of animals to produce antibodies.We measured the levels of IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM in serum from 129, I-Aβ-EGFP, and I-Aβ-K225R-EGFP mice . No diffIn order to assess antigen-specific immunoglobulin production, mice were immunized with Ova and hen egg lysozyme (HEL). Serum was isolated from mice prior to immunization, again after a 4-week course of immunization, and was then analyzed for the presence of HEL- and Ova-specific IgG . The antIn order to test the ability of the I-Aβ-K225R-EGFP mice to generate an antibody response under more physiologically relevant infection conditions we assessed their ability to produce antibodies in response to infection with influenza. The influenza-specific antibody response was measured 14 days after inoculation with the influenza virus. There was no difference in the generation of influenza-specific IgG between I-Aβ-EGFP and I-Aβ-K225R-EGFP mice . In concPrevious studies have shown that class II MHC is ubiquitinated on the β-chain, which helps control the localization and expression of class II MHC molecules In order to understand the role of class II MHC ubiquitination in the context of the immune system we generated a mouse in which the endogenous I-Aβ locus was replaced with a mutant version of I-Aβ that lacks the cytosolic lysine residue that is essential for ubiquitination (I-Aβ-K225R-EGFP). As expected, we found that the K225R mutation abolishes ubiquitination of the class II MHC β chain in splenocytes. I-Aβ-K225R-EGFP mice have increased class II MHC levels due to decreased degradation, and show an altered distribution of class II MHC between endolysosomal compartments and plasma membrane in immature bone marrow-derived dendritic cells.Several possibilities can be envisioned for how altered trafficking dynamics might affect class II MHC function: altered dynamics of class II MHC recycling may affect the ability to present antigens to T cells, alterations in the levels and efficiency of class II MHC molecules in the thymus could lead to skewing of the overall CD4 T cell population. In the context of an immune response, the ability of animals to produce antibodies in response to antigen challenge could be altered.However, altered localization and trafficking of class II MHC in bone marrow derived dendritic cells from I-Aβ-K225R-EGFP mice did not affect antigen presentation to T cells, notwithstanding increased levels of class II MHC at the plasma membrane. Perhaps the population of class II MHC that is ubiquitinated is intended for degradation, or destined for some other fate that is not immediately relevant for the efficiency of antigen presentation in BMDCs.A recent report by Ohmura-Hoshino et al. The differences in antigen presentation between splenic and bone marrow-derived DCs observed by Ohmura-Hoshino et al. may be partially attributable to differences in DC development. In addition, MARCH I may well target substrates other than class II MHC. Using our in vitro differentiation protocol we have bypassed the effects of DC interaction with the splenic microenvironment, and thus we can argue that our system allows us to directly investigate the effect of class II MHC ubiquitination on antigen presentation, rather than on dendritic cell development.We found that development and numbers of CD4 and CD8 T cells are normal in the I-Aβ-K225R-EGFP mice. If the dendritic cells in the thymus were defective in antigen presentation a change in the number of CD4 T cells would be expected Additionally, we find that antibody responses are normal in mice that cannot ubiquitinate class II MHC. Antibody generation requires efficient class II MHC-mediated antigen presentation by both B cells and dendritic cells, and thus the ability of the I-Aβ-K225R-EGFP to generate antibody responses indicates that in vivo the level of antigen presentation by class II MHC is sufficient at least to support the antibody response.In conclusion, our experiments yielded no evidence that the alterations in class II MHC levels and their trafficking affected efficiency of antigen presentation per se, or the ability of mice to generate antibody responses. As previously published results imply a role for class II MHC ubiquitination in the regulation of dendritic cell functions in vivo C57BL/6J mice were obtained from The Jackson Laboratory. I-Aβ-EGFP mice Bone marrow dendritic cells (BMDCs) were prepared by isolating cells from the femurs and tibias of mice. Cells were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS, penicillin/streptomycin, 10 ng/ml recombinant mouse GM-CSF (Peprotech), and 1 ng/ml recombinant mouse IL-4. Media was partially replaced every two days. BMDCs were used for experiments after 5 days in culture.CCGTCACAGGAGTCAGAGAGGTGAGGAGCTC, rev-GAGCTCCTCACCTCTCTGACTCCTGTGACGG. The long arm of homology was inserted upstream of the ACN cassette, while the short arm of homology was inserted downstream of the ACN cassette. The diptheria toxin (DT) negative selection marker was cloned from this pACN-TV and a second DT gene was added into the construct after the short arm of homology so that the targeting construct was flanked by DT selection genes. Linear targeting constructs were introduced into C57BL/6J/129 F1 ES cells GATTCGCAGCGCATCGCCTTCTATC, rev-GTCAGCTGCACCACTTCTGTTAGGTCTCAC. The positive ES cell lines were further screened by southern blotting for the correctly targeted long arm of homology using the neomycin resistance gene as a template for probe generation. The generation of the I-Aβ-K225R-EGFP mice was confirmed by sequencing of exon 4 of I-Aβ using the following primers to amplify the region, and the fwd primer to sequence the region: fwd-TATACACTGGGGCCCTGGAACTTG and rev-CAGCTCCTCGCCCTTGCTCAC.The targeting vector pACN-TV 2, 0.5% NP40) supplemented with protease inhibitor cocktail (Roche) and 10 mM N-Ethylmaleimide (NEM). The I-Aβ complex was immunoprecipitated with JV1 antiserum (directed against the class II MHC α chain) and ProtA beads (Repligen). The protein complex was eluted off the beads by addition of a small volume NP40 buffer with 1% SDS, 20 mM DTT and10 mM NEM. The eluate was subjected to a second round of immunoprecipitation with an antibody directed against GFP (Abcam) and ProtA beads. Total lysates and immunoprecipitates were subjected to SDS-PAGE and immunoblot analysis.I-Aβ-EGFP mice Virus preparation and transductions were performed as described previously Images were acquired using a spinning disk confocal microscope and a 3-W water-cooled laser with an acoustic-optic tunable filter (Prairie Technologies). The system incorporated a Nikon TE2000-U inverted microscope using a Nikon ×100 magnification, 1.4 numerical aperture, differential interference contrast oil lens. Nikon type A immersion oil was used (Nikon). Images were acquired and processed using Metamorph software (Molecular Devices). Images were subsequently cropped in Adobe Photoshop and figures were constructed in Adobe Illustrator.bβ-PE (BD Pharmingen). Data were collected on an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TriStar).Cells were incubated with Fc block antibody (BD Pharmingen) before FACS staining. Antibodies used for FACS staining were anti B220-PerCP-Cy5.5, anti B220-APC, anti CD11c-APC, anti CD3-PE, anti CD4-APC, anti CD8-PerCP-Cy5.5, CD69-PE, anti-I-A35S]-methionine/cystine (Perkin-Elmer) for 30 minutes. After labeling, cells were incubated in chase medium containing unlabeled methionine (2.5 mM) and cysteine (0.5 mM). At the end of each chase interval, cells were lysed in NP-40 lysis buffer  supplemented with protease inhibitor cocktail (Roche). Lysates were then analyzed by immunoprecipitation with rabbit anti-I-Aα serum (JV1) Pulse-chase experiments were performed as described Cells were lysed in NP-40 lysis buffer supplemented with protease inhibitor cocktail. The protein concentrations of the supernatants were determined by BCA assay (Pierce). Samples were boiled in SDS-PAGE sample buffer  with β-ME and separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes, blocked in 2% (wt/vol) milk in PBS, and immunoblotted with the indicated antibodies and appropriate horseradish peroxidase-conjugated secondary antibodies. Antibodies used are rabbit anti-GFP antibody (Abcam), rabbit anti-I-Aα serum (JV1), rabbit anti-I-Aβ serum (JV2) Day 5 BMDCs were incubated with ovalbumin at the concentrations and times indicated, BMDCs were subsequently fixed with 3% paraformaldehyde and then extensively washed. T cells were isolated from the lymph nodes and spleens of OT-II mice using the Pan T Cell Isolation Kit (Milteni Biotec). T cells were incubated with fixed BMDCs overnight. Cells were harvested by pipetting and were stained for flow cytometry with vβ5.1+5.2-FITC, CD4-FITC, CD69-PE (BD Pharmingen). Data was collected on FACS Calibur flow cytometer (BD Biosciences) and analyzed with FlowJo software (TriStar). Cells were gated on CD4 positive cells and analyzed for surface expression of CD69.On day 1 mice were immunized intraperitoneally with 10 µg each of Ovalbumin (Ova) and lysozyme (HEL) mixed with complete Freund's adjuvant (Sigma) and on day 21 with 10 µg each of Ova and HEL mixed with incomplete Freund's adjuvant. Sera were collected on day 0 and 28 for analysis by ELISA.Mice were anesthetized by a single dose of 0.4–0.6 mg/g body weight of avertin , delivered by intraperitoneal injection. Infections were accomplished by intranasal instillation of influenza virus (Influenza A/PR8/34 or Influenza A/WSN/33) in up to 50 µl of PBS over a 10 minutes period. Weight of infected animals was monitored daily. Blood was extracted from the tail vein or by cardiac puncture after the animals were sacrificed.Plates were coated with either anti-mouse Ig antibodies (Southern Biotech), Ova, HEL or whole influenza particles. Bound antibodies were detected with HRP-conjugated isotype specific antibodies (Southern Biotech). ELISA plates were read using SpectraMax M2 microplate reader (Molecular Devices).