A Coxsackievirus B1-mediated nonlytic Extracellular Vesicle-to-cell mechanism of virus transmission and its possible control through modulation of EV release.

Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced 'medium-sized' EVs (CVB1i-mEVs). These mEVs (100-300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection.

Loss of thymidine phosphorylase activity disrupts adipocyte differentiation and induces insulin-resistant lipoatrophic diabetes.

BACKGROUND: Thymidine phosphorylase (TP), encoded by the TYMP gene, is a cytosolic enzyme essential for the nucleotide salvage pathway. TP catalyzes the phosphorylation of the deoxyribonucleosides, thymidine and 2'-deoxyuridine, to thymine and uracil. Biallelic TYMP variants are responsible for Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE), an autosomal recessive disorder characterized in most patients by gastrointestinal and neurological symptoms, ultimately leading to death. Studies on the impact of TYMP variants in cellular systems with relevance to the organs affected in MNGIE are still scarce and the role of TP in adipose tissue remains unexplored. METHODS: Deep phenotyping was performed in three patients from two families carrying homozygous TYMP variants and presenting with lipoatrophic diabetes. The impact of the loss of TP expression was evaluated using a CRISPR-Cas9-mediated TP knockout (KO) strategy in human adipose stem cells (ASC), which can be differentiated into adipocytes in vitro. Protein expression profiles and cellular characteristics were investigated in this KO model. RESULTS: All patients had TYMP loss-of-function variants and first presented with generalized loss of adipose tissue and insulin-resistant diabetes. CRISPR-Cas9-mediated TP KO in ASC abolished adipocyte differentiation and decreased insulin response, consistent with the patients' phenotype. This KO also induced major oxidative stress, altered mitochondrial functions, and promoted cellular senescence. This translational study identifies a new role of TP by demonstrating its key regulatory functions in adipose tissue. CONCLUSIONS: The implication of TP variants in atypical forms of monogenic diabetes shows that genetic diagnosis of lipodystrophic syndromes should include TYMP analysis. The fact that TP is crucial for adipocyte differentiation and function through the control of mitochondrial homeostasis highlights the importance of mitochondria in adipose tissue biology.

Biomarkers in Rare Diseases 2.0.

It is estimated that there are over 7000 rare diseases, collectively affecting more than 350 million individuals worldwide [...].

Biomarkers in Rare Diseases.

There is no single global definition of a rare disease, and for different geographical areas the definition is based on the disease occurrence in that population [...].

Circulating miRNAs as Biomarkers for Mitochondrial Neuro-Gastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disease for which there are currently no validated outcome measures for assessing therapeutic intervention efficacy. The aim of this study was to identify a plasma and/or serum microRNA (miRNA) biomarker panel for MNGIE. Sixty-five patients and 65 age and sex matched healthy controls were recruited and assigned to one of four study phases: (i) discovery for sample size determination; (ii) candidate screening; (iii) candidate validation; and (iv) verifying the performance of the validated miRNA panel in four patients treated with erythrocyte-encapsulated thymidine phosphorylase (EE-TP), an enzyme replacement under development for MNGIE. Quantitative PCR (qPCR) was used to profile miRNAs in serum and/or plasma samples collected for the discovery, validation and performance phases, and next generation sequencing (NGS) analysis was applied to serum samples assigned to the candidate screening phase. Forty-one differentially expressed candidate miRNAs were identified in the sera of patients (p < 0.05, log2 fold change > 1). The validation cohort revealed that of those, 27 miRNAs were upregulated in plasma and three miRNAs were upregulated in sera (p < 0.05). Through binary logistic regression analyses, five plasma miRNAs (miR-192-5p, miR-193a-5p, miR-194-5p, miR-215-5p and miR-34a-5p) and three serum miRNAs (miR-192-5p, miR-194-5p and miR-34a-5p) were shown to robustly distinguish MNGIE from healthy controls. Reduced longitudinal miRNA expression of miR-34a-5p was observed in all four patients treated with EE-TP and coincided with biochemical and clinical improvements. We recommend the inclusion of the plasma exploratory miRNA biomarker panel in future clinical trials of investigational therapies for MNGIE; it may have prognostic value for assessing clinical status.

Differences in collagen ultrastructure of human first trimester decidua basalis and parietalis: implications for trophoblastic invasion of the placental bed.

AIM: The human embryo-maternal interface in the first trimester of pregnancy is an area of extensive tissue remodeling. Because collagen is the most abundant constituent of the extracellular matrix of the placental bed, successful invasion must involve its rapid turnover. We compared the nature and distribution of collagen fibrils in decidua basalis and parietalis. METHODS: We used a direct-vision hysteroscopic technique to obtain biopsies of the decidua basalis and parietalis from 11 women undergoing pregnancy termination in the first trimester. The biopsies were subjected to light, transmission and scanning electron microscopy, and immunohistochemical studies using mouse monoclonal antibodies against cytokeratin 7 and collagen types I, III and V. RESULTS: Collagen fibrils in the stroma of decidua basalis were significantly thicker when compared to those in decidua parietalis (56.48 ± 1.37 nm vs 45.64 ± 0.85 nm; P < 0.0001 [mean ± standard error]) between 9 and 12 weeks gestation, but this difference in thickness was not observed at gestations below 9 weeks. In basalis, the fibrils appeared disrupted at most places surrounding the decidual/trophoblast cells while a uniform regular arrangement was preserved throughout most of parietalis. CONCLUSION: There are differences in the ultrastructure of collagen fibrils between basalis and parietalis, with thicker and disrupted fibrils within abundant amorphous tissue in basalis, and thinner uniform fibrils in parietalis. These differences may reflect an adaptive response by decidua or a direct consequence of the invading trophoblast cells.

Mitochondrial neurogastrointestinal encephalomyopathy: approaches to diagnosis and treatment.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disease caused by mutations in TYMP, the gene encoding for the enzyme thymidine phosphorylase. The resulting enzyme deficiency leads to a systemic accumulation of thymidine and 2'-deoxyuridine and ultimately mitochondrial failure due to a progressive acquisition of secondary mitochondrial DNA (mtDNA) mutations and mtDNA depletion. MNGIE is characterised by gastrointestinal dysmotility, cachexia, peripheral neuropathy, ophthalmoplegia, ptosis and leukoencephalopathy. The disease is progressively degenerative and leads to death at an average age of 37.6 years. Patients invariably encounter misdiagnoses, diagnostic delays, and non-specific clinical management. Despite its rarity, MNGIE has invoked much interest in the development of therapeutic strategies, mainly because it is one of the few mitochondrial disorders where the molecular abnormality is metabolically and physically accessible to manipulation. This review provides a resume of the current diagnosis and treatment approaches and aims to increase the clinical awareness of MNGIE and thereby facilitate early diagnosis and timely access to treatments, before the development of untreatable and irreversible organ damage.

Erythrocytes as Carriers of Therapeutic Enzymes.

Therapeutic enzymes are administered for the treatment of a wide variety of diseases. They exert their effects through binding with a high affinity and specificity to disease-causing substrates to catalyze their conversion to a non-noxious product, to induce an advantageous physiological change. However, the metabolic and clinical efficacies of parenterally or intramuscularly administered therapeutic enzymes are very often limited by short circulatory half-lives and hypersensitive and immunogenic reactions. Over the past five decades, the erythrocyte carrier has been extensively studied as a strategy for overcoming these limitations and increasing therapeutic efficacy. This review examines the rationale for the different therapeutic strategies that have been applied to erythrocyte-mediated enzyme therapy. These strategies include their application as circulating bioreactors, targeting the monocyte-macrophage system, the coupling of enzymes to the surface of the erythrocyte and the engineering of CD34+ hematopoietic precursor cells for the expression of therapeutic enzymes. An overview of the diverse biomedical applications for which they have been investigated is also provided, including the detoxification of exogenous chemicals, thrombolytic therapy, enzyme replacement therapy for metabolic diseases and antitumor therapy.

Pregnancy in MNGIE: a clinical and metabolic honeymoon.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an inherited disease caused by a deficiency in thymidine phosphorylase and characterized by elevated systemic deoxyribonucleotides and gastrointestinal (GI) and neurological manifestations. We report the clinical and biochemical manifestations that were evaluated in a single patient before, during, and after pregnancy, over a period of 7 years. GI symptoms significantly improved, and plasma deoxyribonucleotide concentrations decreased during pregnancy. Within days after delivery, the patient's digestive symptoms recurred, coinciding with a rapid increase in plasma deoxyribonucleotide concentrations. We hypothesize that the clinico-metabolic improvements could be attributed to the enzyme replacement action of the placental thymidine phosphorylase.

Quantification of Plasma and Urine Thymidine and 2'-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2'-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2'-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2'-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10-10,000 ng/mL for plasma, and 1-50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP.

Organs to Cells and Cells to Organoids: The Evolution of in vitro Central Nervous System Modelling.

With 100 billion neurons and 100 trillion synapses, the human brain is not just the most complex organ in the human body, but has also been described as "the most complex thing in the universe." The limited availability of human living brain tissue for the study of neurogenesis, neural processes and neurological disorders has resulted in more than a century-long strive from researchers worldwide to model the central nervous system (CNS) and dissect both its striking physiology and enigmatic pathophysiology. The invaluable knowledge gained with the use of animal models and post mortem human tissue remains limited to cross-species similarities and structural features, respectively. The advent of human induced pluripotent stem cell (hiPSC) and 3-D organoid technologies has revolutionised the approach to the study of human brain and CNS in vitro, presenting great potential for disease modelling and translational adoption in drug screening and regenerative medicine, also contributing beneficially to clinical research. We have surveyed more than 100 years of research in CNS modelling and provide in this review an historical excursus of its evolution, from early neural tissue explants and organotypic cultures, to 2-D patient-derived cell monolayers, to the latest development of 3-D cerebral organoids. We have generated a comprehensive summary of CNS modelling techniques and approaches, protocol refinements throughout the course of decades and developments in the study of specific neuropathologies. Current limitations and caveats such as clonal variation, developmental stage, validation of pluripotency and chromosomal stability, functional assessment, reproducibility, accuracy and scalability of these models are also discussed.

Safety and Efficacy of Erythrocyte Encapsulated Thymidine Phosphorylase in Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare autosomal recessive disorder of nucleoside metabolism that is caused by mutations in the nuclear thymidine phosphorylase gene (TYMP) gene, encoding for the enzyme thymidine phosphorylase. There are currently no approved treatments for MNGIE. The aim of this study was to investigate the safety, tolerability, and efficacy of an enzyme replacement therapy for the treatment of MNGIE. In this single centre study, three adult patients with MNGIE received intravenous escalating doses of erythrocyte encapsulated thymidine phosphorylase (EE-TP; dose range: 4 to 108 U/kg/4 weeks). EE-TP was well tolerated and reductions in the disease-associated plasma metabolites, thymidine, and deoxyuridine were observed in all three patients. Clinical improvements, including weight gain and improved disease scores, were observed in two patients, suggesting that EE-TP is able to reverse some aspects of the disease pathology. Transient, non-serious adverse events were observed in two of the three patients; these did not lead to therapy discontinuation and they were managed with pre-medication prior to infusion of EE-TP. To conclude, enzyme replacement therapy with EE-TP demonstrated biochemical and clinical therapeutic efficacy with an acceptable clinical safety profile.

Erythrocyte Encapsulated Thymidine Phosphorylase for the Treatment of Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy: Study Protocol for a Multi-Centre, Multiple Dose, Open Label Trial.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder which primarily affects the gastrointestinal and nervous systems. This disease is caused by mutations in the nuclear TYMP gene, which encodes for thymidine phosphorylase, an enzyme required for the normal metabolism of deoxynucleosides, thymidine, and deoxyuridine. The subsequent elevated systemic concentrations of deoxynucleosides lead to increased intracellular concentrations of their corresponding triphosphates, and ultimately mitochondrial failure due to progressive accumulation of mitochondrial DNA (mtDNA) defects and mtDNA depletion. Currently, there are no treatments for MNGIE where effectiveness has been evidenced in clinical trials. This Phase 2, multi-centre, multiple dose, open label trial without a control will investigate the application of erythrocyte-encapsulated thymidine phosphorylase (EE-TP) as an enzyme replacement therapy for MNGIE. Three EE-TP dose levels are planned with patients receiving the dose level that achieves metabolic correction. The study duration is 31 months, comprising 28 days of screening, 90 days of run-in, 24 months of treatment and 90 days of post-dose follow-up. The primary objectives are to determine the safety, tolerability, pharmacodynamics, and efficacy of multiple doses of EE-TP. The secondary objectives are to assess EE-TP immunogenicity after multiple dose administrations and changes in clinical assessments, and the pharmacodynamics effect of EE-TP on clinical assessments.

The development of an in vitro cerebral organoid model for investigating the pathomolecular mechanisms associated with the central nervous system involvement in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE).

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disorder caused by mutations in the thymidine phosphorylase gene (TYMP), leading to secondary aberrations to the mitochondrial genome. The disease is characterised by gastrointestinal dysmotility, sensorimotor peripheral neuropathy and leukoencephalopathy. The understanding of the molecular mechanisms that underlie the central nervous system (CNS) is hindered by the lack of a representative disease model; to address this we have developed an in vitro 3-D cerebral organoid of MNGIE. Induced pluripotent stem cells (iPSCs) generated from peripheral blood mononuclear cells (PBMCs) of a healthy control and a patient with MNGIE were characterised to ascertain bona fide pluripotency through the evaluation of pluripotency markers and the differentiation to the germ layers. iPSC lines were differentiated into cerebral organoids. Thymidine phosphorylase expression in PBMCs, iPSCs and Day 92 organoids was evaluated by immunoblotting and intact organoids were sampled for histological evaluation of neural markers. iPSCs demonstrated the expression of pluripotency markers SOX2 and TRA1-60 and the plasticity to differentiate into the germ layers. Cerebral organoids stained positive for the neural markers GFAP, O4, Tuj1, Nestin, SOX2 and MBP. Consistent with the disease phenotypes, MNGIE cells did not display thymidine phosphorylase expression whereas control PBMCs and Day 92 organoids did. Remarkably, control iPSCs did not stain positive for thymidine phosphorylase. We have established for the first time a MNGIE iPSC line and cerebral organoid model, which exhibited the expression of cells relevant to the study of the disease, such as neural stem cells, astrocytes and myelinating oligodendrocytes.

Discovery profiling and bioinformatics analysis of serum microRNA in Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE).

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare and fatal inherited metabolic disorder due to mutations in the nuclear TYMP gene and leads to a deficiency in the enzyme thymidine phosphorylase. This results in an accumulation of the deoxynucleosides, thymidine and deoxyuridine in the cellular and extracellular compartments, ultimately leading to mitochondrial failure. The understanding of the precise molecular mechanisms that underlie the disease pathology is limited, being hampered by the rarity of the disorder. Expression profiling of serum based mircoRNAs and subsequent bioinformatical analyses provide an approach to facilitate the identity of dysregulated genes and signalling pathways potentially involved in the pathogenesis of MNGIE.

Mitochondrial Neurogastrointestinal Encephalomyopathy: Into the Fourth Decade, What We Have Learned So Far.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare metabolic autosomal recessive disease, caused by mutations in the nuclear gene TYMP which encodes the enzyme thymidine phosphorylase. The resulting enzyme deficiency leads to a systemic accumulation of the deoxyribonucleosides thymidine and deoxyuridine, and ultimately mitochondrial failure due to a progressive acquisition of secondary mitochondrial DNA (mtDNA) mutations and mtDNA depletion. Clinically, MNGIE is characterized by gastrointestinal and neurological manifestations, including cachexia, gastrointestinal dysmotility, peripheral neuropathy, leukoencephalopathy, ophthalmoplegia and ptosis. The disease is progressively degenerative and leads to death at an average age of 37.6 years. As with the vast majority of rare diseases, patients with MNGIE face a number of unmet needs related to diagnostic delays, a lack of approved therapies, and non-specific clinical management. We provide here a comprehensive collation of the available knowledge of MNGIE since the disease was first described 42 years ago. This review includes symptomatology, diagnostic procedures and hurdles, in vitro and in vivo disease models that have enhanced our understanding of the disease pathology, and finally experimental therapeutic approaches under development. The ultimate aim of this review is to increase clinical awareness of MNGIE, thereby reducing diagnostic delay and improving patient access to putative treatments under investigation.

Validation of an Immunoassay for Anti-thymidine Phosphorylase Antibodies in Patients with MNGIE Treated with Enzyme Replacement Therapy.

Erythrocyte encapsulated thymidine phosphorylase is recombinant Escherichia coli thymidine phosphorylase encapsulated within human autologous erythrocytes and is under development as an enzyme replacement therapy for the ultra-rare inherited metabolic disorder mitochondrial neurogastrointestinal encephalomyopathy. This study describes the method validation of a two-step bridging electrochemiluminescence immunoassay for the detection of anti-thymidine phosphorylase antibodies in human serum according to current industry practice and regulatory guidelines. The analytical method was assessed for screening cut point, specificity, selectivity, precision, prozone effect, drug tolerance, and stability. Key findings were a correction factor of 129 relative light units for the cut-point determination; a specificity cut point of 93% inhibition; confirmed intra-assay and inter-assay precision; assay sensitivity of 356 ng/mL; no matrix or prozone effects up to 25,900 ng/mL; a drug tolerance of 156 ng/mL; and stability at room temperature for 24 hr and up to five freeze-thaws. Immunogenicity evaluations of serum from three patients who received erythrocyte encapsulated thymidine phosphorylase under a compassionate treatment program showed specific anti-thymidine phosphorylase antibodies in one patient. To conclude, a sensitive, specific, and selective immunoassay has been validated for the measurement of anti-thymidine phosphorylase antibodies; this will be utilized in a phase II pivotal clinical trial of erythrocyte encapsulated thymidine phosphorylase.

Drug-loaded erythrocytes: on the road toward marketing approval.

Erythrocyte drug encapsulation is one of the most promising therapeutic alternative approaches for the administration of toxic or rapidly cleared drugs. Drug-loaded erythrocytes can operate through one of the three main mechanisms of action: extension of circulation half-life (bioreactor), slow drug release, or specific organ targeting. Although the clinical development of erythrocyte carriers is confronted with regulatory and development process challenges, industrial development is expanding. The manufacture of this type of product can be either centralized or bedside based, and different procedures are employed for the encapsulation of therapeutic agents. The major challenges for successful industrialization include production scalability, process validation, and quality control of the released therapeutic agents. Advantages and drawbacks of the different manufacturing processes as well as success key points of clinical development are discussed. Several entrapment technologies based on osmotic methods have been industrialized. Companies have already achieved many of the critical clinical stages, thus providing the opportunity in the future to cover a wide range of diseases for which effective therapies are not currently available.

Unexplained gastrointestinal symptoms: think mitochondrial disease.

Defects in mitochondrial function are increasingly recognised as central to the pathogenesis of many diseases, both inherited and acquired. Many of these mitochondrial defects arise from abnormalities in mitochondrial DNA and can result in multisystem disease, with gastrointestinal involvement common. Moreover, mitochondrial disease may present with a range of non-specific symptoms, and thus can be easily misdiagnosed, or even considered to be non-organic. We describe the clinical, histopathological and genetic findings of six patients from three families with gastrointestinal manifestations of mitochondrial disease. In two of the patients, anorexia nervosa was considered as an initial diagnosis. These cases illustrate the challenges of both diagnosing and managing mitochondrial disease and highlight two important but poorly understood aspects, the clinical and the genetic. The pathophysiology of gastrointestinal involvement in mitochondrial disease is discussed and emerging treatments are described. Finally, we provide a checklist of investigations for the gastroenterologist when mitochondrial disease is suspected.

The development and validation of an immunoassay for the measurement of anti-thymidine phosphorylase antibodies in mouse and dog sera.

Erythrocyte encapsulated thymidine phosphorylase (EE-TP) is under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a fatal metabolic disorder resulting from an inherited deficiency of the enzyme thymidine phosphorylase. We report here the development and validation of a sensitive electrochemiluminescent (ECL) bridging immunoassay to support Good Laboratory Practice (GLP)-compliant preclinical safety studies of EE-TP in the mouse and dog. Affinity-purified rabbit anti-E. coli thymidine phosphorylase (TP) antibody was used as a calibrator standard with an effective working range of 2.5-7500 ng/mL. The minimum required dilution (MRD) for both mouse and dog sera was 1:10. The mean analytical recoveries for anti-TP antibodies spiked into serum at 70 ng/mL and 7000 ng/mL were 117.9% and 93.2%, respectively for mouse, and 112.0% and 104.3%, respectively for dog. The intra-assay precision (coefficient of variation, CV) ranged between 1.1% and 8.0% in mouse serum, and 1.9% and 2.5% in dog serum. Inter-assay precision ranged between -1.6% and 6.7% in mouse serum, and -13.0% and -2.5% in dog serum. Assay cut-point/screening cut-point correction factors were 201.37 and 44.4, respectively for mouse and dog sera. For future analysis of positive test samples, less than 37.12% (mouse) and 31.41% (dog) inhibition of the assay signal in the confirmation assay will confer anti-TP antibody specificity. Assay drift and hook effects (prozone) were not observed. The intra-assay and inter-assay accuracy for robustness were within ±20%.