Sex differences in spontaneous sleep amount are largely dependent on reproductive hormones; however, in mice some sex differences in sleep amount during the active phase are preserved after gonadectomy and may be driven by non-hormonal factors. In this study, we sought to determine whether or not these sex differences are driven by sex chromosome complement. Mice from the four core genotype (FCG) mouse model, whose sex chromosome complement  is independent of phenotype , were implanted with electroencephalographic (EEG) and electromyographic (EMG) electrodes for the recording of sleep-wake states and underwent a 24-hr baseline recording followed by six hours of forced wakefulness. During baseline conditions in mice whose gonads remained intact, males had more total sleep and non-rapid eye movement sleep than females during the active phase. Gonadectomized FCG mice exhibited no sex differences in rest-phase sleep amount; however, during the mid-active-phase (nighttime), XX males had more spontaneous non-rapid eye movement (NREM) sleep than XX females. The XY mice did not exhibit sex differences in sleep amount. Following forced wakefulness there was a change in the factors regulating sleep. XY females slept more during their mid-active phase siestas than XX females and had higher NREM slow wave activity, a measure of sleep propensity. These findings suggest that the process that regulates sleep propensity is sex-linked, and that sleep amount and sleep propensity are regulated differently in males and females following sleep loss. Reproductive hormones, particularly in females, exert regulatory influences on the sleep-wake cycle. As early as 1969, Colvin et al. In 2006, we reported that male mice exhibited more daily NREM sleep than female mice and that gonadectomy (GDX) in males and females eliminated this sex difference Sleep propensity is an inclination to sleep that is driven by: 1) the circadian timing system and 2) homeostatic pressure to sleep that builds during extended wakefulness. Although sleep amount is an indicator of sleep propensity, the most reliable measure is NREM slow wave activity (SWA), which measures the spectral power of delta (.5–4 Hz) oscillations in electroencephalographic (EEG) recordings during NREM sleep. In 2006, we reported that GDX reduced sex differences in absolute values of NREM SWA, but that the dissipation of SWA during recovery from sleep deprivation occurred more quickly in GDX males than in GDX females.In the current study, we sought to examine the role of sex linkage in regulating sleep phenotypes by testing the hypothesis that sex differences that remain in GDX mice are driven by sex chromosome complement. To accomplish our goal we took advantage of the four core genotype (FCG) mouse line in which the sex chromosome complement is independent of gonadal sex Sry” from the Y chromosome of MF-1 outbred mice and the subsequent insertion of the Sry transgene onto an autosome Sry develop ovaries. When XX mice have an Sry autosomal transgene, (these mice are designated XXM) they develop normal functioning testes. Conversely, XYF mice lack the Sry autosomal transgene and consequently develop ovaries. The FCG mouse line is comprised of four primary genotypes: XXF , XYF  and XYM (XY mice with Sry and testes). This study examined gonadally intact (XYM (n = 3), XYF (n = 3), XXM (n = 3) and XXF (n = 3), and GDX (XYM (n = 7), XYF (n = 8), XXM (n = 10) and XXF (n = 10)) mice. Food and water were available ad libitum.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols and procedures were approved by the Morehouse School of Medicine Institutional Animal Care and Use Committee (NIH OLAW Assurance Number A3944-01). All efforts were made to minimize pain and suffering. Adult FCG breeder mice on a C57BL/6J background were kindly donated by Dr. Arthur Arnold  and the line was bred at Morehouse School of Medicine and maintained on a 12-hour light:12-hour dark (12L:12D) schedule throughout the study. This mouse line was initiated by a spontaneous deletion of the testis determining gene “Animals (8–10 weeks of age) were deeply anesthetized with an intraperitoneal injection of ketaset (80 mg/kg) and xylazine (8 mg/kg). In male mice, a vertical incision was made at the midline of the abdomen, and fatty and connective tissue were removed to expose the inner sacs that encase the testicles. A second incision was made through each casing, and the testes fat pad and epididymes were expressed to the outside and removed. The area was cleaned, and the incisions were closed using silk suture. In female mice, an incision was made dorsally through the skin and muscle just above the location of the ovary. The ovary was expressed to the surface and ligated using a silk suture. The ovary was removed, the uterine horn was returned to the cavity, and the muscle and skin layers were sutured.Immediately following GDX, EEG and EMG electrodes for polysomnographic recording of sleep–wake states were implanted in anesthetized mice. A prefabricated head mount  was used to position four stainless steel epidural screw electrodes. The first 2 electrodes  were located 1.5 mm anterior to Bregma and 1.5 mm on either side of the central suture whereas the second 2 electrodes  were located 3.5 mm posterior to bregma and 1.5 mm on either side of the central suture. Electrical continuity between the screw electrodes and headmount was aided by the use of silver epoxy. EMG activity was monitored using stainless-steel Teflon coated wires integrated into the headmount and inserted bilaterally into the nuchal muscle. The headmount (integrated 2x3 pin grid array) was secured to the skull with dental acrylic. In order to control for pain, over the next 36 h mice were given Buprenex  once every 12 h. All procedures were performed on a heating pad and mice were allowed to recover for at least 14 days before being transferred to sleep recording chambers. A separate cohort of FCG mice with intact gonads was also implanted with recording electrodes.Following recovery (at least 14 days), the mice were placed in a sleep-recording chamber and connected to a lightweight tether connected to a low-resistance commutator mounted over the cage . This enabled complete freedom of movement throughout the cage. Except for the recording tether, conditions in the recording chamber were identical to those in the home cage. Mice were allowed a minimum of 7 additional days to acclimate to the tether. Recording of EEG and EMG waveforms began at zeitgeber time (ZT) 0 (light onset in 12:12 LD). Data acquisition was performed on a PC running Sirenia Acquisition software , a software system designed specifically for polysomnographic recording in rodents. EEG signals were low-pass filtered with a 40 Hz cutoff and collected continuously at a sampling rate of 400Hz. After collection, all waveforms were classified in two-second epochs by a trained observer (using both EEG leads and EMG) as wake , NREM sleep  or rapid-eye movement (REM) sleep . EEG epochs determined to have artifact  were excluded from analysis. Artifact comprised less than five percent of all recordings used for analysis. Analysis of slow wave activity (SWA) was accomplished by applying a Fast Fourier Transformation to raw EEG waveforms. Only epochs classified as NREM sleep were included in this analysis. SWA was measured as spectral power in the 0.5 to 4 Hz frequency range.Following a 24-hr baseline recording, mice were sleep deprived during the first 6 hours of the light phase (ZT 0–6) by gentle handling  and allowed an18-hr recovery opportunity (ZT 6–0). Four of the animals from the baseline recording were excluded from recovery analysis because of deterioration of the EEG or EMG signal .c = .025), since there were only two groups to compare. To compare across the FCG genotypes, repeated measures ANOVAs were used to detect between-  and within-  factor differences. Since these analyses included more than two groups they required multiple comparisons, therefore post hoc analysis was conducted using Tukey tests to follow-up variance of main effects and interactions. All t-statistics are reported as the absolute value. 2×2 comparisons  were conducted in gonadally-intact animals and were analyzed using two-way ANOVAs with phase (light or dark) as the within-subjects variable. Post-hoc analyses of these 2×2 comparisons were conducted using paired t-tests with a Bonferroni correction , and 65.5 min. more NREM sleep  than females (XYF & XXF), but no differences in REM sleep amount  and NREM sleep  revealed that these sex differences were predominant during the active (dark) phase. Follow-up paired t-tests revealed the sex differences during the dark phase  but not the light phase . These effects of gonadal sex are similar to those previously reported in wild-type mice First we investigated sex differences in mice with intact gonads. For this analysis, 2×2 comparisons of sex and chromosome complement were sufficient. During 24-hr recording of baseline sleep-wake states, males (XYM & XXM) had 69.2 min. more total sleep (FWe then investigated the effect of genetic sex (sex chromosome complement) in intact FCG mice by comparing XY mice (XYM & XYF) to XX mice (XXM &XXF) during 24-hr baseline sleep recording. There were no significant effects of sex chromosome complement on total sleep, NREM sleep, or REM sleep during baseline recording . These r3,31 = 3.2; p = .04), but not on NREM sleep , or REM sleep . Post hoc analyses of total sleep averaged into 2-hr intervals revealed no differences between any of the four genotypes during the 12-hr light phase. Post-hoc analyses revealed that during a single 2-hr interval between ZT 16–18 just prior to the peak in active-phase sleep amount, GDX XXMs had 39.3 min. more total sleep  than GDX XXFs  or NREM sleep , but there was a main effect of genotype on REM sleep . Posthoc analysis of REM sleep revealed that at ZT 12–14 XXMs had 4.1 min. more REM sleep than XXFs , NREM recovery sleep  or REM recovery sleep ; however, there were genotype x time interactions for total sleep  and NREM sleep . Post hoc analysis revealed that during 2 hrs at the peak of active phase siesta sleep, XYFs had 38.2 min. more total recovery sleep  and 35.9 min. more NREM recovery sleep  than XXFs  during recovery from forced wakefulness. There were no main effects of genotype on total recovery sleep  and a genotype x time interaction . Posthoc analysis revealed effects at four time intervals , during which GDX XY females had almost 40 minutes more recovery sleep than GDX XX females. This time interval and the adjacent intervals are of particular interest because it is when mice exhibit the most active-phase sleep and elevated active-phase SWA. The mid-late active phase is also when humans are most likely to experience elevated propensity for siesta sleep FCG mice with intact gonads in the current study exhibited sex differences similar to previous reports in wild-types Sry gene on an XX background is sufficient to increase mid-active phase sleep amount. The second is that there may be lingering effects of gonadal hormones, or some effects of sex phenotype, that only express themselves in XX mice. Such effects are commonly associated with organizational effects of reproductive hormones. The observation that this sex difference is not observed after sleep deprivation suggests that under conditions of high homeostatic sleep pressure, direct effects of sex chromosomes on sleep propensity mask the phenotypic sex differences observed during baseline sleep.The observation that GDX males continue to sleep more than GDX females during the mid-active phase of baseline recording, but only in the XX mice, results from two potential possibilities. The first is that expressing the Sex chromosome complement had a number of revealing influences on NREM SWA during recovery from sleep loss. NREM SWA measures homeostatic sleep pressure, which builds with increasing duration of wakefulness and dissipates during subsequent NREM sleep. When mice are sleep deprived during the first six hours of the light phase, the most rapid dissipation of SWA typically occurs during the remaining six hours of the light phase. During this span, SWA in XX females declined more slowly than any other genotypes. This finding in similar to our previous report which indicates that after sleep loss SWA declines more slowly in GDX females than in males. This suggests that the presence of the Y chromosome, or a male phenotype, enhances SWA dissipation after sleep loss. In fact, while SWA began increasing during the late dark phase in the other genotypes, it continued to fall in the XX females, who ended the recovery period with lower SWA than the other genotypes. This suggests that the homeostatic ability to dissipate sleep pressure after sleep loss is regulated differently in mice that lack the male genotype or phenotype.Over the past several years a number of studies have reported that women are more likely than men to suffer negative health outcomes from insufficient daily sleep.


To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for  Using luciferase-expressing genetically engineered bacteria to enable a rapid and non-destructive reporter for infection burden has been applied for a variety of bacterial infections in vivo studies have used two classes of luciferase, either from luminous beetles including Photinus pyralis firefly luciferase  Pyrophorus plagiophtalamus click beetle luciferase  Photobacterium and Vibro genera  Gaussia princeps has not been successfully applied for in vivo bacterial studies.The detection and quantification of bacterial colony-forming units (CFU) within animal infection models is critical for both basic research of host-pathogen interactions and pre-clinical evaluation of antibacterial agents and vaccines. However, longitudinal monitoring of CFU from Gaussia princeps luciferase  from marine copepod catalyzes the oxidation of its substrate, coelenterazine (CTZ), producing blue luminescence concomitantly. As the smallest known luciferase, Gluc in either cell-associated form or secreted form has been used for in vivo monitoring of a variety of mammalian cell behaviors including primary tumor growth in vivo detection of certain nonmammalian organisms such as the fungus pathogen Candida albicansChlamydomonas reinhardtiiRenilla reniformis luciferase  Gaussia princeps using its native secretion signal (SS), enabling reporting from both the cells themselves and their extracelluar environment; Third, Gluc exhibits good stability under adverse conditions including low pH, hydrogen peroxide, high temperature and even β-mercaptoethanol in vivo environments that are expected within sites of infection. Last, unlike the firefly luciferase (FFluc) or bacterial luciferase (LuxCDABE) 2 for its reaction. The independence of Gluc activity from these metabolites avoids declines in detection sensitivity associated with the decreased concentration of bacterial metabolite, occuring upon stationary phase The in vitro studies, such as monitoring Mycobacterium smegmatis (M. smegmatis) in vitro growth under hostile conditions in vitro culture of Salmonella enterica Serovar Typhimurium (S.Typhimurium) M. smegmatis or Lactococcus lactis (L. lactis) to stably express Gluc are illustrative of the challenge for making in vivo bacterial reporter. Although Gluc was robustly expressed by M. smegmatis in vitro, in vivo imaging of M. smegmatis in mice lungs was not successful in vivo and ex vivo imaging of Gluc-producing L. lactis did not generate any signal above the background either in vivo bacteria detection using ex situ sampling (eg. body fluid and plasma), we prepared an engineered pathogenic E. coli that can stably secrete Gluc and tested whether Gluc can function as a CFU reporter within a persistent tissue cage model of infection.Despite the many potential advantages of Gluc-based bacterial reporter systems, successful attempts have only been demonstrated for in vivo emergence of bacterial resistance during antibiotic treatment of persistent infections s.c.) on the back of mice to form tissue cages wherein interstitial fluid/tissue cage fluid (TCF) accumulates. Infection is initiated by injection of bacteria into the cage cavity via percutaneous puncture (p.p.). This model enables the study of a persistent infection, for up to 3 weeks, wherein the host cellular immune responses are in effect. As long as the initial inoculum size is controlled, tissue cage infection normally does not spread systemically but instead results in an abscess milieu within the cage The subcutaneous tissue cage infection model E. coli enables a convenient CFU reporter for bacterial infection in mice. Gluc empowers in vivo imaging of stationary phase bacteria in tissue cage infection. Furthermore, CFU burden in TCF and secreted Gluc activity in TCF correlate to a high degree. Lastly, stationary phase bacterial burden in tissue cage even can be indicated by secreted Gluc in the blood samples of mice bearing tissue cage infection.Here we report for the first time that the secretion of Gluc by pathogenic E. coli for their ability to promote secretion in vitro. These included the full length Gluc which contains the native 16 amino acid N-terminal secretion signal (SS) or Gluc with these residues removed. Two further fusion proteins were designed from the latter construct, Gluc appended to the 21 N-terminal amino acid secretion signal from pectate lyase B (PelB), and also a construct with the 60 amino acid secretion signal of Hemolysin A (HlyA) appended at the C-terminus of the Gluc protein. The cell lysates of E. coli harboring pCOLDI based plasmid encoding these different versions were analyzed by Western-blotting (E. coli. This result highlights the differing efficiency of Gluc secretion between E. coli and Mycobacterium smegmatis (M. smegmatis), noting that almost 100% of Gluc was detected in the culture supernatant of M. smegmatis expressing Gluc without a secretion signal E. coli cultures expressing Gluc with native secretion signal (SS) to reach the same cell density as other E. coli strains (Data not shown), suggesting fitness cost with this construct. Additional bands for A total of four Gluc-derived proteins with varied secretion signaling peptides were tested in blotting . The majblotting , but notblotting . Similarblotting , indicatE. coli, the gluc gene with codon usage optimised for Gram-negative bacteria was integrated site-specifically into the E. coli chromosome under the control of a constitutive promoter. Three different promoters that have been reported to have varying strengths were tested gluc with either the pelB or the native secretion signal (SS) were examined. The three promoter sequences each fused to tagged gluc ORFs were cloned into pSMM25, a derivative of the vector pKD4 pelB tagged or the SS tagged gluc gene driven by each of the three different promoters was generated. These plasmids were used as PCR templates for generating the DNA fragments used for site-specific recombination with the chromosome. These DNA fragments were integrated into the chromosome using the λ Red recombinase gluc gene driven by either strong or intermediate promoters to make template plasmids, multiple mutations were repetitively observed in the promoter region of these template plasmids (data not shown), suggesting that constitutive expression of secreted forms of Gluc at elevated levels is toxic to E. coli. As represented in pelB tagged gluc ORF or the SS tagged gluc ORF.In order to achieve a stable expression and secretion of Gluc from lacZ locus within the genome was selected as the integration site of the gluc gene based on two considerations. First, LacZ is not required for E. coli replication and pathogenesis, avoiding the fitness cost caused by the substitution of lacZ with gluc. Second, the substitution of lacZ with gluc enables a fast X-gal Blue/White screening for targeted integration of gluc in lacZ locus. Integration of the gluc gene into lacZ locus was performed in both the opposite orientation of the lacZ ORF  to confirm the stability of the gluc integration after elimination of the kanamycin resistance gene. These studies demonstrated that elimination of the kanamycin resistance cassette did not affect the stability of chromosomal integration of the gluc gene in the lacZ locus  in either LB medium (data not shown) or in Tryptic Soy Broth (TSB) was compared against the parental strain ATCC25922  showing stable integration of TCC25922 . No evido growth .in vitro passage test for the stability of chromosomal integration of the gluc gene, we also examined the stability of the gluc integration after in vivo growth of the recombinant E. coli strain. Comparison of the Gluc secretion levels of the recombinant E. coli clone (ML6189) before and after in vivo thigh infection was performed using a Gluc assay kit from New England Biolab  substrate was assessed over four different concentrations up to the solubility limit (118 µM) of CTZ in biological samples relative to the NEB kit conditions . Using aE. coli recombinant clone could endure a typical tissue cage infection course (greater than a week), mice with tissue cages infected by strain ML6189 were imaged at both 11 and 18 days post-infection, when CFU burdens are fully established at ≥108 CFU/ml tissue cage fluid (TCF). The background level of luminescence signal was determined by imaging mice infected with ATCC25922 parental strain.To assess whether the Gluc secreting capability of i.p.), tail vein/intravenous (i.v.) and intra-tissue cage (i.t.) were tested using a high concentration of CTZ (4mg/kg body weight), while avoiding solubility issues of CTZ in physiological fluid. Neither i.p. nor i.v. injection of substrate produced sufficient luminescence signal for imaging detection in tissue cages (Data not shown). In contrast, i.t. injection gave rise to prominent luminescence signal immediately after substrate administration , followed by a reduced level plateau stage (2–6 min post CTZ injection) that gradually decreases over time (6–30 min post CTZ injection) where the signal decays by ∼30–70% with signal in some mice returning to background level , a windoin vivo, the growth rate and time dependency of CFU burden were assessed for the Gluc secreting strain ML6189 and the parental strain ATCC25922 over 18 days in the tissue cage model. In addition, both CFU in TCF and the secreted Gluc activity present in TCF were monitored simultaneously over 18 days of tissue cage infection to profile the correlation between CFU burden and detection of secreted Gluc. For tissue cages inoculated with 105 CFU, both strains demonstrated the same CFU increase (about 3 logs) within the first 3 days and the maximal CFU burden was reached by day 3 (59 hr) post-infection (≥108 CFU/ml TCF). At this time point, there was no statistically significant difference between the two strains in establishing the infection , where the infections in most mice were fully established with both strains by day 3 (E. coli burden for the remaining course (18 days) of the tissue cage infection .For l strain . For tish of CFU . First, observed  except tobserved  and S5 sEx vivo measurement of secreted Gluc for established tissue cage infection presented a signal to background ratio of three logs, a quantification window larger than the one generated by imaging. More importantly, measuring samples in vitro with a luminometer equipped with a substrate injector avoids the timing complications encountered in whole-animal imaging, thereby aiding sensitivity of detection. Lastly, ex vivo measurements required much less CTZ substrate than the amount infused into whole animal for imaging experiment. In summary, ex vivo measurements of Gluc in TCF requires sampling of TCF, but provides a sensitive method of detecting secreted Gluc as a reporter for E. coli replication.E. coli could be detected in the circulating blood and used for quantification of CFU burden in tissue cage infection loci. Tissue cage infections initiated with either 103 CFU , the potential for real time quantitation of in vivo bacterial burden using this luciferase reporter is especially useful.Despite the extensive use of Gluc expression in mammalian cells for monitoring E. coli that stably secretes Gluc, it was necessary to optimize both the expression promoter and the secretion tag. Our trials of making plasmids expressing Gluc driven by strong promoters repeatedly induced multiple mutations in the promoter region. In addition, high expression and or secretion of Gluc with its native (eukaryotic) secretion signal by pCOLDI expression plasmid severely affected the growth of E. coil in culture. These results are in contrast with what seen in tumor cells where Gluc with its native secretion signal driven by the strong CMV promoter demonstrated a consistent, high expression and secretion of Gluc in vivo detection, a promoter reported to have moderate strength E. coli codon-optimized gluc gene. In addition, it was found that plasmid encoding Gluc in Lactobacillus plantarum (L. plantarum) was not stable L. plantarum. Hence, in contrast to expression of Gluc from plasmid in either M. smegmatis or L. lactisgluc gene was stably integrated on the E. coli chromosome in the non-essential lacZ locus  is not only bigger than the range (1–1.5 logs) provided by Gluc in vivo imaging (in vivo imaging of FFluc expressing M. smegmatis (2–2.5 logs) Our results showed that Gluc expressed by  imaging . While tvantages  and 8. I imaging  and S5, in vivo and ex vivo luminescence during the plateau stage of tissue cage infection  was not well represented by Gluc activity in TCF for all animals sampled  injection of 80 mg/kg Ketamine and 10 mg/kg Xylazine during tissue cage surgery, and all efforts were made to minimize suffering.This research was performed by strict following the guidelines of Public Health Service Policy on Humane Care of Laboratory Animals and the mouse research protocol used here was approved by the Institutional Animal Care and Use Committee of AstraZeneca (IACUC Permit Number: 11-01-i). Mice were anesthetized with intraperitoneal  medium for 16 hr at 200 rpm at 37°C. The culture was centrifuged for 2 min at 16,000×g and the supernatant collected. For cultivation of bacteria for mouse infections, E. coli clone ML6189 or ATCC25922 was grown in TSB medium for 16 hr at 200 rpm at 37°C. Then the overnight culture was diluted 1∶10 in TSB, the OD600 determined. The calculated volume of the overnight culture was pipetted into the appropriate volume of saline to obtain the target inoculum of either 103 CFU/100 µl per tissue cage, 105 CFU/100 µl per tissue cage or 106 CFU/100 µl per mouse thigh.All strains/clones used and or generated in this study can be found in E. coli harboring pCOLDI construct was grown in LB medium at 37°C with shaking at 200 rpm until OD600 reached 0.5. A portion of bacteria was pelleted  to provide the pre-induction sample . The post-induction supernatant samples were concentrated by chloroform-methanol precipitation 8 cells were loaded into each well of protein gel and detected by Western as described above.e “B” in . The rese “B” in  and the e “B” in . The remE. coli codon-optimized gluc open reading frame (ORF) without secretion signal (Gluc), with HlyA secretion signal (Gluc-HlyA), with PelB secretion signal (PelB-Gluc) and with SS secretion signal (SS-Gluc) were synthesized  and cloned in pBlueScript (II)SK(-) gluc or the PelB tagged gluc was cloned into pSMM25 through a blunt-sticky ligation using Eco53kI/BamHI digested promoter driven gluc construct and BmgI/BamHI digested pSMM25 to generate two template plasmids, pJWW3 and pJWW6 respectively (List S2 in All primers used in this study can be found in List S1 of pir-dependent R6kg replication origin of the pKD4 parent plasmid. The presence of the introduced restriction sites were verified by restriction digest of purified plasmid with Eco RI and Bam HI.pSMM25 is the derivative of pKD4 gluc (with ss tag or pelB tag) together with a kanamycin resistance cassette from pJWW3 or pJWW6. Integration of either the pelB tagged gluc or the ss tagged gluc into lacZ locus of ATCC25922 chromosome by λ red recombinase strategy was performed as described previously gluc in the same orientation as the original lacZ ORF. The DNA fragment produced from the primer pair of OlacZ_ForwardL_MYL and OlacZ_ReverseL_MYL was used to integrate gluc in the orientation opposite to the original lacZ ORF. Successful targeted integration events were selected for white colonies on X-gal plates, kanamycin resistance (KanR), and ampicillin sensitivity (AmpS for the expulsion of the λ red recombinase expressing helper plasmid) and were validated by PCR tests using locus specific primers  injection of 80 mg/kg Ketamine and 10 mg/kg Xylazine. When Stage III anesthesia was achieved, as determined by lack of pedal reflex, buprenorphine (0.1 mg/kg) was subcutaneously (s.c.) administrated to mice. A sterile tissue cage was s.c. implanted on the mouse back using aseptic techniques All animal protocols were conducted under approved institutional guidelines (IACUC protocol number 11-01-i). Mice were kept in a specific pathogen free (SPF) facility of AstraZeneca Boston R&D. Eight weeks old female CD1 mice  were anesthetized with intraperitoneal (p.p.) using a sterile 25-gauge needle. For determination of CFU burden in TCF, 20 µl of TCF was 1∶10 serially diluted in saline and plated on TSA plates. Colonies were counted after 16 hr incubation at 37°C. The remaining TCF sample was subjected to a 4°C centrifugation of 7,500×g for 15 min to yield a clear supernatant that was frozen at −80°C until use. About 50–100 µl blood was drawn from mice by submandibular bleeding 2EDTA containing plastic tubes  to avoid clotting. Blood then was centrifuged at 2,000×g for 25 min at 4°C to harvest plasma sample that was frozen at −80°C until use.Mice were anesthetized by inhalation of isofluorane and about 20–40 µl interstitial fluid (TCF) in tissue cage was drawn by percutaneous puncture  was reconstituted in methanol to a concentration of 11.8 mM (5 mg/ml) and stored at −80°C until use i.v., i.p., or intra-tissue cage (i.t.) route, at a dose of 4 mg/kg body weight. In vivo imaging was performed at the indicated time after CTZ administration using an IVIS Spectrum system . Mice were positioned within the imaging chamber and 2.5% isofluorane in air was administered via nose cones through the IXG8 gas anesthesia system . Images used for quantification analysis for i.t. route administration of CTZ were all acquired using 16 second integration times. For assessment of Gaussia luminescence intensity at a given location by the Living image software (version 4.3.1), pseudocolor images were generated and represented with different colors according to the color scale bar . Total luminescence quantification (photons second−1) at each time point was done by the region of interest (ROI) tool of the Living image software.via a built-in substrate injector in luminometer . Gluc activity in TCF (CPS per µl TCF) or Gluc activity in plasma (CPS per µl plasma) was determined by the luminescence signal falling into the linear measurement range of the luminometer. It was found that 118 µM CTZ yield better results than 39 µM. Consequently, 118 µM CTZ was used for all final quantification analysis.Clear supernatant of TCF or plasma samples  was initially diluted 50-fold in D-PBS then further 1∶ 10 serially diluted. For each dilution, 50 µl sample was loaded into each well of 96-well plates . The plates were assessed by adding 50 µl of CTZ substrate to each well at the final concentration of either 118 µM or 39 µM t-test was used for all two comparisons with one variable (indicated by the connected lines in the figure), P<0.01 was considered as statistically significant.Unpaired and two tailed Student's Figure S1Gluc without secretion signal expressed by E. coli is associated with bacterial cell pellet but not culture supernatant.(TIF)Click here for additional data file.Figure S2Integration of the pelB tagged gluc gene in the same orientation as the original lacZ ORF on E. coli chromosome reproducibly generates higher level of secretion of Gluc to bacterial culture supernatant than other integration orientations.(TIF)Click here for additional data file.Figure S3Integration of the pelB tagged gluc gene into chromosomal lacZ locus is stable in vitro.(TIF)Click here for additional data file.Figure S4Comparison of growth rate and bioluminescence production for a PelB tagged Gluc expressing strain ML6189 and the parental strain ATCC25922 in vitro.(TIF)Click here for additional data file.Figure S5Secreted Gluc activity in tissue cage fluid (TCF) correlates with CFU burden of the recombinant E. coli in TCF.(TIF)Click here for additional data file.File S1includes supplementary figure legend, primer sequences and plasmids used in this study.(DOCX)Click here for additional data file.


We identified a family in which pitted hypomineralized amelogenesis imperfecta (AI) with premature enamel failure segregated in an autosomal recessive fashion. Whole-exome sequencing revealed a missense mutation  in the I-domain of integrin-β6 (ITGB6), which is consistently predicted to be pathogenic by all available programmes and is the only variant that segregates with the disease phenotype. Furthermore, a recent study revealed that mice lacking a functional allele of Itgb6 display a hypomaturation AI phenotype. Phenotypic characterization of affected human teeth in this study showed areas of abnormal prismatic organization, areas of low mineral density and severe abnormal surface pitting in the tooth's coronal portion. We suggest that the pathogenesis of this form of AI may be due to ineffective ligand binding of ITGB6 resulting in either compromised cell–matrix interaction or compromised ITGB6 activation of transforming growth factor-β (TGF-β) impacting indirectly on ameloblast–ameloblast interactions and proteolytic processing of extracellular matrix proteins via MMP20. This study adds to the list of genes mutated in AI and further highlights the importance of cell–matrix interactions during enamel formation. Enamel is the hardest human tissue and when formed correctly has the capacity to remain functional in to very old age. Amelogenesis imperfecta (AI) is the collective term for a heterogeneous group of conditions characterized by genetically determined defects in tooth enamel biomineralization which lead to premature clinical failure. Typically, all teeth of the primary and secondary dentitions are affected, with variations in phenotype being influenced by the underlying genotypes . The imp10[PO4]6[OH]2) (HAP). The physical properties of mature enamel are a result of its high mineral content and the complex, though ordered, spatial inter-relationship and orientation of the enamel prisms. For correct enamel formation to occur, ameloblasts must undergo four main stages: pre-secretory, secretory, transition and maturation  , enamel matrix proteolytic enzymes , an ion transporter  and a putative crystal nucleator  (FAM83H (MIM 611927) and WDR72 (MIM 613214), which are of unknown functions, shows that much remains to be understood  . HoweverR72 MIM 6214, whic829 . HowITGB4 (MIM 147557) and LAMB3 (MIM 150310), have been identified in patients with isolated and syndromic AI (Integrin-β6 (Itgb6) null mouse was described with a hypomaturation AI phenotype  and subsequent MMP20 activation via Runx2.Here, we report findings from a consanguineous family with autosomal recessive pitted hypomineralized AI. We show that a missense mutation of a highly conserved residue in ITGB6 is the cause of the condition in this family, and that the enamel phenotype is similar to that described for the We identified a consanguineous family (AI-23) living locally, but originating from Pakistan, in which pitted hypomineralized AI segregates with an autosomal recessive mode of inheritance in the absence of any other co-segregating diseases Fig. . AffecteTo identify the genetic basis of AI in this family, individuals V:3 and V:5 were genotyped using Affymetrix 6.0 SNP microarrays, and shared regions of homozygosity were identified using AutoSNPa . Six regGenomic DNA from individual V:3 was subjected to whole-exome sequencing using a 100 bp paired-end protocol on an Illumina Hi-Seq 2000 sequencer. Sequence reads obtained were aligned to the human reference sequence (GRCh37) using Bowtie2 software. The resulting alignment was processed in the SAM/BAM format using the SAMtools, Picard and GATK programs in order to correct alignments around indel sites and mark potential PCR duplicates. Following post-processing and duplicate removal, a mean depth of 64 reads was achieved for targeted exons in the homozygous regions, with 98.2% of these bases covered by at least five reads.http://www.ncbi.nlm.nih.gov/projects/SNP/), any variants present in dbSNP 129, together with those variants present in dbSNP 137 with a minor allele frequency (MAF) ≥1%, were then excluded. The 138 remaining variants were annotated using the SeattleSeq Variation Annotation server v.137 (http://snp.gs.washington.edu/SeattleSeqAnnotation137/), which identified 134 as either synonymous or deep intronic variants. Of the remaining four variants, two are present in dbSNP with an MAF of <1.0%. These are rs147066399 (NM_213621:c.736C>T [p.R246W] in HTR3A (MIM 182139)) with an MAF of 1/4545; and rs113262393 (NM_033394.2:c.1413T>G [p.S471R] in TANC1 (MIM 611397)) with an MAF of 9/2190. In addition, rs147066399 in HTR3A has been observed once in 87 South Asian samples in an in-house exome dataset. A third missense variant, p.W879S in LRP2 (NM_004525:c.2636C>G (MIM 600073)), has also been observed in heterozygous form twice in 87 South Asian samples in our in-house exome dataset. Furthermore, these three variants do not segregate fully with the disease phenotype in the AI-23 family.Indel and single-nucleotide variants within the six candidate regions were called in the VCF format using the Unified Genotyper function of the GATK program, revealing a total of 1847 variants. Using the dbSNP database at NCBI (ITGB6 (NM_000888:c.586C > A) .The remaining variant, p.P196T in  A) Fig. A, was noITGB6 were therefore performed. No further pathogenic variants were identified.In light of these findings, we investigated a panel of 44 unrelated individuals diagnosed with hypomineralized AI from diverse backgrounds. PCR amplification and Sanger sequencing of all the coding exons and intron–exon boundaries of 3  . The affThe integrins are a major family of cell surface-adhesion receptors involved in cell–cell, cell–matrix and cell–pathogen interactions . Each inMatrix–cell binding proteins such as fibronectin are important in the epithelial–mesenchymal interactions during the pre-secretory-stage amelogenesis , but areItgb6, with both falling within the hypomineralized AI spectrum. The cause of hypomineralization in affected teeth could be a reduction in prism density per se or prism density may be normal but the individual enamel crystallites within each prism may have failed to grow to their normal maximum dimensions. The laboratory phenotyping of affected human enamel presented here provides insight into why the enamel is prone to premature failure post-eruption. Although enamel of normal thickness is produced during amelogenesis of affected individuals and the normal elements of enamel architecture; i.e. enamel prisms are present, µCT and SEM indicate that the enamel is compromised by defects running from abnormal enamel surface defects (pits) towards the enamel–dentine junction. In addition, the affected teeth appear to contain localized regions of hypomineralization; especially in the cuspal regions. There is also indication that the macro-organization of enamel prisms in affected teeth is abnormal as evidenced by the SEM data which would be expected to impact the mechanical properties of the enamel that depend on the organization of prism cohorts. In normal enamel development, the synchronous movement of cohorts of ameloblasts gives rise to equivalent cohorts of prisms that follow a sinusoidal track from the enamel–dentine junction to the enamel surface. This gives rise to the Hunter–Schreger bands which serve to inhibit crack propagation in the enamel. Based on our preliminary phenotyping, we suggest that affected enamel is structurally compromised by pitting, localized hypomineralization and disturbed prism architecture, all of which undermine the ability of the enamel to resist the stresses generated during mastication. In summary, the enamel in affected members of this family is consistent with hypomineralized AI with focal hypoplasia in the form of pits.The AI phenotype in this family shares some similarity to that of mice lacking a functional allele of ITGB6 binding to LAP of TGF-β1 activates the cytokine . TGF-β1aMMP20 has also been implicated in controlling ameloblast–ameloblast cell contact by cleaving an extracellular domain of cadherin, which in turn controls the ability of ameloblasts to execute their movements relative to each other. These movements are essential to generate the correct prism architecture ,33 needeThe distribution of enamel defects in AI resulting from a mutation in ITGB6 was not even throughout the anatomical crown. Pitting and areas of hypomineralization were localized to the coronal portion of the tooth, the thinner cervical enamel being spared and of normal mineral density and appearance. Ameloblasts travel a shorter distance in elaborating cervical tissue and their activity takes place over a shorter timescale (as apposition rates are similar irrespective of the location on the tooth) . MathemaThe mutation in ITGB6 would be predicted to inhibit activation of TGFβ due to its failure to bind to LAP.Itgb6-null mouse.This, in turn, would inhibit expression of MMP20 which is essential for matrix processing to permit secondary crystal growth to occur. Failure to process matrix would lead to hypomineralization as observed in our study and in the Itgb6-null mouse), including the disruption of Hunter–Schreger banding.MMP20 is also necessary for correct ameloblast–ameloblast cell contact due to its role in cadherin processing. Failure in cell–cell contacts will compromise the ability of ameloblasts to move relative to one another, leading to the abnormal prism decussation seen here  or via venous blood samples using conventional techniques.Members of a family (AI-23), originating from Pakistan, were recruited following informed consent in accordance with the principles outlined in the declaration of Helsinki, with local ethical approval. DNA samples were obtained from family members using either OrageneGenomic DNAs from two affected individuals were genotyped using Affymetrix 6.0 SNP microarrays by AROS Applied Biotechnology . The resulting CEL files were annotated and analysed using autoSNPa to identify shared regions of homozygosity between both the affected individuals .Whole-exome sequencing was performed on genomic DNA using the Nextera Exome Enrichment system . In brief, 50 ng of genomic DNA was tagged and fragmented using the Illumina Tagmentation system. Following a clean-up step, tagged DNA was PCR amplified and the subsequent library validated. Validated samples were pooled six per lane with sequencing performed on an Illumina Hi-Seq 2000 using a 100 bp paired-end protocol. Data analysis was performed using Bowtie2, SAMtools, Picard and the Genome Analysis Toolkit (GATK) –41.ITGB6 were designed using ExonPrimer (http://ihg.gsf.de/ihg/ExonPrimer.html) and are found in Supplementary Table 2.Segregation of variants identified by whole-exome sequencing and screening of additional AI families was performed by PCR and Sanger sequencing using standard protocols. Primers to amplify the exons and exon–intron boundaries of An extracted, dried AI-23 deciduous canine was compared with an extracted, dried control deciduous canine using µCT and SEM. The control tooth was obtained with patient consent and ethical approval from a registered tissue bank operated by the School of Dentistry University of Leeds. Teeth were scanned in air together with a calibration phantom comprising a segment of mouse incisor enamel and dentine of known mineral density using a Skyscan 1172 high-resolution X-ray CT scanner  operated at 55 kV; 10watts power with an image pixel size of 2–2.2 µm. The teeth were rotated through 180° and shadow X-ray images captured at 0.27° intervals. The X-rays were filtered through a 0.5 mm Al/Cu filter to reduce beam hardening effects. The shadow images were reconstructed using Skyscan Recon software to generate an image stack comprising sections through the teeth. The Recon software was also used to further correct ring artefacts and beam hardening. The sectional images were further analysed using ImageJ software  and calibrated with respect to mineral density using the mouse incisor as a calibrator.For SEM, teeth were cut along the buccal–lingual midline or transversely through the cusp region with a diamond cutting disk. The cut surfaces were lightly polished using 2000 grade carborundum paper and etched for 20 s by gentle agitation in 30% phosphoric acid to remove any smear layer. Samples were rinsed in copious amounts of distilled water and dried under vacuum overnight prior to sputter coating with gold. SEM images were obtained using a Hitachi S-3400 operating in secondary electron emission mode at an accelerating voltage of 20 kV and an emission current of 22.1 µA.HMG onlineSupplementary Material is available at .Wellcome Trust (grant no. 093113). J.K. is supported by the NIHR Leeds Musculoskeletal Biomedical Research Unit. Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust.The project was funded by the


Conserved upstream open reading frames (uORFs) are found within many eukaryotic transcripts and are known to regulate protein translation. Evidence from genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of human diseases. A genetic mouse model has recently provided proof-of-principle support for the physiological relevance of uORF-mediated translational control in mammals. The targeted disruption of the uORF initiation codon within the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) gene resulted in deregulated C/EBPβ protein isoform expression, associated with defective liver regeneration and impaired osteoclast differentiation. The high prevalence of uORFs in the human transcriptome suggests that intensified search for mutations within 5′ RNA leader regions may reveal a multitude of alterations affecting uORFs, causing pathogenic deregulation of protein expression. Defective translational control of protein expression is increasingly recognized as an important mechanism in the etiology of human diseases A growing body of evidence obtained from bioinformatic and genetic studies suggests that, in particular, uORF-mediated translational control may serve as a comprehensive mechanism of protein expression control. Recently, the targeted genetic ablation of the translational start site in the uORF of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) validated the physiological relevance of uORF-mediated translational control in an animal model This paper aims to provide a brief overview on uORF-mediated translational control in general. Moreover, we show how aberrant uORF regulation may translate into (patho)physiology, as illustrated by data obtained from analyses of C/EBP transcription factors. Finally, we outline how contemporary sequencing technologies may help to unravel the implications of uORF-mediated translational control in a multitude of as-yet-unexplained human diseases.iMet) ternary complex and additional eIFs, engages with the 7-methyl-guanosine (m7G) mRNA cap structure located at the 5′ end of a transcript iMet anticodon matches a functional AUG codon. Joining of the 60S ribosomal subunit completes the assembly of a fully functional ribosome and permits initiation of translation. Initially it was assumed that scanning ribosomes would generally initiate translation at the m7G-cap proximal AUG initiation codon Translation of eukaryotic transcripts follows a coordinated sequence of events, as summarized in the ribosomal scanning model of translation The yeast transcription factor GCN4 represents the best-studied example of uORF-mediated translational control and illustrates how uORFs can facilitate the paradoxical induction of GCN4 protein expression under conditions of reduced global translation Mechanistically, the expression of GCN4 is determined by the combined effects of leaky scanning and reinitiation events, which are sensitive to changing global translational conditions. Data obtained from the GCN4 transcript showed that the length of a uORF, the sequence context adjacent to its termination codon and the distance to the downstream initiation codon modulates the inhibitory effect of the uORF on ribosomal reinitiation in vivo and confirmed changing translation rates of the uORFs and the MCS of GCN4 in response to altered nutritional conditions Bioinformatic surveys have now identified uORFs in 35–49% of human and rodent transcripts 7G-cap, terminating upstream or downstream of the MCS initiation codon), sequence (no common uORF sequence motif has been identified) and secondary structure. In approximately half of the uORF-bearing transcripts, a single uORF precedes the MCS initiation codon uORFs are extremely diverse in both structural features and regulatory functions. As exemplified for humans, uORFs vary in length (average of 48 nt), number per transcript (0–13), position  and the full-length isoforms p42 and LAP)  and LAP RCCAUGG (Kozak consensus sequence), with a purine base in position −3 and a guanine base in position +4 as most important for initiation In most tissues, C/EBPα-p42 and C/EBPβ-LAP are the most abundant protein isoforms, despite the presence of two preceding translational initiation codons and despite a suboptimal initiation codon context . An optie.g. the differentiation of osteoclasts Several lines of evidence showed that the C/EBPα and β uORFs integrate the signaling status of a cell to modulate the expression ratio of isoforms. One key component in adjusting the activity of the translational machinery to environmental changes is the mammalian target of rapamycin kinase (mTOR). Many nutritional and signaling pathways downstream of growth factor-, cytokine-, or hormone receptors alter the activity of the mTOR kinase. Activated mTOR signaling is associated with enhanced global translational conditions and increased activity of important eIFs, including eIF4E Together, these data suggested that the uORF initiation codon may serve as a physiologically important sensor of global translational conditions, shifting the isoform expression ratio toward the truncated isoforms under favorable conditions and to the full-length isoforms under unfavorable conditions. This function may be due to the suboptimal Kozak context that surrounds the uORF initiation codon , which aΔuORF), now confirmed the concept of uORF-mediated isoform expression in vivo and contributed to a deeper understanding of how changes in the isoform ratio of C/EBPβ affect mammalian physiology ΔuORF mice were generated using homologous recombination into the endogenous c/ebpβ gene locus. The ΔuORF mutation eliminates the uORF initiation codon and thus disrupts its function as molecular switch to induce the truncated LIP isoform, without alteration of the amino acid sequence of C/EBPβ. Data obtained from the C/EBPβΔuORF homozygous mice showed that the C/EBPβ isoform production becomes unresponsive to extracellular stimuli, such as lipopolysaccharide treatment, which normally increases the LIP to LAP ratio LIP), the endogenous c/ebpβ gene locus was replaced by the coding sequence of the LIP isoform only, resulting in complete loss of expression of LAP* and LAP. The exclusive expression of LIP in these animals rescued both the expression of cell cycle genes and the entry of hepatocytes into S phase LIP mice displayed enhanced differentiation of bone-resorbing osteoclasts, while in turn, the decreased LIP to LAP isoform ratio in C/EBPβΔuORF mice showed an impaired osteoclast differentiation. The C/EBPβ LAP isoform was found to induce the expression of the transcription factor MafB in monocytes. MafB inhibits or sequesters other transcription factors that are known to mediate osteoclastic differentiation, including Fos, Nfatc1, and Mitf. In contrast, LIP downregulates MafB expression, resulting in increased availability of those osteoclastic transcription factors ΔuORF mouse comprises the first genetic animal model that confirms the physiological significance of uORF translation in vivo and its action as a molecular switch driving cell fate decisions by modulating isoform expression ratios. The in vivo data support the idea that the abundance of individual C/EBPβ isoforms is regulated by uORF-mediated integration of cellular signals, resulting in tissue-specific functions that depend on the cellular context.The recent generation of genetically altered mice, carrying a single nucleotide exchange of the ATG uORF initiation codon to TTG in the C/EBPβ gene  start sites are conserved, but the uORFhyp terminates five bases upstream of the p30 initiation codon only in humans, while in cow and mouse it overlaps the p30-coding sequence by 85 nucleotides. Given that all three potential uORF start sites are surrounded by intermediate or favorable Kozak consensus sequences, uORF-mediated translational control might be an additional level of C/EBPɛ expression regulation.Another example of how isoform expression ratios affect cell fate decisions comes from C/EBPɛ, the third C/EBP family member that is produced as various N-terminally truncated isoforms. The C/EBPɛ gene differs structurally from C/EBPα and β in that it contains introns. In addition to alternative translational initiation, the expression of four alternative C/EBPɛ isoforms  was attributed to differential promoter usage and alternative splicing. Similar to the short C/EBPα and β isoforms, the short C/EBPɛ isoforms display less trans-activation potential, with the shortest isoform (p14) virtually lacking trans-activating domains ΔuORF mice, naturally occurring uORF mutations in other genes may cause physiological alterations by deregulating translation of the affected transcript. Such mutations could either change the presence of a uORF by generating or deleting an initiation codon upstream of the MCS start site, or could affect translational control by changing one of the structural features of an existing uORF physiological importance of uORF translation in mice added a new level of significance to this cis-regulatory mechanism of translational control. C/EBPα and β transcription factors represent well-established examples of how translational control by uORFs may affect cell fate decisions. Accumulating evidence suggests that deregulated uORF function might be a widespread mechanism underlying the development of human diseases. The rapid progress in advanced sequencing technologies will permit screening approaches to identify causative uORF mutations in primary material derived from patients. Malignancies of the blood might be among the most suitable types of diseases to start such an analysis, as cell samples are readily accessible. One would, proteins . Given t


Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process.The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism,  Differentiated cells, tissues and organs must be replenished throughout adult life in response to normal growth, aging, and injury through regenerative processes. However the potential and source of regeneration vary amongst both organisms and in individual tissues Botryllus schlosseri. Ascidians such as Botryllus can be found in shallow waters and harbors throughout the world Botryllus has been the focus of numerous genetic, developmental, and regenerative studies including studies of asexual development Botryllus is highly regenerative and will replace entire portions of differentiated tissue, on the order of several cm2, within 24–48 hours following surgical ablation Here we investigate the source of the regenerative potential exhibited by the extracorporeal vasculature of the colonial ascidian Botryllus consists of two major structures, an internal plot of sinuses and lacunae of mesenchymal cells that surround the major organs and tissues of individual animals in the colony known as zooids, and a large extracorporeal vasculature consisting of ramified mono-layered vessels embedded within an extracellular matrix made of both cellulose and protein components, known as the tunic Botryllus. For the latter, when ampullae of two individuals come into contact, they initiate a reaction that results in either anastamosis of the two vasculatures at the site of contact forming a blood chimera, or an inflammatory reaction which blocks vessel fusion. Proteins involved in this reaction are differentially expressed on the tips of the ampullae and absent from the vasculature, including the fuhcsec and fuhctmThe vascular system of Botryllus is clear, the cellular identity and molecular source of this regenerative potential is unknown.The regeneration of these differentiated vascular tissues has been shown to occur through a sprouting mechanism with the participation of angiogenic factors: VEGF, FGF-2, and EGF Botryllus vasculature makes Botryllus a unique model for studies on vascular biology. Here we use a novel vascular cell lineage tracing technique to both trace and isolate cells that participate in the vascular regeneration of the extracorporeal vasculature and differentiation of ampullae. In this study we gain insights into the bi-potentiality of these vascular tissue resident cells and determine their regenerative proliferation, mobility, as well as their cellular and molecular contributions to regenerating differentiated vascular tissue.The large and experimentally accessible vasculature, natural parabiosis that occurs between histocompatible colonies, and vast regeneration potential of the Botryllus schlosseri colonies were collected from the yacht harbor in Santa Barbara, CA, spawned and cultured in laboratory conditions at 18–20°C according to . The recombinant GFP and mCherry protein contained N- & C- termini 6xHis tag. Expression of recombinant GFP and mCherry was induced with a final concentration of 1 mM IPTG when bacterial culture reached an optical density at 600 nm (OD600 nm) absorbance of 0.5 to 0.8 at 37°C with shaking at 250 RPM. Induced cultures grew at 37°C for 18 to 24 hours with shaking at 250 RPM.Recombinant GFP and mCherry fluorescent protein were produced in a BL21(DE3)pLysE  bacterial cell line through a pET28A expression vector  under a lac operon expression system. The GFP and mCherry genes (Clontech) were cloned into the pET28A vector with BamHI and XhoI which were introduced through primer design of PCR with Recombinant protein was extracted from the bacteria via sonication in Equilibrium/Wash Buffer as described in the HisPur Cobalt Resin protocol  and supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Bacterial debris were removed via centrifugation and extracted proteins were incubated with HisPur Cobalt Resin overnight at 4°C. Resin with bound recombinant GFP or mCherry protein was collected in a gravity flow column then washed and eluted as described in the manufacturer's protocol . Recombinant GFP and mCherry protein was further concentrated with an Amicon column with a 3 kDa MWCO . Final concentrations of protein were determined by Bradford assay following the manufacturer's guidelines  with BSA used to generate the standard curve.Recombinant GFP and mCherry proteins along with Fluorescein conjugated Dextran (FITC-Dextran) (3000MW) , Alexa Fluor 488 conjugated BSA , and Alexa Fluor 594 conjugated BSA  were diluted in PBS to a final concentration of 0.1 µg/ml. While we injected a range between 0.01 µg/ml to 1 µg/ml of these macromolecule fluorophores to label vasculature cells, our analysis indicated that 1 µl injections of 0.1 µg/ml per system (5-8 zooids) of macromolecule fluorophore per system gave the strongest vascular labeling without residual fluorophore in the blood stream at time points of 24 hours and beyond. To determine the presence of lysosomes in these tissues we used LysoTracker Green DND-26  at 1∶100 in Seawater and soaked the animals for 30 minutes.Chimeric assays were conducted using multiple single systems of genetically identical colonies that were injected with 1 µl of 0.1 µg/ml of either mCherry protein, FITC-Dextran, Alexa Fluor 488 conjugated BSA, or Alexa Fluor 594 conjugated BSA to label vasculature cells. Single systems were allowed to incubate 24 hours prior to additional system contact to prevent transfer of the fluorescent macromolecules and allow for cell labeling to occur. Colonies with labeled vasculatures were then placed on slides in close proximity to each other and allowed to undergo vascular anastamosis. Once common blood flow was established, chimeric animals were then allowed to undergo a takeover event prior to being separated by ampullaectomy surgery. In both single systems and chimeric colonies peripheral ampullae and parts of the marginal vessels were surgically removed by ampullaectomy surgeries as described by Whole-mount in situ hybridization was performed with digoxigenin (DIG)-labeled probes as described by B. schlosseri colonies were anesthetized using MS-222  until oral siphons opened and were unresponsive. Systems were then fixed with 4% paraformaldyhyde  with 0.5 M NaCl at 4°C for 12 hours. Following fixation, systems were bleached using 6% H202 in methanol under light for 1 hour at room temperature to quench auto-fluorescence. Systems were then washed through a series graded methanol and PBS with 0.1% Tween-20 (PBT) and then blocked with 5% heat inactivated horse serum  with 2 mg/ml IgG-free BSA  in PBT at room temperature for 4 hours. Systems were then incubated with either a Rabbit pan-Cadherin antibody  (1∶500) or Rabbit pHH3 antibody  (1∶1000) in blocking buffer and incubated for 48 hours at 4°C. Following primary incubation, system were washed with PBT and incubated with an Alexa Fluor 488 Goat Anti-Rabbit secondary antibody  (1∶200) for 24 hours at 4°C. Systems were then washed with PBT for 6 hours at room temperature to remove unbound secondary antibody and flat mounted in Vectashield mounting medium for fluorescence with DAPI .Single systems of control and ampullaectomized in situ hybridization was imaged on a 1000 Fluoview Spectral Confocal (Olympus).Live imaging of single and chimaeric colonies was carried out using a MZ16FA (Leica) equipped with epifluorescence capabilities to image vascular labeling. Fixed tissue for both immunohisotchemistry and Botryllus schlosseri vascular cells were isolated for sorting as outlined in previous studies . Briefly, animals were injected with 1 µl of 1 µg/ml FITC-Dextran per system. Cells were then extracted by mincing whole animals followed by 70 µm and 40 µm filtration into ice-cold Botryllus buffer  in sterile seawater containing 2% isotonic horse serum . Cells were peleted at 500 g for10 minutes at 4°C and then resuspended in 500 µl of Botryllus buffer.Fluorescently Activated Cell Sorting (FACS) was carried out using a FACSAria (BD Biosciences). Samples were manually compensated and gated as positive or negative based on uninjected control animal fluorescence. Analysis was the performed using BD FACS Diva software (BD Biosciences) and positive cells were sorted. Sorted cells were sorted directly into Lysis buffer for immediate RNA extraction. RNA was extracted using magnetic mRNA isolation kit  and then reverse transcribed to cDNA using oligo (dT) primer  and Superscript II Reverse Transcriptase  according to manufacture guidelines.−ΔΔCt method. Each experiment was analyzed in triplicate from different genotypes (n = 3) and multiple systems (n = 4) for both control and ampullaectomy (AMPX) colonies.Quantitative RT-PCR (QRT-PCR) was carried out using a LightCycler 480 II (Roche) and LightCycler DNA Master SYBR Green I detection . The thermocycling profile was as follows: 5 minutes at 95°C, 45 cycles of 95°C for 10 sec, 50-60°C for 10 sec, and 72°C for 10 sec. Primers for CD133: 5′-atagtagatcacacatgtaaccccatc-3′ and 5′-tgtataatcagtaccgtgtgacaaaat-3′which amplified a 227 bp fragment, VEGFR-2 primers: 5′-gaagctttgatggatcgtaagatagcacc-3′ and 5′-agtcataatgcaactcgtttatctcaaagt-3′ which amplified a 234 bp fragment and Cadherin primers: 5′-ttttcctatcactgttttcaatttacc-3′ and 5′- aattttgacttcgatcatatactttgg-3′ which amplified a 179 bp fragment were used in our analysis. All gene expression data was normalized to elongation factor 1-α (EF1-α) as a reference gene and reported as relative expression using the 2Botryllus we first sought to identify the source of new vascular tissue by analyzing the source of cell proliferation following surgical ablation of the extracorporeal vasculature compared to unmanipulated controls. Regeneration of vascular tissue is induced by removing the entire peripheral vasculature, consisting of marginal vessels and ampullae in a procedure known as an ampullaectomy (AMPX), as previously described To characterize the cellular and molecular mechanisms regulating vascular regeneration in + cells per ampullae with an s.e.m. of 0.42 compared to control ampullae with an average of 1.75 pHH3+ cells per ampullae with an s.e.m. of 0.21 (The location and levels of cell proliferation were characterized using immunohistochemistry to identify the cell division metaphase marker phospho-Histone H3 (pHH3) in normal versus regenerating vascular tissue. In unmanipulated control vasculature, peripheral ampullae and marginal vessels were largely quiescent with a limited number of randomly distributed proliferating cells within blood vessels and peripheral ampullae in control animals . However of 0.21 .+ cells in individual zooid gut with an s.e.m of 1.36) and AMPX  colonies. The lack of proliferative response by another somatic tissue such as the gut coupled with the stochastic active cell proliferation of vascular tissue during vascular regeneration indicated that resident vascular cells were capable of regenerative proliferation following injury.To determine if the increase in vascular cell proliferation during regeneration was due to a global somatic proliferation response to injury or was a specific regenerative response by vascular tissue, we examined the number of proliferative cells in the gut of individual zooids in these colonies. We found that although the gut is a highly proliferative tissue there was no significant increase in cell proliferation between control , a macromolecule that is also fluorescently stable in low pH environments, into the peripheral vasculature of Botryllus in a similar manner as the mCherry protein. We found that the injection of both mCherry protein    to label, n = 6) . Within , n = 6) . The vas, n = 6) . The mCh, n = 6) . To conf, n = 6) .Botryllus homologs of vertebrate genes, whose involvement in both vascular progenitors (CD133 and VEGFR-2) and differentiated vascular cell markers (VE-Cadherin) has been described in vertebrate vascular regeneration, may be regulated in Botryllus vascular resident cells during vascular regeneration. To test this hypothesis we used in situ hybridization for CD133 and VEGFR to localize these genes in regenerating vascular tissue. We found that CD133 was expressed by newly budding ampullae and a small subset of blood cells in Botryllus  analysis. FACS was used to isolate labeled vascular cells in both control and regenerating conditions prior to takeover events to limit the amount of phagocytic cell labeling  fold up regulation of CD133 in AMPX animals, 2.2 (+/− 0.35) fold up regulation of VEGFR and 0.74 (+/− 0.88) fold down regulation of Cadherin in AMPX animals,  when compared to the stage-matched controls   . CompariBotryllus might be limited. To assess the potential mobility of vascular cells during vascular regeneration we analyzed the cell lineage of regenerated vascular cells in parabiosed blood chimeras. To establish the chimeric vascular-labeled colonies needed for these experiments we injected sub-clones of genetically identical individuals with either Alexa Fluor 488 conjugated BSA or Alexa Fluor 594 conjugated BSA, waited 24 hours for vascular labeling to occur and then set up fusion assays as previously described sec, fuhctm, fester and uncle fester The contribution of pre-existing vascular resident cells and the differential regulation of vascular progenitor and differentiated vascular cell markers suggested the mobility of vascular cells contributing to the regeneration of vascular tissue in Following the vascular anastamosis of the two separately labeled colonies, the parabiosed colonies were left for 3–5 days, allowing both colonies to undergo a takeover event. This allowed us to also label the phagocytic cells, which could then be used to confirm blood chimerism. At that point we surgically removed the vasculature at the base of each individual, as close to the zooids as possible shown in , and monIn contrast, the labeled phagocytic cells had transferred between the two individuals, and contained label of the other partner. In addition, we saw a number of double-labeled phagocytic cells, indicating they had engulfed dying cells in both individuals, consistent with previous result using double-labeled phagocytic populations In addition to the animal specific regenerative potential of blood vessels and ampullae, the lack of double-labeled vascular cells in these chimeric animals also confirmed that injected fluorophores used to label vascular tissue was not being recycled through phagocytic cells but rather the observed regenerative labeling arose from the contribution of specifically labeled resident vascular cells.To further delineate the source of the differentiated ampullae, we repeated this experiment, but instead of cutting the ampullae at the base of each individual, we surgically separated the parabiosed individuals by slicing out a section across the median of the fused vessels, such that a subset of vessels was chimeric, with the distal portion being from one juxtaposed partner, and the proximal from the other . We founIn summary, these experiments reveal that individual cells adopt either vessel or ampullae fates depending on their original location following surgical manipulation, with the distal portion becoming the distal tip of the ampullae, and the proximal region retaining vessel or proximal ampullae identity. Thus, immobile, resident vascular cells regenerate new vessels and have the ability to reversibly switch between vessel and ampullae cell fates during vascular remodeling events.Botryllus vasculature is fundamental to understanding how these basal chordates regenerate and maintain differentiated vascular tissue involved in colony growth, adherence and allorecognition capabilities. Through a novel vascular cell lineage tracing method we show that the regenerative proliferation of resident cells within the vascular tissue of Botryllus have the potential to contribute to both regenerated blood vessels as well as regenerated differentiated vascular tissue known as ampullae. These cells express both progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the vascular regenerative process and represent an immobile cell population in chimeric animals that maintain individual tissue identity during vascular regeneration. Furthermore, these cells can reversibly adopt two differentiated phenotypes, those of the vessel wall, and those of the ampullae, which can be differentiated by the expression of four allorecognition proteins.Identifying the source of regeneration of the Botryllus, we found that the production of additional cells contributing to the regeneration of vascular tissues occurs through the stochastic proliferation of pre-existing vascular resident cells. This stochastic regenerative proliferation indicates that the regenerative potential of this tissue is due to the proliferation and differentiation of pre-existing vascular resident cells, rather than a regenerative proliferation within a specific niche of undifferentiated vascular resident stem cells. Such an increase in the stochastic proliferation observed in pre-existing vascular resident cells suggests that the regenerative potential of vascular tissue in Botryllus occurs through an epimorphoric regenerative process where cells within the vascular tissue maintain the potential to both proliferate and differentiate. However, while there is no canonical spatial pattern to proliferation, this does not address whether there is still heterogeneity within the vascular cell population. The same can be said for differentiation between vascular and ampullae tissue, while the creation of chimeric ampullae indicates that pre-existing vascular cells have the potential to differentiate into ampullae, there still could be only a minor population of labeled vascular resident cells that do so.Following injury, early signaling events stimulate the production of additional cells that are capable of rebuilding lost tissues in vivo method for cell lineage tracing in vascular tissue through lysosomes. Fluorophores that exhibit pH-stability have been used extensively to study lysosomal protein activity because of their fluorescent stability in low pH environments Botryllus vasculature allows for the tracking of pre-existing vascular resident cells and their mitotic contributions to regenerating tissue. While it has yet to be determined what the specific uptake mechanism of these macromolecules is for vascular cells, it is clear that vascular cells in Botryllus have the ability to preferentially uptake macromolecules that have been introduced into the blood stream and provide a novel way to trace vascular cell lineages in this animal.While the stochastic proliferation of pre-existing vascular resident cells suggested that these cells were capable of rebuilding surgically ablated blood vessels and ampullae, confirming resident vascular cells as the source of vascular regeneration required cell lineage tracing. Here we show that the use of pH-stable macromolecules can be used as an Botryllus and observed the contribution of these cells to both regenerating blood vessels and differentiated ampullae tissue. Similar to Botryllus, the identification of pre-existing tissue resident cells as the source of regeneration and their lineage restriction following injury has been observed in a number of regenerating tissues in vertebrates Botryllus also seem to be lineage-restricted, as we observed that vascular cells solely gave rise to ectodermally derived blood vessels and ampullae. However the ability of these pre-existing blood vessel cells to differentiate into ampullae cells that express allorecognition proteins indicates that these vascular resident cells maintain lineage-restricted bi-potentiality in Botryllus.Using this vascular cell lineage tracing technique we were able to identify pre-existing vascular tissue resident cells as the source of vascular regeneration in While the source of regeneration and lineage potential has been established for a number of regenerating vertebrate tissues such as the skin, liver, and kidney, the source of neo-vascularization in vertebrates is still largely debated in vitro+CD133+VEGFR-2+ molecular signature were able to home to and differentiate at sites of neo-vascularization suggesting the presence of mobile endothelial progenitor cells In mammals, the mobile vascular progenitor hypothesis first arose from studies showing that cells isolated from peripheral blood using CD34 and VEGFR-2 antibodies had the potential to differentiate into mature endothelial cells + precursors contribute to the vascular endothelium and that neo-vascularization does not require bone marrow-derived endothelial progenitors While the above studies suggested that mobile vascular progenitors have been implicated in neo-vascularization, other studies have shown that no bone marrow derived VEFGR-2Botryllus homologs for genes implicated in vertebrate endothelial neo-vascularization were also regulated during the regeneration of ectodermally derived vascular tissue in this basal chordate. While we were unable to identify a homolog to CD34 in Botryllus, homologs for CD133, VEGFR-2, and VE-Cadherin were expressed by the Botryllus vasculature. In congruence with previous studies on the regeneration of Botryllus vascular tissue Botryllus vascular regeneration showed that ampullae injury caused local increase in Cadherin protein in damaged epithelial tissue, a reaction that resembled expression patterns of endothelial VE-Cadherin during injury of normal blood vessel Within the context that tissue resident cells may be evolutionary conserved units of regeneration we sought to determine if the Botryllus ampullae differentiation from blood vessels.While both regenerating and mature blood vessel and ampullae tissue expressed VEGFR-2 and Cadherin, we found that CD133 was only expressed by newly forming/regenerating ampullae and a small subset of blood cells. In addition, CD133 was also significantly up-regulated in isolated resident vascular cells during regeneration, suggesting that CD133 may be expressed by newly forming ampullae during the regenerative process as they differentiate from blood vessels to ampullae. While CD133 was originally described as a somatic stem cell marker Drosphila trachea system was replaced by a VEGF/VEGFR-mediated endothelial vascular system in the vertebrates g vascular regeneration in Botryllus.The regulation of VEGFR-2, Cadherin, and CD133 by resident vascular cells in an ectodermally derived vascular system suggests the potential for the conservation of the regulation of genes involved in endothelial angiogenesis in vertebrates during vascular regeneration in a basal chordate. It has been suggested that endothelial angiogenesis in vertebrates and the evolution of the vertebrate endothelial vascular system arose through the cooption of a hypoxia-sensing mechanism shown to be involved in branching morphogenesis in invertebrates Botryllus allorecognition system. The highly polymorphic allorecognition system in Botryllus likely exists to strictly limit the possibility of vascular anastamosis, as stem cells in blood-based chimeras can move from one individual to the next, and once transplanted, these stem cells are in competition for contribution to somatic and germline tissues Botryllus blood chimeras we observed that the regeneration of ampullae tissue maintained individual identity, thus while a fusion event can result in a chimeric germline for one of the individuals, it will not result in a chimeric vasculature. It may be that evolutionary pressures also played a role in limiting the source of vascular regeneration to an immobile precursor.While the study of vascular regeneration and the conservation of regenerative mechanisms in a basal chordate have implications into the evolution of the vertebrate vascular system, the identification of the source of vascular regeneration has additional significance in the context of the


The long-term consequences of unsuccessful interferon-α based hepatitis C treatment on liver disease progression and survival have not been fully explored.We performed retrospective analyses to assess long-term clinical outcomes among treated and untreated patients with hepatitis C virus in two independent cohorts from a United States Veterans Affairs Medical Center and a University Teaching Hospital. Eligible patients underwent liver biopsy during consideration for interferon-α based treatment between 1992 and 2007. They were assessed for the probability of developing cirrhosis and of dying during follow-up using Cox proportional hazards models, stratified by pretreatment liver fibrosis stage and adjusted for known risk factors for cirrhosis and characteristics affecting treatment selection. The major predictor was a time-dependent covariate for treatment outcome among four patient groups: 1) patients with sustained virological response to treatment; 2) treatment relapsers; 3) treatment nonresponders; and 4) never treated patients. Treatment nonresponders in both cohorts had a statistically significantly increased hazard of cirrhosis compared to never treated patients, as stratified by pretreatment liver fibrosis stage and adjusted for clinical and psychosocial risk factors that disproportionately affect patients who were ineligible for treatment . Despite their increased risk for liver disease progression, the overall survival of nonresponders in both cohorts was not significantly different from that of never treated patients.These unexpected findings suggest that patients who receive interferon-α based therapies but fail to clear the hepatitis C virus may have an increased hazard of cirrhosis compared to untreated patients. More than 3.2 million people in the United States (1% of the population) are chronically infected with hepatitis C virus (HCV) 3, suggests that failed IFNα-based therapy might have either beneficial, null, or detrimental effects on liver related outcomes in HCV treatment failures Among treatment failures, it has been postulated that transient reductions in viral load during treatment or anti-fibrotic effects of IFNα may attenuate liver disease progression This study was conducted in accordance with the ethical principles stated in the Declaration of Helsinki and is consistent with good clinical practice and applicable regulatory requirements We conducted a medical records review of patients with chronic HCV who were first seen at the San Francisco Veterans Affairs (SFVA) Medical Center Liver Clinic between January, 1992 and July 2007. Most patients had been prospectively consented at the time of liver biopsy for inclusion in a longitudinal database. Eligible patients were ≥18 years of age, had documented chronic HCV, underwent a pre-treatment liver biopsy, received follow-up care at the SFVA Liver Clinic for at least one year after the initial visit, and had at least one follow-up liver imaging study, biopsy or clinic visit. Patients were excluded if they were co-infected with either HIV-1 or hepatitis B virus or if they had decompensated cirrhosis, hepatocellular carcinoma, or liver transplantation prior to their first clinic visit. The study was replicated using an independent cohort of HCV patients from the UCSF Liver Clinic to which the same selection criteria were applied. Each cohort is comprised of all patients meeting study eligibility criteria who were evaluated and followed in these two clinics during this time period.The SFVA and UCSF electronic medical records were the major sources of data for the study. Deaths were confirmed by cross reference with the national Social Security Death Index (SSDI). Data abstraction was performed by two teams, each including one clinician and one research staff member, using a standardized search algorithm. Two additional raters performed data validation on a random sample of patient charts to confirm the reliability of values for select variables.Clinical parameters including body mass index (BMI), alanine aminotransferase (ALT), and HCV viral load were collected from the electronic medical record on the date closest to the initial liver clinic visit. Follow-up ALT was obtained from the laboratory test taken closest to 52 weeks following the end of treatment for treated patients. To obtain a comparable ALT value for the never treated patients, we used the ALT closest to 170.7 weeks following initial liver clinic visit. This interval corresponds to the average time between the first liver clinic visit and the completion of one year of post therapy follow-up among treated patients.Psychosocial, demographic, and behavioral risk factors were assessed and recorded by clinic staff at the time of first liver clinic visit. This assessment and subsequent progress notes were used to determine the presence of risk factors affecting treatment eligibility such as current injection drug use, other substance abuse, history of depression and post-traumatic stress disorder (PTSD). Heavy alcohol use was defined as five or more years of daily alcohol use exceeding the equivalent of four to five drinks per day by patient self-report The Batts-Ludwig system was used to assess fibrosis stage and inflammatory grade from liver biopsies Patients were categorized into four HCV treatment-related groups using previously described standard definitions: SVR, relapsers, nonresponders, and never treated The primary outcome variables were time-to-cirrhosis and time-to-all-cause-death during the follow-up period. Never treated patients comprised the reference category. Time zero for all time-to-event analyses was the date of first liver clinic visit. To overcome the temporal bias associated with variations in treatment start time, we constructed a time-dependent covariate, using standard methods as previously described All analyses were performed using SAS version 9.2 . Chi square tests were performed for categorical data analysis, and the Student’s t test or Wilcoxon rank sum test was used to evaluate the association of continuous predictors on categorical dependent variables (such as patient treatment group). Cox proportional hazards models were used to analyze both univariate and multivariate effects on the outcomes of interest. Since differences in baseline fibrosis stage likely have nonlinear influences on long-term fibrosis progression, and because the distribution of fibrosis stage violated the proportional hazards assumption, all Cox proportional hazards models were stratified by fibrosis stage groupings . Time-to-cirrhosis analysis was repeated using an alternate fibrosis stratification strategy to allow a closer examination of advanced stage 3 compared to stages 0–2.We used two strategies to adjust for the non-random distribution of characteristics differentiating treated from untreated patients, including age at initial liver biopsy, race/ethnicity, HCV genotype, history of heavy alcohol use, other substance use, psychiatric comorbidities, and social stability. First, these factors were assessed individually in univariate hazards models and incorporated into the full multivariate model through backward stepwise regression, as described in p<0.01).Among SFVA patients screened, 358  met the study inclusion criteria, and 159 patients (44.4%) received antiviral treatment for HCV. Approximately 80% were between the ages of 45 and 65 at initial liver clinic visit, with a mean age of 51 . This agp<0.0001) and treatment nonresponders and relapsers had more follow-up liver imaging (or liver biopsies) than never treated patients (p<0.0001) (p = 0.04), more likely to be African American and more likely to be infected with “difficult to clear” genotypes HCV genotypes 1 or 4 than 2 or 3  . Never tn 2 or 3 . Treatedn 2 or 3 . The mosn 2 or 3 .p = 0.15, data not shown), although the SVR group had a marginally higher baseline value as compared to the other treatment outcome groups (p = 0.05) (data not shown). In further analysis we found that 69.2% of nonresponders and relapsers had a decrease in ALT ≥25% following treatment as compared to 45.2% of never treated patients, while 19.2% of nonresponders and relapsers and 41.9% of never treated patients had an increase in ALT of ≥25% (p = 0.03) (data not shown).No statistically significant difference was found in mean baseline ALT between treated and never treated groups (e groups . We compp = 0.03) as were patients with HCV genotypes 1 or 4 (p = 0.007) and those with higher pretreatment fibrosis stage (p = 0.004). No significant difference was noted in treatment success between the small number of SFVA patients who received IFNα monotherapy and those treated with IFNα or pegylated IFNα/RBV combination therapy  and employing the time dependent covariate for SFVA treatment group, nonresponders were twice as likely to develop cirrhosis when compared to never treated patients . The mean follow-up time was 7.7 years as compared to 10 years for the SFVA (p<0.0001)  . DescripCumulative incidence of cirrhosis among UCSF patients with baseline fibrosis stages 2 and 3 was examined using age adjusted proportional hazards curves , which ip = 0.006) (= 0.006) , panel BCox proportional hazards models were developed to examine the contribution of multiple risk factors on time-to-death . Time-toTime-to-death or liver transplantation analyses were repeated for the UCSF cohort. Proportionally fewer UCSF patients died and more underwent liver transplantation than SFVA patients . We examThe present study measured long-term outcomes in patients with chronic HCV in two independent cohorts followed over the course of 7.7 to 10 years. Cohort patients were heterogeneous with regard to demographic and psychosocial characteristics, representing typical clinical practice, and data collection methods were optimized to maximize validity and measure known confounders. Unlike previously published studies, SVR was not associated with significant protection against cirrhosis in either cohort, even after stratifying for baseline levels of liver fibrosis and adjusting for liver inflammation Although our study is not intended to identify an explanatory mechanism for this finding, it raises the question of whether hepatic inflammation and fibrosis could be increased by immunostimulatory IFNα-based antiviral therapies in cases where HCV is not eradicated. IFNα/RBV can trigger broad and robust antiviral T cell responses, which are beneficial when they result in SVR, but might contribute to worsened inflammation and scarring (cirrhosis) in the continued presence of viral antigens The long-term effects of IFNα-based anti-HCV treatment on liver disease progression in noncirrhotic patients have been difficult to quantify from previous studies. In a meta-analysis of HCV cohort studies with greater than one year of follow-up, nearly 70% tracked subjects for less than seven years, whereas the mean duration of follow-up among patients in our SFVA cohort was 10 years 3 trials which enrolled previously treated patients with advanced fibrosis. These two prospective studies examined histologic effects of low dose maintenance pegylated IFNα in prior HCV treatment failures with METAVIR F2 and F3 fibrosis at study initiation 3 study showed no statistically significant differences between METAVIR fibrosis scores of the treated and observation groups at the end of the study period In recent years, there has been an emphasis on studies of IFNa-based retreatment in previous nonresponders and relapsers and their outcomes compared to those achieving SVR post hoc analysis of primary data presented in this report found no significant difference in cirrhosis development among patients without SVR and untreated patients In one of the few large-scale studies to compare outcomes between IFNα treated and untreated patients, the Japanese IHIT Study Group followed patients who had been previously treated with IFNα monotherapy over a median period of 3.7 years, using paired biopsies to compare liver fibrosis progression among SVR patients, patients without SVR and untreated patients, stratified by fibrosis stage at initial biopsy Unlike the patients in the Japanese cohort, the majority of our patients were treated with IFNα/RBV combination therapy, rather than with IFNα monotherapy, and our follow-up period was more than twice as long. Our study included few Asian patients, who have a higher probability of achieving SVR, but also are more likely to progress to cirrhosis 3 studies, found a significant increase in all-cause mortality in IFNα maintenance patients and concluded that patients with severe fibrosis who failed previous IFNα treatment did not derive a survival benefit from further therapy with pegylated IFNα Previously published studies also found that never treated HCV patients had a greater mortality risk than patients who achieve SVR, and in some cases, those who fail treatment These studies did not assess the effects of clinical and behavioral risk factors on liver disease outcomes as comprehensively as ours did. Our multivariate time-to-death analyses reveal that, even though nonresponders had more than twice the hazard of cirrhosis, their survival was not significantly different from that of never treated patients. The effects of cirrhosis on survival in our cohort may be offset by the relatively younger age and more beneficial clinical and psychosocial risk factor profile of nonresponders compared with never treated patients. Our findings suggest that some previously reported benefits of therapy among treatment failures might be attributable to the lower concomitant risks associated with treatment candidacy, rather than to disease modifying benefits of pharmacologic therapy As is the case in most nonrandomized studies, the presence of bias by indication can be difficult to resolve Our results suggest the possibility that treatment with IFNα-based regimens without viral clearance may be associated with progressive liver disease. Although these data reflect the long-term outcomes for two entire patient cohorts at independent institutions, they should be interpreted with caution as they are derived from retrospective chart reviews. If confirmed, these results make a compelling case for the enhanced use of sensitive diagnostic and predictive tools, including recently described genetic tests, to identify patients most likely to benefit from IFNα-based treatment Figure S1Cumulative incidence of cirrhosis among UCSF patients with baseline fibrosis stages 2 and 3. Age adjusted proportional hazards curves indicate that there were no differences in the incidence of cirrhosis among treatment groups, unadjusted for other risk factors.(TIF)Click here for additional data file.Figure S2Proportion remaining alive among UCSF patients with baseline liver fibrosis stage 0–1 (panel A) and stage 2–4 (panel B). Age adjusted proportional hazards curves indicate that there were no deaths among treated patients with baseline fibrosis stage 0–1 (panel A). Amongst patients with baseline fibrosis stage 2–4, relapsers and never treated a greater proportion died during follow-up than among SVR and nonresponders patients, but these differences were not statistically significant.(TIF)Click here for additional data file.Table S1Demographic and Clinical Characteristics of the UCSF Cohort.(DOC)Click here for additional data file.Table S2Risk Factors of Treated and Untreated Patient Groups (UCSF Cohort).(DOC)Click here for additional data file.Table S3Predictors of Development of Cirrhosis Stratified by Fibrosis Stage (UCSF Cohort).(DOC)Click here for additional data file.Table S4Predictors of Death or Liver Transplant Stratified by Fibrosis Stage (UCSF Cohort).(DOC)Click here for additional data file.Statistical Methods S1Details of Construction and Reduction of Statistical Models.(DOC)Click here for additional data file.Replication Cohort S1UCSF Medical Center Liver Clinic.(DOC)Click here for additional data file.