Glycogen storage disease type III: a mixed-methods study to assess the burden of disease.

Background: Glycogen storage disease type III (GSD III) is a rare inherited disorder that results from a glycogen debranching enzyme deficiency. Objectives: The purpose of this research was to collect data on the signs, symptoms, and impacts of GSD III from the perspective of adult patients and caregivers of individuals with GSD III. Design: Online survey and qualitative interviews. Methods: Following institutional review board approval, adult patients and caregivers of children with GSD III were recruited through advocacy networks and clinical sites. If eligible, participants were consented, screened, and sent a survey and/or participated in a 60-min interview. The survey and interview included questions about family history, diagnosis, signs and symptoms, impacts, and management of GSD III. Conceptual models were developed following the analysis of results. Results: In all, 29 adults and 46 caregivers completed the online survey and/or the interviews with 73 survey and 19 interview respondents. Adults and caregivers reported digestive, musculoskeletal, growth and physical appearance, and cardiac signs and symptoms. Liver conditions were reported by most respondents (83%). Adults and caregivers frequently reported impacts such as difficulty keeping up with peers (77%) and difficulty exercising/difficulty with physical activity (53%). Hypoglycemia was frequently reported in both adults and children, with more than half reporting hospitalizations due to hypoglycemia. Caregivers focused on hypoglycemia when reporting signs/symptoms that most interfere with their child's life and prevention of hypoglycemia as a desired outcome for an effective therapy. Adults most often reported muscle weakness as a top interfering symptom and the most important goal of a potential therapy. Impacts were also reported in activities of daily living, cognitive, emotional, work/school, and sleep domains. Conclusion: Individuals with GSD III experience a broad spectrum of symptoms and disease impacts. There is an unmet need for therapies that improve metabolic control, reduce the burden of dietary management, reduce fatigue and liver problems, and improve muscle strength and function.

In vivo characterization of glycogen storage disease type III in a mouse model using glycoNOE MRI.

PURPOSE: Glycogen storage disease type III (GSD III) is a rare inherited metabolic disease characterized by excessive accumulation of glycogen in liver, skeletal muscle, and heart. Currently, there are no widely available noninvasive methods to assess tissue glycogen levels and disease load. Here, we use glycogen nuclear Overhauser effect (glycoNOE) MRI to quantify hepatic glycogen levels in a mouse model of GSD III. METHODS: Agl knockout mice (n = 13) and wild-type controls (n = 10) were scanned for liver glycogen content using glycoNOE MRI. All mice were fasted for 12 to 16 h before MRI scans. GlycoNOE signal was quantified by fitting the Z-spectrum using a four-pool Voigt lineshape model. Next, the fitted direct water saturation pool was removed and glycoNOE signal was estimated from the integral of the residual Z spectrum within -0.6 to -1.4 ppm. Glycogen concentration was also measured ex vivo using a biochemical assay. RESULTS: GlycoNOE MRI clearly distinguished Agl knockout mice from wild-type controls, showing a statistically significant difference in glycoNOE signals in the livers across genotypes. There was a linear correlation between glycoNOE signal and glycogen concentration determined by the biochemical assay. The obtained glycoNOE maps of mouse livers also showed higher glycogen levels in Agl knockout mice compared to wild-type mice. CONCLUSION: GlycoNOE MRI was used successfully as a noninvasive method to detect liver glycogen levels in mice, suggesting the potential of this method to be applied to assess glycogen storage diseases.

Efficacy, safety, and pharmacokinetics of teduglutide in adult Japanese patients with short bowel syndrome and intestinal failure: two phase III studies with an extension.

PURPOSE: The short- and long-term efficacy, safety, and pharmacokinetics of teduglutide were analyzed in adult Japanese patients with short bowel syndrome and intestinal failure (SBS-IF). METHODS: Patients received teduglutide 0.05 mg/kg/day in clinical trials (TED-C14-004, SHP633-306, and extension SHP633-307). Data were analyzed at 24 weeks and an interim data cut-off of 4.5 years. RESULTS: The parenteral support (PS) volume decreased by ≥ 20% for 9/18 patients at 24 weeks and in all 11 patients by data cut-off in SHP633-307. The mean (standard deviation) PS volume decreased from baseline at 24 weeks in TED-C14-004 (-30.1 ± 25.9%) and SHP633-306 (-25.6 ± 25.5%), and at data cut-off in SHP633-307 (-57.08 ± 28.49%). Teduglutide was absorbed quickly. The adverse events were consistent with the underlying disease and known adverse drug reactions. Anti-teduglutide antibody titers declined with long-term treatment. CONCLUSIONS: In Japanese adults with SBS-IF, teduglutide treatment was associated with clinically meaningful reductions in PS requirements, similar to findings in prior international studies. No new safety concerns specific to the Japanese SBS-IF patient population were identified with short- or long-term teduglutide treatment. Anti-teduglutide antibody titers disappeared in most Japanese adults with long-term treatment. These results constitute the longest evaluation of teduglutide treatment within clinical trials reported to date.

Safety Findings in Pediatric Patients During Long-Term Treatment With Teduglutide for Short-Bowel Syndrome-Associated Intestinal Failure: Pooled Analysis of 4 Clinical Studies.

BACKGROUND: This analysis assessed combined safety data from 4 clinical studies of teduglutide in pediatric patients with short-bowel syndrome-associated intestinal failure (SBS-IF). METHODS: Safety data from teduglutide-treated patients in 4 clinical trials were pooled. The completed 12-week and 24-week phase 3 core studies (NCT01952080/EudraCT 2013-004588-30 and NCT02682381/EudraCT 2015-002252-27) enrolled children aged 1-17 years with SBS-IF. Patients could elect to enroll in ongoing open-label extensions (NCT02949362/EudraCT 2016-000863-17 and NCT02954458/EudraCT 2016-000849-30). Interim data from ongoing studies were included. RESULTS: Safety data are reported for 89 pediatric patients treated with teduglutide for a median (range) of 51.7 (5.0-94.7) weeks. Adverse events (AEs) were reported in all patients; the most common were vomiting (51.7%), pyrexia (43.8%), upper respiratory tract infection (41.6%), and cough (33.7%). Thirty-five patients (39.3%) had AEs considered related to teduglutide treatment; abdominal pain and vomiting were most frequent (5.6% each). Three serious AEs in 3 patients (3.4%) were considered related to teduglutide treatment: ileus, d-lactic acidosis, and gastrointestinal obstruction due to hard stools. All 3 events resolved. One cecal polyp was detected, which was not biopsied or found on repeat colonoscopy. No cases of neoplasia occurred. CONCLUSION: Based on integrated data from 4 clinical studies, including long-term follow-up for ≤161 weeks, teduglutide had a safety profile consistent with the individual core pediatric studies and as expected for pediatric patients with SBS-IF who never received teduglutide. The most frequent AEs reflected treatment with teduglutide, complications of the underlying disease, and typical childhood illnesses.

Safety and Efficacy of Teduglutide in Pediatric Patients With Intestinal Failure due to Short Bowel Syndrome: A 24-Week, Phase III Study.

BACKGROUND: This study evaluated the safety and efficacy of teduglutide in pediatric patients with short bowel syndrome-associated intestinal failure (SBS-IF). METHODS: A 24-week, phase III trial with 2 randomized, double-blind teduglutide dose groups and a nonblinded standard of care (SOC) arm was used; patients received 0.025 mg/kg or 0.05 mg/kg teduglutide once daily. Safety end points included treatment-emergent adverse events (TEAEs) and growth parameters. The primary efficacy/pharmacodynamic end point was the number of patients who achieved a ≥20% reduction in parenteral support (PS) from baseline at week 24. RESULTS: All 59 enrolled patients completed the study (0.025 mg/kg, n = 24; 0.05 mg/kg, n = 26; SOC, n = 9). Baseline demographics and disease characteristics were comparable among groups. TEAEs were reported by 98% and 100% of patients in the teduglutide and SOC groups, respectively. The most common TEAEs in the teduglutide-treated groups were pyrexia and vomiting. The primary end point was achieved by 13 (54.2%), 18 (69.2%), and 1 (11.1%) patients who received 0.025 mg/kg teduglutide, 0.05 mg/kg teduglutide, and SOC, respectively (P < 0.05 vs SOC). Both 0.025-mg/kg and 0.05-mg/kg teduglutide groups showed clinically significant reductions in PS volume (P < 0.05 vs SOC), PS calories, days per week and hours per day of PS infusions, and increases in enteral nutrition and plasma citrulline at week 24 compared with baseline. Two (8.3%, 0.025 mg/kg teduglutide) and 3 patients (11.5%, 0.05 mg/kg teduglutide) achieved enteral autonomy. CONCLUSION: The safety profile of teduglutide was similar to that reported previously in children and adults. Treatment with teduglutide was associated with significant reductions in PS for pediatric patients with SBS-IF over 24 weeks.

Correction: HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days-Implications for HIV Remission.

[This corrects the article DOI: 10.1371/journal.ppat.1005000.][This corrects the article DOI: 10.1371/journal.ppat.1005740.][This corrects the article DOI: 10.1371/journal.ppat.1005679.].

In Vivo Hepatic Reprogramming of Myofibroblasts with AAV Vectors as a Therapeutic Strategy for Liver Fibrosis.

Liver fibrosis, a form of scarring, develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. Initially, collagen produced by myofibroblasts (MFs) functions to maintain the integrity of the liver, but excessive collagen accumulation suppresses residual hepatocyte function, leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 capsid as effective in transducing MFs in a mouse model of liver fibrosis. We then showed in lineage-tracing mice that AAV6 vector-mediated in vivo hepatic reprogramming of MFs generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis.

HIV-1 Mutation and Recombination Rates Are Different in Macrophages and T-cells.

High rates of mutation and recombination help human immunodeficiency virus (HIV) to evade the immune system and develop resistance to antiretroviral therapy. Macrophages and T-cells are the natural target cells of HIV-1 infection. A consensus has not been reached as to whether HIV replication results in differential recombination between primary T-cells and macrophages. Here, we used HIV with silent mutation markers along with next generation sequencing to compare the mutation and the recombination rates of HIV directly in T lymphocytes and macrophages. We observed a more than four-fold higher recombination rate of HIV in macrophages compared to T-cells (p < 0.001) and demonstrated that this difference is not due to different reliance on C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 5 (CCR5) co-receptors between T-cells and macrophages. We also found that the pattern of recombination across the HIV genome (hot and cold spots) remains constant between T-cells and macrophages despite a three-fold increase in the overall recombination rate. This indicates that the difference in rates is a general feature of HIV DNA synthesis during macrophage infection. In contrast to HIV recombination, we found that T-cells have a 30% higher mutation rate than macrophages (p < 0.001) and that the mutational profile is similar between these cell types. Unexpectedly, we found no association between mutation and recombination in macrophages, in contrast to T-cells. Our data highlights some of the fundamental difference of HIV recombination and mutation amongst these two major target cells of infection. Understanding these differences will provide invaluable insights toward HIV evolution and how the virus evades immune surveillance and anti-retroviral therapeutics.

Assessing the therapeutic potential of lab-made hepatocytes.

Hepatocyte transplantation has potential as a bridge or even alternative to whole-organ liver transplantation. Because donor livers are scarce, realizing this potential requires the development of alternative cell sources. To be therapeutically effective, surrogate hepatocytes must replicate the complex function and ability to proliferate of primary human hepatocytes. Ideally, they are also autologous to eliminate the need for immune suppression, which can have severe side effects and may not be sufficient to prevent rejection long term. In the past decade, several methods have been developed to generate hepatocytes from other readily and safely accessible somatic cells. These lab-made hepatocytes show promise in animal models of liver diseases, supporting the feasibility of autologous liver cell therapies. Here, we review recent preclinical studies exemplifying different types of lab-made hepatocytes that can potentially be used in autologous liver cell therapies. To define the therapeutic efficacy of current lab-made hepatocytes, we compare them to primary human hepatocytes, focusing on engraftment efficiency and posttransplant proliferation and function. In addition to summarizing published results, we discuss animal models and assays effective in assessing therapeutic efficacy. This analysis underscores the therapeutic potential of current lab-made hepatocytes, but also highlights deficiencies and uncertainties that need to be addressed in future studies aimed at developing liver cell therapies with lab-made hepatocytes. (Hepatology 2016;64:287-294).

Estimating the in-vivo HIV template switching and recombination rate.

BACKGROUND: HIV recombination has been estimated in vitro using a variety of approaches, and shows a high rate of template switching per reverse transcription event. In-vivo studies of recombination generally measure the accumulation of recombinant strains over time, and thus do not directly estimate a comparable template switching rate. METHOD: To examine whether the estimated in-vitro template switching rate is representative of the rate that occurs during HIV infection in vivo, we adopted a novel approach, analysing single genome sequences from early founder viruses to study the in-vivo template switching rate in the env region of HIV. RESULTS: We estimated the in-vivo per cycle template switching rate to be between 0.5 and 1.5/1000 nt, or approximately 5-14 recombination events over the length of the HIV genome. CONCLUSION: The in-vivo estimated template switching rate is close to the in-vitro estimated rate found in primary T lymphocytes but not macrophages, which is consistent with the majority of HIV infection occurring in T lymphocytes.

HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days--Implications for HIV Remission.

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to 'purge' the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different 'reactivation founder' viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.

A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

BACKGROUND: Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. METHOD: Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. RESULTS: We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. CONCLUSIONS: Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection.

CD8(+) T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8(+) T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8(+) T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8(+) T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8(+) T cells reach 0.5% of total CD8(+) T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8(+) T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8(+) T cells.

Measuring turnover of SIV DNA in resting CD4+ T cells using pyrosequencing: implications for the timing of HIV eradication therapies.

Resting CD4+ T cells are a reservoir of latent HIV-1. Understanding the turnover of HIV DNA in these cells has implications for the development of eradication strategies. Most studies of viral latency focus on viral persistence under antiretroviral therapy (ART). We studied the turnover of SIV DNA resting CD4+ T cells during active infection in a cohort of 20 SIV-infected pigtail macaques. We compared SIV sequences at two Mane-A1*084:01-restricted CTL epitopes using serial plasma RNA and resting CD4+ T cell DNA samples by pyrosequencing, and used a mathematical modeling approach to estimate SIV DNA turnover. We found SIV DNA turnover in resting CD4+ T cells was slow in animals with low chronic viral loads, consistent with the long persistence of latency seen under ART. However, in animals with high levels of chronic viral replication, turnover was high. SIV DNA half-life within resting CD4 cells correleated with viral load (p = 0.0052) at the Gag KP9 CTL epitope. At a second CTL epitope in Tat (KVA10) there was a trend towards an association of SIV DNA half-life in resting CD4 cells and viral load (p = 0.0971). Further, we found that the turnover of resting CD4+ T cell SIV DNA was higher for escape during early infection than for escape later in infection (p = 0.0084). Our results suggest viral DNA within resting CD4 T cells is more labile and may be more susceptible to reactivation/eradication treatments when there are higher levels of virus replication and during early/acute infection.

Fifteen to twenty percent of HIV substitution mutations are associated with recombination.

HIV undergoes high rates of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we used a system of silent marker mutations in HIV and a single round of infection in primary T lymphocytes combined with a high-throughput sequencing and mathematical modeling approach to directly estimate the viral recombination and mutation rates. From >7 million nucleotides (nt) of sequences from HIV infection, we observed 4,801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1,000 nt, respectively). We used experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We found that the single-cycle virus-induced mutation rate is 4.6 × 10(-5) mutations per nt after correction. By sorting of our data into recombined and nonrecombined sequences, we found a significantly higher mutation rate in recombined regions (P = 0.003 by Fisher's exact test). We used a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination and is not simply colocated in the genome. By comparing mutation rates in recombined and nonrecombined regions, we found that recombination-associated mutations account for 15 to 20% of all mutations occurring during reverse transcription.

Identifying recombination hot spots in the HIV-1 genome.

UNLABELLED: HIV-1 infection is characterized by the rapid generation of genetic diversity that facilitates viral escape from immune selection and antiretroviral therapy. Despite recombination's crucial role in viral diversity and evolution, little is known about the genomic factors that influence recombination between highly similar genomes. In this study, we use a minimally modified full-length HIV-1 genome and high-throughput sequence analysis to study recombination in gag and pol in T cells. We find that recombination is favored at a number of recombination hot spots, where recombination occurs six times more frequently than at corresponding cold spots. Interestingly, these hot spots occur near important features of the HIV-1 genome but do not occur at sites immediately around protease inhibitor or reverse transcriptase inhibitor drug resistance mutations. We show that the recombination hot and cold spots are consistent across five blood donors and are independent of coreceptor-mediated entry. Finally, we check common experimental confounders and find that these are not driving the location of recombination hot spots. This is the first study to identify the location of recombination hot spots between two similar viral genomes with great statistical power and under conditions that closely reflect natural recombination events among HIV-1 quasispecies. IMPORTANCE: The ability of HIV-1 to evade the immune system and antiretroviral therapy depends on genetic diversity within the viral quasispecies. Retroviral recombination is an important mechanism that helps to generate and maintain this genetic diversity, but little is known about how recombination rates vary within the HIV-1 genome. We measured recombination rates in gag and pol and identified recombination hot and cold spots, demonstrating that recombination is not random but depends on the underlying gene sequence. The strength and location of these recombination hot and cold spots can be used to improve models of viral dynamics and evolution, which will be useful for the design of robust antiretroviral therapies.

Trivalent live attenuated influenza-simian immunodeficiency virus vaccines: efficacy and evolution of cytotoxic T lymphocyte escape in macaques.

There is an urgent need for a human immunodeficiency virus (HIV) vaccine that induces robust mucosal immunity. CD8(+) cytotoxic T lymphocytes (CTLs) apply substantial antiviral pressure, but CTLs to individual epitopes select for immune escape variants in both HIV in humans and SIV in macaques. Inducing multiple simian immunodeficiency virus (SIV)-specific CTLs may assist in controlling viremia. We vaccinated 10 Mane-A1*08401(+) female pigtail macaques with recombinant influenza viruses expressing three Mane-A1*08401-restricted SIV-specific CTL epitopes and subsequently challenged the animals, along with five controls, intravaginally with SIV(mac251). Seroconversion to the influenza virus vector resulted and small, but detectable, SIV-specific CTL responses were induced. There was a boost in CTL responses after challenge but no protection from high-level viremia or CD4 depletion was observed. All three CTL epitopes underwent a coordinated pattern of immune escape during early SIV infection. CTL escape was more rapid in the vaccinees than in the controls at the more dominant CTL epitopes. Although CTL escape can incur a "fitness" cost to the virus, a putative compensatory mutation 20 amino acids upstream from an immunodominant Gag CTL epitope also evolved soon after the primary CTL escape mutation. We conclude that vaccines based only on CTL epitopes will likely be undermined by rapid evolution of both CTL escape and compensatory mutations. More potent and possibly broader immune responses may be required to protect pigtail macaques from SIV.

A novel transcript of cyclin-dependent kinase-like 5 (CDKL5) has an alternative C-terminus and is the predominant transcript in brain.

The X-linked cyclin-dependent kinase-like 5 (CDKL5) gene is an important molecular determinant of early-onset intractable seizures with infantile spasms and Rett syndrome-like phenotype. The gene encodes a kinase that may influence components of molecular pathways associated with MeCP2. In humans there are two previously reported splice variants that differ in the 5' untranslated exons and produce the same 115 kDa protein. Furthermore, very recently, a novel transcript including a novel exon (16b) has been described. By aligning both the human and mouse CDKL5 proteins to the orthologs of other species, we identified a theoretical 107 kDa isoform with an alternative C-terminus that terminates in intron 18. In human brain and all other tissues investigated except the testis, this novel isoform is the major CDKL5 transcript. The detailed characterisation of this novel isoform of CDKL5 reveals functional and subcellular localisation attributes that overlap greatly, but not completely, with that of the previously studied human CDKL5 protein. Considering its predominant expression in the human and mouse brain, we believe that this novel isoform is likely to be of primary pathogenic importance in human diseases associated with CDKL5 deficiency, and suggest that screening of the related intronic sequence should be included in the molecular genetic analyses of patients with a suggestive clinical phenotype.

A nutrient-sensitive interaction between Sirt1 and HNF-1α regulates Crp expression.

Silent information regulator 2 (Sir2) orthologs are an evolutionarily conserved family of NAD-dependent protein deacetylases that regulate aging and longevity in model organisms. The mammalian Sir2 ortholog Sirt1 regulates metabolic and stress responses through the deacetylation of many transcriptional regulatory factors. To elucidate the mechanism by which Sirt1 controls gene expression in response to nutrient availability, we devised a bioinformatic screen combining gene expression analysis with phylogenetic footprinting to identify transcription factors as new candidate partners of Sirt1. One candidate target was HNF-1α, a homeodomain transcription factor that regulates pancreatic ß-cell and hepatocyte functions and is commonly mutated in patients with maturity-onset diabetes of the young (MODY). Interestingly, Sirt1 physically interacts with HNF-1αin vitro but does so in vivo only in nutrient-restricting conditions. This interaction requires 12-24 h of nutrient restriction and is dependent on protein synthesis. Both nutrient restriction and Sirt1 suppress HNF-1α transcriptional activity and the expression of one of its target genes, C-reactive protein (Crp), in mouse primary hepatocytes. Pharmacological inhibition of Sirt1 blocks the suppression of Crp by nutrient restriction. Similarly, Crp expression is also suppressed in fasted and diet-restricted liver. Furthermore, Sirt1 and HNF-1α co-localize on two HNF-1α binding sites on the Crp promoter, leading to decreased acetylation of lysine 16 of histone H4 at these sites only in response to nutrient restriction. These findings reveal a novel nutrient-dependent interaction between Sirt1 and HNF-1α and provide important insight into the molecular mechanism by which Sirt1 mediates the anti-aging effects of diet restriction.

Ultrastructural basis of enhanced antitumor cytotoxicity of cord blood-derived CTLs: a comparative analysis with peripheral blood and bone marrow.

Umbilical cord blood cells (UCBC) are a rich source of immature immune effector and accessory cells, including dendritic cells. UCBC-derived cytotoxic T lymphocytes (CTLs) generated against human breast cancer or neuroblastoma have shown an increased tumor-specific cytotoxicity compared to peripheral blood (PB)-derived CTLs. The precise mechanism of this increased cytotoxicity is not known. Since dendritic cells (DCs) play a central role in the immunostimulation, we compared the ultrastructure and antigen presenting nature of DCs from UCBC, PB and bone marrow (BM) at various stages of maturation using scanning and transmission electron microscopy as well as fluorescent microscopy to elucidate the mechanism underlying the increased cytotoxicity of UCBC-derived CTLs. DCs were examined for their immunophenotype nuclear morphology, dendritic processes and cytoplasmic endosomal vesicles after 0, 3, 7 and 10 days in culture with antigen priming on day 6. Results showed that there were smaller and more vesicles in UCB-DCs compared to DCs from the other two sources, while the endosomal vesicles in PB-DCs were heterogenous in size. The antigen processing ability of the UCB-DCs showed an increase in antigen-positive endosomes compared to PB-DCs as determined by the fluorescent microscopy. Thus, our results provided the comparative analyses of DCs from cord blood, peripheral blood and bone marrow, and suggested that UCBC-DCs might have better antigen presenting ability leading to increased CTL-mediated antitumor cytotoxicity.