Herpes simplex virus 2 is an important human pathogen as the causative agent of genital herpes, neonatal herpes, and increased risk of HIV acquisition and transmission. Nevertheless, the only genomic sequence that has been completed is the attenuated HSV-2 HG52 laboratory strain. In this study we defined the genomic sequence of the HSV-2 SD90e low passage clinical isolate and a plaque-purified derivative, SD90-3P. We found minimal sequence differences between SD90e and SD90-3P. However, in comparisons with the HSV-2 HG52 reference genome sequence, the SD90e genome ORFs contained numerous point mutations, 13 insertions/delections (indels), and 9 short compensatory frameshifts. The indels were true sequence differences, but the compensatory frameshifts were likely sequence errors in the original HG52 sequence. Because HG52 virus is less virulent than other HSV-2 strains and may not be representative of wildtype HSV-2 strains, we propose that the HSV-2 SD90e genome serve as the new HSV-2 reference genome.
Paracoccidiodomycosis (PCM) is a clinically important fungal disease that can acquire serious systemic forms and is caused by the thermodimorphic fungal Paracoccidioides spp. PCM is a tropical disease that is endemic in Latin America, where up to ten million people are infected; 80% of reported cases occur in Brazil, followed by Colombia and Venezuela. To enable genomic studies and to better characterize the pathogenesis of this dimorphic fungus, two reference strains of P. brasiliensis (Pb03, Pb18) and one strain of P. lutzii (Pb01) were sequenced . While the initial draft assemblies were accurate in large scale structure and had high overall base quality, the sequences had frequent small scale defects such as poor quality stretches, unknown bases (N's), and artifactual deletions or nucleotide duplications, all of which caused larger scale errors in predicted gene structures. Since assembly consensus errors can now be addressed using next generation sequencing (NGS) in combination with recent methods allowing systematic assembly improvement, we re-sequenced the three reference strains of Paracoccidioides spp. using Illumina technology. We utilized the high sequencing depth to re-evaluate and improve the original assemblies generated from Sanger sequence reads, and obtained more complete and accurate reference assemblies. The new assemblies led to improved transcript predictions for the vast majority of genes of these reference strains, and often substantially corrected gene structures. These include several genes that are central to virulence or expressed during the pathogenic yeast stage in Paracoccidioides and other fungi, such as HSP90, RYP1-3, BAD1, catalase B, alpha-1,3-glucan synthase and the beta glucan synthase target gene FKS1. The improvement and validation of these reference sequences will now allow more accurate genome-based analyses. To our knowledge, this is one of the first reports of a fully automated and quality-assessed upgrade of a genome assembly and annotation for a non-model fungus.
The fungal genus Paracoccidioides is the causal agent of paracoccidioidomycosis (PCM), a neglected tropical disease that is endemic in several countries of South America. Paracoccidioides is a pathogenic dimorphic fungus that is capable of converting to a virulent yeast form after inhalation by the host. Therefore the molecular biology of the switch to the yeast phase is of particular interest for understanding the virulence of this and other human pathogenic fungi, and ultimately for reducing the morbidity and mortality caused by such fungal infections. We here present the strategy and methods we used to update and improve accuracy of three reference genome sequences of Paracoccidioides spp. utilizing state-of-the-art Illumina re-sequencing, assembly improvement, re-annotation, and quality assessment. The resulting improved genome resource should be of wide use not solely for advancing research on the genetics and molecular biology of Paracoccidioides and the closely related pathogenic species Histoplasma and Blastomyces, but also for fungal diagnostics based on sequencing or molecular assays, characterizing rapidly changing proteins that may be involved in virulence, SNP-based population analyses and other tasks that require high sequence accuracy. The genome update and underlying strategy and methods also serve as a proof of principle that could encourage similar improvements of other draft genomes.
Spindle length varies dramatically across species and during early development to segregate chromosomes optimally. Both intrinsic factors, such as regulatory molecules, and extrinsic factors, such as cytoplasmic volume, determine spindle length scaling. However, the properties that govern spindle shape and whether these features can be modulated remain unknown. Here, we analyzed quantitatively how the molecular players which regulate microtubule dynamics control the kinetics of spindle formation and shape. We find that, in absence of Clasp1 and Clasp2, spindle assembly is biphasic due to unopposed inward pulling forces from the kinetochore-fibers and that kinetochore-fibers also alter spindle geometry. We demonstrate that spindle shape scaling is independent of the nature of the molecules that regulate dynamic microtubule properties, but is dependent on the steady-state metaphase spindle length. The shape of the spindle scales anisotropically with increasing length. Our results suggest that intrinsic mechanisms control the shape of the spindle to ensure the efficient capture and alignment of chromosomes independently of spindle length.
K-fiber; Clasp; Microtubules; Mitosis; Spindle
Advances in modern sequencing technologies allow us to generate sufficient data to analyze hundreds of bacterial genomes from a single machine in a single day. This potential for sequencing massive numbers of genomes calls for fully automated methods to produce high-quality assemblies and variant calls. We introduce Pilon, a fully automated, all-in-one tool for correcting draft assemblies and calling sequence variants of multiple sizes, including very large insertions and deletions. Pilon works with many types of sequence data, but is particularly strong when supplied with paired end data from two Illumina libraries with small e.g., 180 bp and large e.g., 3–5 Kb inserts. Pilon significantly improves draft genome assemblies by correcting bases, fixing mis-assemblies and filling gaps. For both haploid and diploid genomes, Pilon produces more contiguous genomes with fewer errors, enabling identification of more biologically relevant genes. Furthermore, Pilon identifies small variants with high accuracy as compared to state-of-the-art tools and is unique in its ability to accurately identify large sequence variants including duplications and resolve large insertions. Pilon is being used to improve the assemblies of thousands of new genomes and to identify variants from thousands of clinically relevant bacterial strains. Pilon is freely available as open source software.
Microsporidia comprise a phylum of obligate intracellular pathogens related to fungi. Microsporidia Nematocida sp1 strain ERTm6 was isolated from wild-caught Caenorhabditis briggsae and causes a lethal intestinal infection in Caenorhabditis nematodes. We report the genome sequence of N. sp1 ERTm6, which will facilitate study of the Nematocida genus and other Microsporidia.
Draft genome sequences of seven enterohepatic Helicobacter species, H. bilis, H. canadensis, H. canis, H. cinaedi, H. winghamensis, H. pullorum, and H. macacae, are presented. These isolates were obtained from clinical patients and a nonhuman primate. Due to potential zoonotic risks, we characterized antibiotic resistance markers and Helicobacter virulence factors.
LuCaP serially transplantable xenografts derived from primary and metastatic human prostate cancer encompass the molecular and cellular heterogeneity of the disease and are an invaluable resource for in vivo preclinical studies. A limitation of this model, however, has been the inability to establish and passage cell cultures derived from the xenografts. Here, we describe a novel spheroid culture system that supports long-term growth of LuCaP cells in vitro.
Xenografts were minced and digested with collagenase. Tissue dissociation was terminated while the majority of cells remained as clusters rather than single cells. The cell clusters were suspended in StemPro medium supplemented with R1881 and Y-27632, a Rho kinase inhibitor, and placed in ultralow attachment dishes for spheroid culture. Serial passage was achieved by partial digestion to small clusters with trypsin/EDTA in the presence of Y-27632. Cell viability, growth and phenotype were monitored with LIVE-DEAD®, MTS, qRT-PCR and immunocytochemical assays.
Cells from six LuCaP xenografts formed proliferating spheroids that were serially passaged a minimum of 3 times and cryopreserved. Two of the cell lines, LuCaP 136 and LuCaP 147, were further passaged and characterized. Both expressed biomarkers characteristic of the xenografts of origin, were determined to be of independent origin by STR fingerprinting, and were free of mycoplasma. LuCaP 147 formed tumors similar to the original xenograft when injected into mice.
The ability to culture LuCaP cells affords new opportunities for fast, cheap, and efficient preclinical studies and extends the value of the LuCaP xenograft models.
prostate cancer; preclinical model; spheroids
Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.
Horizontal chromosome transfer introduces host-specific pathogenicity among members of the Fusarium oxysporum species complex and is responsible for some of the most destructive and intractable plant diseases. This paper reports the genome sequence of F. oxysporum f. sp. melonis (NRRL 26406), a causal agent of Fusarium wilt disease on melon.
Sporothrix schenckii is a pathogenic dimorphic fungus that grows as a yeast and as mycelia. This species is the causative agent of sporotrichosis, typically a skin infection. We report the genome sequence of S. schenckii, which will facilitate the study of this fungus and of the Sporothrix schenckii group.
The isotope-labeled intravenous glucose tolerance test (IVGTT) combined with computer modeling is widely used to derive parameters related to glucose metabolism in vivo. Most of these methods involve use of either 2H2-labeled or 13C1-labeled D-glucose as a tracer with GC-MS to measure the isotope enrichment. These methods are challenging, both technologically and economically. We have developed a novel approach that is suitable for labeled-IVGTT studies involving a large cohort of individuals.
The tracer, D-[13C6]glucose, is a low-cost alternative with the significant advantage that the sixth isotope of natural glucose has virtually zero natural abundance, which facilitates isotopomer analysis with <1% labeled glucose in the infusate. After deproteinization of plasma samples collected at various times, glucose is converted to a stable derivative, purified by solid-phase extraction (SPE), and analyzed by HPLC–electrospray ionization mass spectrometry to accumulate the isotope-abundance data for the A+2, A+3, and A+6 ions of the glucose derivative. A 2-pool modeling program was used to derive standard kinetic parameters.
With labeled-IVGTT data from 10 healthy male individuals, the values for insulin sensitivity, glucose effectiveness, and the plasma clearance rate estimated with the 2-pool minimal model compared well with values obtained via traditional methods.
The relative simplicity and robustness of the new method permit the preparation and analysis of up to 48 samples/day, a throughput equivalent to 2 complete IVGTT experiments, and this method is readily adaptable to existing 96 well–format purification and analytical systems.
The formation of discrete elastin bands at the tips of secondary alveolar septa is important for normal alveolar development, but the mechanisms regulating the lung elastogenic program are incompletely understood. JNK suppress elastin synthesis in the aorta and is important in a host of developmental processes. We sought to determine whether JNK suppresses pulmonary fibroblast elastogenesis during lung development.
Alveolar size, elastin content, and mRNA of elastin-associated genes were quantitated in wild type and JNK-deficient mouse lungs, and expression profiles were validated in primary lung fibroblasts. Tropoelastin protein was quantitated by Western blot. Changes in lung JNK activity throughout development were quantitated, and pJNK was localized by confocal imaging and lineage tracing.
By morphometry, alveolar diameters were increased by 7% and lung elastin content increased 2-fold in JNK-deficient mouse lungs compared to wild type. By Western blot, tropoelastin protein was increased 5-fold in JNK-deficient lungs. Postnatal day 14 (PND14) lung JNK activity was 11-fold higher and pJNK:JNK ratio 6-fold higher compared to PN 8 week lung. Lung tropoelastin, emilin-1, fibrillin-1, fibulin-5, and lysyl oxidase mRNAs inversely correlated with lung JNK activity during alveolar development. Phosphorylated JNK localized to pulmonary lipofibroblasts. PND14 JNK-deficient mouse lungs contained 7-fold more tropoelastin, 2,000-fold more emilin-1, 800-fold more fibrillin-1, and 60-fold more fibulin-5 than PND14 wild type lungs. Primarily lung fibroblasts from wild type and JNK-deficient mice showed similar differences in elastogenic mRNAs.
JNK suppresses fibroblast elastogenesis during the alveolar stage of lung development.
Lung development; Elastin; c-terminal Jun kinase; Rho kinase
Enzyme replacement therapy with alglucosidase alfa for infantile Pompe disease has improved survival creating new management challenges. We describe an emerging phenotype in a retrospective review of long-term survivors.
Inclusion criteria included ventilator-free status and age ≤6 months at treatment initiation, and survival to age ≥5 years. Clinical outcome measures included invasive ventilator-free survival and parameters for cardiac, pulmonary, musculoskeletal, gross motor and ambulatory status; growth; speech, hearing, and swallowing; and gastrointestinal and nutritional status.
Eleven of 17 patients met study criteria. All were cross-reactive immunologic material-positive, alive, and invasive ventilator-free at most recent assessment, with a median age of 8.0 years (range: 5.4 to 12.0 years). All had marked improvements in cardiac parameters. Commonly present were gross motor weakness, motor speech deficits, sensorineural and/or conductive hearing loss, osteopenia, gastroesophageal reflux disease, and dysphagia with aspiration risk. Seven of 11 patients were independently ambulatory and four required the use of assistive ambulatory devices. All long-term survivors had low or undetectable anti-alglucosidase alfa antibody titers.
Long-term survivors exhibited sustained improvements in cardiac parameters and gross motor function. Residual muscle weakness, hearing loss, risk for arrhythmias, hypernasal speech, dysphagia with risk for aspiration, and osteopenia were commonly observed findings.
acid maltase deficiency; enzyme replacement therapy; glycogen storage disease type II; neuromuscular diseases; Pompe disease
Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin.
We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls.
DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease.
We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen. (Funded by the National Cancer Institute and others.)
Oxidative stress has been implicated in Down syndrome (DS) pathology. This study compares DS individuals and controls on their urinary levels of allantoin and 2,3-dinor-iPF2α-III; these biomarkers have been previously validated in a clinical model of oxidative stress. Urine samples were collected from 48 individuals with DS and 130 controls. Biomarkers were assayed by ultra-performance liquid chromatography-tandem mass spectrometry, normalized by urinary creatinine concentration. After adjusting for age and gender, mean allantoin levels were lower among DS individuals versus controls (p = 0.04). The adjusted mean levels of 2,3-dinor-iPF2α-III were similar in DS individuals and controls (p = 0.7). Our results do not support the hypothesis that DS individuals have chronic systemic oxidative stress.
Down syndrome; biomarker; urine; tandem mass spectrometry
Microsporidia from the Encephalitozoonidae are obligate intracellular parasites with highly conserved and compacted nuclear genomes: they have few introns, short intergenic regions, and almost identical gene complements and chromosome arrangements. Comparative genomics of Encephalitozoon and microsporidia in general have focused largely on the genomic diversity between different species, and we know very little about the levels of genetic diversity within species. Polymorphism studies with Encephalitozoon are so far restricted to a small number of genes, and a few genetically distinct strains have been identified; most notably, three genotypes (ECI, ECII, and ECIII) of the model species E. cuniculi have been identified based on variable repeats in the rRNA internal transcribed spacer (ITS). To determine if E. cuniculi genotypes are genetically distinct lineages across the entire genome and at the same time to examine the question of intraspecies genetic diversity in microsporidia in general, we sequenced de novo genomes from each of the three genotypes and analyzed patterns of single nucleotide polymorphisms (SNPs) and insertions/deletions across the genomes. Although the strains have almost identical gene contents, they harbor large numbers of SNPs, including numerous nonsynonymous changes, indicating massive intraspecies variation within the Encephalitozoonidae. Based on this diversity, we conclude that the recognized genotypes are genetically distinct and propose new molecular markers for microsporidian genotyping.
Enterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand how E. faecium emerged as a leading hospital pathogen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation.
Enterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections.
Auditory hallucinations (AH) are a cardinal feature of schizophrenia spectrum disorders. They are not disease specific, however, and can occur in other conditions, including affective psychoses.
In this descriptive, cross-sectional study, we examined AH in relation to other psychotic symptoms, mood symptoms, illness severity, and functional status in 569 patients with psychosis (n=172 schizophrenia, n=153 schizoaffective disorder, n=244 bipolar disorder with psychotic features).
323 (56.7%) patients reported a lifetime history of AH (75.6% of patients with schizophrenia, 71.9% schizoaffective disorder, and 34.0% bipolar disorder). The mean score for the hallucinations item (P3) of the Positive and Negative Syndrome Scale (PANSS) in the AH group was 3.66 ± 1.79, indicating mild to moderate state hallucinations severity. AH were strongly associated with hallucinations in other sensory modalities and with the first-rank symptoms of delusions of control, thought insertion, and thought broadcasting. Multivariate analysis showed that AH were associated with lower education even after controlling for diagnosis, age, and gender. There was no association between AH and functional status as measured by the Multnomah Community Ability Scale (MCAS).
AH are associated with specific clinical features across the continuum of both schizophrenic and affective psychoses independent of DSM-IV diagnosis.
Hallucinations, auditory; Psychotic disorders; Schizophrenia; Bipolar disorder
Deficiency of acid alpha glucosidase (GAA) causes Pompe disease, which is usually fatal if onset occurs in infancy. Patients synthesize a non-functional form of GAA or are unable to form native enzyme. Enzyme replacement therapy with recombinant human GAA (rhGAA) prolongs survival in infantile Pompe patients but may be less effective in cross-reactive immunologic material (CRIM)-negative patients. We retrospectively analyzed the influence of CRIM status on outcome in 21 CRIM-positive and 11 CRIM-negative infantile Pompe patients receiving rhGAA. Patients were from the clinical setting and from clinical trials of rhGAA, were ≤6 months of age, were not invasively ventilated, and were treated with IV rhGAA at a cumulative or total dose of 20 or 40 mg/kg/2 weeks. Outcome measures included survival, invasive ventilator-free survival, cardiac status, gross motor development, development of antibodies to rhGAA, and levels of urinary Glc4.
Following 52 weeks of treatment, 6/11 (54.5%) CRIM-negative and 1/21 (4.8%) CRIM-positive patients were deceased or invasively ventilated (p < 0.0001). By age 27.1 months, all CRIM-negative patients and 4/21 (19.0%) CRIM-positive patients were deceased or invasively ventilated. Cardiac function and gross motor development improved significantly more in the CRIM-positive group. IgG antibodies to rhGAA developed earlier and serotiters were higher and more sustained in the CRIM-negative group. CRIM-negative status predicted reduced overall survival and invasive ventilator-free survival and poorer clinical outcomes in infants with Pompe disease treated with rhGAA. The effect of CRIM status on outcome appears to be mediated by antibody responses to the exogenous protein.
Pompe disease; Glycogen storage disease; Enzyme replacement therapy; Lysosomal storage disease; Acid alpha glucosidase; GAA; Cross-reactive immunologic material; CRIM; Antibody
Infantile Pompe disease resulting from a deficiency of lysosomal acid α-glucosidase (GAA) requires enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA). Cross-reactive immunologic material negative (CRIM-negative) Pompe patients develop high-titer antibody to the rhGAA and do poorly. We describe successful tolerance induction in CRIM-negative patients.
Two CRIM-negative patients with preexisting anti-GAA antibodies were treated therapeutically with rituximab, methotrexate, and gammaglobulins. Two additional CRIM-negative patients were treated prophylactically with a short course of rituximab and methotrexate, in parallel with initiating rhGAA.
In both patients treated therapeutically, anti-rhGAA was eliminated after 3 and 19 months. All four patients are immune tolerant to rhGAA, off immune therapy, showing B-cell recovery while continuing to receive ERT at ages 36 and 56 months (therapeutic) and 18 and 35 months (prophylactic). All patients show clinical response to ERT, in stark contrast to the rapid deterioration of their nontolerized CRIM-negative counterparts.
The combination of rituximab with methotrexate ± intravenous gammaglobulins (IVIG) is an option for tolerance induction of CRIM-negative Pompe to ERT when instituted in the naïve setting or following antibody development. It should be considered in other conditions in which antibody response to the therapeutic protein elicits robust antibody response that interferes with product efficacy.
immune tolerance; methotrexate; Pompe disease; rituximab
We report the clinical course of a patient with severe infantile onset Pompe disease [cross-reactive immunologic material (CRIM) negative, R854X/R854X] who was diagnosed prenatally and received standard dosing of alglucosidase alfa (Myozyme®) enzyme replacement therapy (ERT) from day 10 of life until she passed away at the age of 3 years 9 months. In the immediate neonatal period there was cardiomegaly on chest X-ray, cardiac hypertrophy by echocardiogram, and development of a wide complex tachycardia. CRIM negative (CN) status was suspected based on her family history, and the available data at the time indicated that CN patients had limited survival even with ERT. However, given the opportunity for very early treatment, the treating provider and family elected to initiate treatment with ERT, without immune modulation. By 9 months of age echocardiogram was normal. Early motor development was within normal limits but by 2 years of age her developmental progress had slowed. She seroconverted by the 4th month of ERT, and anti-rhGAA antibody titers peaked at 25,600 in the 27th month. Immunomodulatory therapy was considered but declined by family. She acquired Influenza A at 2 years 6 months, which led to a prolonged hospitalization with invasive respiratory support, and placement of tracheostomy and gastrostomy tube. Her developmental progress ceased, and she died suddenly at home from a presumed cardiac event at age 3 years 9 months. The poor outcomes observed in CN patients have been attributed to the development of high sustained antibody titers. Although this CN patient’s anti-rhGAA response was elevated and sustained, it is unlike any of the 3 patterns that have been previously described: high titer CN, high titer CRIM positive (HTCP), and low titer CP (LTCP) patients. This patient’s clinical course, with achievement of 24 months of motor gains, 30 months of ventilator-free survival and 45 month survival, is like that of only a fraction of ERT treated CN patients, yet it is identical to other reported CN patients in its relentless progression and early fatality. The immunologic response (moderate sustained antibody titers) described here has not been previously reported and may have played a role in the overall pattern of developmental decline. In light of proposed universal newborn screening for Pompe disease, there is an urgent need for improved understanding of the interplay between immunologic responses to the only available treatment, ERT, and the relentless nature of this disease in CN patients.
Glycogen storage disease type II; Pompe disease; Enzyme replacement therapies; Immunologic response; Antibodies
Listeria monocytogenes, a foodborne bacterial pathogen, is comprised of four phylogenetic lineages that vary with regard to their serotypes and distribution among sources. In order to characterize lineage-specific genomic diversity within L. monocytogenes, we sequenced the genomes of eight strains from several lineages and serotypes, and characterized the accessory genome, which was hypothesized to contribute to phenotypic differences across lineages. The eight L. monocytogenes genomes sequenced range in size from 2.85–3.14 Mb, encode 2,822–3,187 genes, and include the first publicly available sequenced representatives of serotypes 1/2c, 3a and 4c. Mapping of the distribution of accessory genes revealed two distinct regions of the L. monocytogenes chromosome: an accessory-rich region in the first 65° adjacent to the origin of replication and a more stable region in the remaining 295°. This pattern of genome organization is distinct from that of related bacteria Staphylococcus aureus and Bacillus cereus. The accessory genome of all lineages is enriched for cell surface-related genes and phosphotransferase systems, and transcriptional regulators, highlighting the selective pressures faced by contemporary strains from their hosts, other microbes, and their environment. Phylogenetic analysis of O-antigen genes and gene clusters predicts that serotype 4 was ancestral in L. monocytogenes and serotype 1/2 associated gene clusters were putatively introduced through horizontal gene transfer in the ancestral population of L. monocytogenes lineage I and II.
Cervical cancer is caused by high-risk, cancer-causing human papillomaviruses (HPV) and is the second highest cause of cancer deaths in women globally. The majority of cervical cancers express well-characterized HPV oncogenes, which are potential targets for immunotherapeutic vaccination. Here we develop a rabbit haemorrhagic disease virus (RHDV) virus-like particle (VLP)-based vaccine designed for immunotherapy against HPV16 positive tumours. An RHDV-VLP, modified to contain the universal helper T cell epitope PADRE and decorated with an MHC I-restricted peptide (aa 48–57) from the HPV16 E6, was tested for its immunotherapeutic efficacy against the TC-1 HPV16 E6 and E7-expressing tumour in mice. The E6-RHDV-VLP-PADRE was administered therapeutically for the treatment of a pre-existing TC-1 tumour and was delivered with antibodies either to deplete regulatory T cells (anti-CD25) or to block T cell suppression mediated through CTLA-4. As a result, the tumour burden was reduced by around 50% and the median survival time of mice to the humane endpoint was almost doubled the compared to controls. The incorporation of PADRE into the RHDV-VLP was necessary for an E6-specific enhancement of the anti-tumour response and the co-administration of the immune modifying antibodies contributed to the overall efficacy of the immunotherapy. The E6-RHDV-VLP-PADRE shows immunotherapeutic efficacy, prolonging survival for HPV tumour-bearing mice. This was enhanced by the systemic administration of immune-modifying antibodies that are commercially available for use in humans. There is potential to further modify these particles for even greater efficacy in the path to development of an immunotherapeutic treatment for HPV precancerous and cancer stages.