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BACKGROUND: Giant axonal neuropathy is a rare, autosomal recessive, pediatric, polysymptomatic, neurodegenerative disorder caused by biallelic loss-of-function variants in GAN, the gene encoding gigaxonin. METHODS: We conducted an intrathecal dose-escalation study of scAAV9/JeT-GAN (a self-complementary adeno-associated virus-based gene therapy containing the GAN transgene) in children with giant axonal neuropathy. Safety was the primary end point. The key secondary clinical end point was at least a 95% posterior probability of slowing the rate of change (i.e., slope) in the 32-item Motor Function Measure total percent score at 1 year after treatment, as compared with the pretreatment slope. RESULTS: One of four intrathecal doses of scAAV9/JeT-GAN was administered to 14 participants - 3.5×1013 total vector genomes (vg) (in 2 participants), 1.2×1014 vg (in 4), 1.8×1014 vg (in 5), and 3.5×1014 vg (in 3). During a median observation period of 68.7 months (range, 8.6 to 90.5), of 48 serious adverse events that had occurred, 1 (fever) was possibly related to treatment; 129 of 682 adverse events were possibly related to treatment. The mean pretreatment slope in the total cohort was -7.17 percentage points per year (95% credible interval, -8.36 to -5.97). At 1 year after treatment, posterior mean changes in slope were -0.54 percentage points (95% credible interval, -7.48 to 6.28) with the 3.5×1013-vg dose, 3.23 percentage points (95% credible interval, -1.27 to 7.65) with the 1.2×1014-vg dose, 5.32 percentage points (95% credible interval, 1.07 to 9.57) with the 1.8×1014-vg dose, and 3.43 percentage points (95% credible interval, -1.89 to 8.82) with the 3.5×1014-vg dose. The corresponding posterior probabilities for slowing the slope were 44% (95% credible interval, 43 to 44); 92% (95% credible interval, 92 to 93); 99% (95% credible interval, 99 to 99), which was above the efficacy threshold; and 90% (95% credible interval, 89 to 90). Between 6 and 24 months after gene transfer, sensory-nerve action potential amplitudes increased, stopped declining, or became recordable after being absent in 6 participants but remained absent in 8. CONCLUSIONS: Intrathecal gene transfer with scAAV9/JeT-GAN for giant axonal neuropathy was associated with adverse events and resulted in a possible benefit in motor function scores and other measures at some vector doses over a year. Further studies are warranted to determine the safety and efficacy of intrathecal AAV-mediated gene therapy in this disorder. (Funded by the National Institute of Neurological Disorders and Stroke and others; ClinicalTrials.gov number, NCT02362438.).
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Técnicas de Transferencia de Gen , Terapia Genética , Neuropatía Axonal Gigante , Niño , Humanos , Proteínas del Citoesqueleto/genética , Terapia Genética/efectos adversos , Terapia Genética/métodos , Neuropatía Axonal Gigante/genética , Neuropatía Axonal Gigante/terapia , Transgenes , Inyecciones EspinalesRESUMEN
While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Autopsia , Oncología Médica , Neoplasias , Humanos , Neoplasias/patología , Neoplasias/mortalidad , Oncología Médica/métodos , Animales , Investigación Biomédica TraslacionalRESUMEN
BACKGROUND: Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. METHODS: Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. RESULTS: While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression (CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1), including those in the COX-2/PGE2 pathway (IL1B, PTGER1/EP1 and PTGER4/EP4). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. CONCLUSIONS: This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.
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Antígenos de Grupos Sanguíneos , Melanoma , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Inhibidores de Puntos de Control Inmunológico , Ciclooxigenasa 2 , Dinoprostona , Inhibidores de la Ciclooxigenasa 2 , Inflamación , Receptores de Antígenos de Linfocitos TRESUMEN
3MC syndromes are rare heterogeneous autosomal recessive conditions previously designated as Mingarelli, Malpuech, Michels, and Carnevale syndromes, characterized by dysmorphic facial features, facial clefts, growth restriction, and intellectual disability. 3MC is secondary to mutations in the MASP1, MASP3, COLEC11, and COLEC10 genes. The number of patients with 3MC syndrome with known mutations in the COLEC11 or MASP1 is, to date, less than 50. At the time this case presented (2015), the only gene identified in Online Mendelian Inheritance in Man to be associated with 3MC syndrome was MASP1. We present, to the best of our knowledge, the first prenatal report of 3MC syndrome, secondary to a homozygous variant in MASP1. Fetal findings included bilateral cleft lip and palate, abnormality of the sacral spine, a right echogenic pelvic kidney, and brachycephaly. 3MC syndrome should be considered as part of the differential diagnosis when fetal ultrasound detects facial clefts and spinal defects, as the risk of recurrence is significant and a molecularly confirmed diagnosis allows for alternate reproductive options.
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Anomalías Múltiples/genética , Labio Leporino/genética , Discapacidad Intelectual/diagnóstico , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Músculos Abdominales/anomalías , Músculos Abdominales/patología , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/patología , Blefaroptosis/genética , Blefaroptosis/patología , Labio Leporino/diagnóstico , Labio Leporino/patología , Fisura del Paladar/genética , Fisura del Paladar/patología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patología , Craneosinostosis/genética , Craneosinostosis/patología , Criptorquidismo/genética , Criptorquidismo/patología , Cara/anomalías , Femenino , Luxación Congénita de la Cadera/genética , Luxación Congénita de la Cadera/patología , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Masculino , Mutación/genética , Embarazo , Estrabismo/genética , Estrabismo/patologíaRESUMEN
Rationale: Remodeling and fibrosis of the right ventricle (RV) may cause RV dysfunction and poor survival in patients with pulmonary hypertension. Objectives: To investigate the consequences of RV fibrosis modulation and the accompanying cellular changes on RV function. Methods: Expression of fibrotic markers was assessed in the RV of patients with pulmonary hypertension, the murine pulmonary artery banding, and rat monocrotaline and Sugen5416/hypoxia models. Invasive hemodynamic and echocardiographic assessment was performed on galectin-3 knockout or inhibitor-treated mice. Measurements and Main Results: Established fibrosis was characterized by marked expression of galectin-3 and an enhanced number of proliferating RV fibroblasts. Galectin-3 genetic and pharmacologic inhibition or antifibrotic treatment with pirfenidone significantly diminished RV fibrosis progression in the pulmonary artery banding model, without improving RV functional parameters. RV fibrotic regions were populated with mesenchymal cells coexpressing vimentin and PDGFRα (platelet-derived growth factor receptor-α), but generally lacked αSMA (α-smooth muscle actin) positivity. Serum levels of galectin-3 were increased in patients with idiopathic pulmonary arterial hypertension but did not correlate with cardiac function. No changes of galectin-3 expression were observed in the lungs. Conclusions: We identified extrapulmonary galectin-3 as an important mediator that drives RV fibrosis in pulmonary hypertension through the expansion of PDGFRα/vimentin-expressing cardiac fibroblasts. However, interventions effectively targeting fibrosis lack significant beneficial effects on RV function.
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Fibrosis/complicaciones , Fibrosis/fisiopatología , Galectina 3/inmunología , Hipertrofia Ventricular Derecha/etiología , Hipertrofia Ventricular Derecha/fisiopatología , Disfunción Ventricular Derecha/etiología , Disfunción Ventricular Derecha/fisiopatología , Animales , Austria , Baltimore , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratas , Función Ventricular Derecha/efectos de los fármacosRESUMEN
Desbuquois dysplasia (DBQD1 [MIM 251450]) is an autosomal recessive chondrodysplasia with micromelia, severe joint laxity and dislocations, and a characteristic radiographic "monkey wrench" appearance at the proximal femur. Type 1 Desbuquois dysplasia is caused by mutations in CANT1 and is distinct from Type 2, caused by mutations in XYLT1, in that the former has unique hand anomalies including accessory phalangeal ossification centers, advanced carpal bone maturation, and/or axial phalangeal deviation. Severe prenatal presentations have been rarely reported. We report a Pakistani female in a consanguineous relationship with a diagnosis of Type 1 Desbuquois dysplasia in three consecutive pregnancies. Multiple similar severe fetal limb anomalies were detected by ultrasound in Pregnancy 1 and 2. Regions of homozygosity within the single nucleotide polymorphism (SNP)-microarray from both terminated fetuses were compared, revealing CANT1 as a likely disease-causing candidate gene. Insufficient fetal DNA precluded confirmatory testing, therefore parents were tested; both had a previously reported heterozygous CANT1 mutation (c.643G>T; Glu215Term). The patient presented with a third pregnancy revealing similarly abnormal limb position and probable polysyndactyly by ultrasound. Targeted testing of CANT1 revealed homozygous c.643G>T CANT1 mutations and this pregnancy was terminated. In clinical situations in which ample DNA is not available or more expensive testing (e.g., whole exome sequencing) with a longer turnaround time is not feasible, utilization of SNP-microarray in consanguineous families at risk for rare autosomal recessive disorders may dramatically narrow the list of candidate genes.
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Anomalías Craneofaciales/diagnóstico , Anomalías Craneofaciales/genética , Enanismo/diagnóstico , Enanismo/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Homocigoto , Inestabilidad de la Articulación/diagnóstico , Inestabilidad de la Articulación/genética , Osificación Heterotópica/diagnóstico , Osificación Heterotópica/genética , Polidactilia/diagnóstico , Polidactilia/genética , Polimorfismo de Nucleótido Simple , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Adulto , Autopsia , Femenino , Humanos , Mutación , Fenotipo , Embarazo , Diagnóstico Prenatal , RadiografíaRESUMEN
Congenital extra-adrenal neuroblastoma is a rare condition, which typically has a favorable prognosis. We present a unique case of extra-adrenal retroperitoneal neuroblastoma diagnosed by fetal magnetic resonance imaging, which ultimately leads to fetal hydrops and neonatal death.
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Imagen por Resonancia Magnética/métodos , Neuroblastoma/congénito , Neuroblastoma/diagnóstico por imagen , Diagnóstico Prenatal/métodos , Neoplasias Retroperitoneales/congénito , Neoplasias Retroperitoneales/diagnóstico por imagen , Adulto , Cesárea , Resultado Fatal , Femenino , Humanos , Recién Nacido , Embarazo , Espacio Retroperitoneal/diagnóstico por imagenRESUMEN
OBJECTIVE: Autoantibodies are clinically useful in phenotyping patients with systemic sclerosis (SSc). Gastrointestinal (GI) function is regulated by the enteric nervous system (ENS) and commonly impaired in SSc, suggesting that the SSc autoimmune response may target ENS antigens. We sought to identify novel anti-ENS autoantibodies with an aim to clinically phenotype SSc GI dysfunction. METHODS: Serum from a patient with SSc with GI dysfunction but without defined SSc-associated autoantibodies was used for autoantibody discovery. Immunoprecipitations performed with murine myenteric plexus lysates were on-bead digested, and autoantigens were identified by mass spectrometry. Prevalence was determined, and clinical features associated with novel autoantibodies were evaluated in a SSc cohort using regression analyses. The expression of gephyrin in human GI tract tissue was examined by immunohistochemistry. RESULTS: We identified gephyrin as a novel SSc autoantigen. Anti-gephyrin antibodies were present in 9% of patients with SSc (16/188) and absent in healthy controls (0/46). Anti-gephyrin antibody-positive patients had higher constipation scores (1.00 vs 0.50, P = 0.02) and were more likely to have severe constipation and severe distention/bloating (46% vs 15%, P = 0.005; 54% vs 25%, P = 0.023, respectively). Anti-gephyrin antibody levels were significantly higher among patients with severe constipation (0.04 vs 0.00; P = 0.001) and severe distention and bloating (0.03 vs 0.004; P = 0.010). Severe constipation was associated with anti-gephyrin antibodies even in the adjusted model. Importantly, gephyrin was expressed in the ENS, which regulates gut motility. CONCLUSION: Gephyrin is a novel ENS autoantigen that is expressed in human myenteric ganglia. Anti-gephyrin autoantibodies are associated with the presence and severity of constipation in patients with SSc.
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Autoanticuerpos , Proteínas de la Membrana , Esclerodermia Sistémica , Proteínas de la Membrana/metabolismo , Autoantígenos/metabolismo , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/fisiopatología , Autoanticuerpos/análisis , Tracto Gastrointestinal/inervación , Tracto Gastrointestinal/fisiopatología , Humanos , Animales , Ratones , Neuronas/metabolismo , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/fisiopatologíaRESUMEN
CONTEXT.: Autopsies performed on COVID-19 patients have provided critical information about SARS-CoV-2's tropism, mechanisms of tissue injury, and the spectrum of disease. OBJECTIVE.: To provide an updated database of postmortem disease in COVID-19 patients, assess relationships among clinical and pathologic variables, evaluate the accuracy of death certification, and correlate disease variables to causes of death. DESIGN.: The 272 postmortem examinations reported in this paper were submitted by 14 pathologists from 9 medical or forensic institutions across the United States. The study spans the eras of the 3 principal COVID-19 strains and incorporates surveyed demographic, clinical, and postmortem data from decedents infected with SARS-CoV-2, including primary and contributing causes of death. It is the largest database of its kind to date. RESULTS.: Demographics of the decedents reported here correspond well to national statistics. Primary causes of death as determined by autopsy and official death certificates were significantly correlated. When specifically cited disease conditions found at autopsy were correlated with COVID-19 versus non-COVID-19 death, only lung findings characteristic of SARS-CoV-2 infection or the absence of lung findings were significantly associated. CONCLUSIONS.: Changes in hospitalization and disease likely stem from longer lifespans after COVID-19 diagnosis and alteration in treatment approaches. Although Omicron variants preferentially replicate in the upper airways, autopsied patients who died of COVID-19 in that time period showed the same lung damage as earlier decedents. Most importantly, findings suggest that there are still unelucidated risk factors for death from COVID-19 including possibly genetic susceptibility.
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BACKGROUND: More than 13,000 children annually in the United States and Canada under the age of 20 will be diagnosed with cancer at a mortality approaching 20% 1,2. Tumor samples obtained by autopsy provide an innovative way to study tumor progression, potentially aiding in the discovery of new treatments and increased survival rates. The purpose of this study was to identify barriers to autopsies and develop guidelines for requesting autopsies for research purposes. PROCEDURE: Families of children treated for childhood cancer were referred by patient advocacy groups and surveyed about attitudes and experiences with research autopsies. From 60 interviews, barriers to autopsy and tumor banking were identified. An additional 14 interviews were conducted with medical and scientific experts. RESULTS: Ninety-three percent of parents of deceased children did or would have consented to a research autopsy if presented with the option; however, only half of these families were given the opportunity to donate autopsy tissue for research. The most significant barriers were the physicians' reluctance to ask a grieving family and lack of awareness about research opportunities. CONCLUSIONS: The value of donating tumor samples to research via an autopsy should be promoted to all groups managing pediatric cancer patients. Not only does autopsy tumor banking offer a potentially important medical and scientific impact, but the opportunity to contribute this Legacy Gift of autopsy tumor tissue also creates a positive outlet for the grieving family. Taking these findings into account, our multidisciplinary team has developed a curriculum addressing key barriers.
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Actitud , Autopsia/estadística & datos numéricos , Investigación Biomédica , Neoplasias , Pediatría/métodos , Adolescente , Adulto , Niño , Preescolar , Familia , Femenino , Pesar , Guías como Asunto , Humanos , Lactante , Recién Nacido , Entrevistas como Asunto , Masculino , Defensa del Paciente , Adulto JovenRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in coagulation activation although it is usually not associated with consumption coagulopathy. D-dimers are also commonly elevated despite systemic hypofibrinolysis. To understand these unusual features of coronavirus disease 2019 (COVID-19) coagulopathy, 64 adult patients with SARS-CoV-2 infection (36 moderate and 28 severe) and 16 controls were studied. We evaluated the repertoire of plasma protease inhibitors (Serpins, Kunitz, Kazal, Cystatin-like) targeting the fibrinolytic system: Plasminogen Activator Inhibitor-1 (PAI-1), Tissue Plasminogen Activator/Plasminogen Activator Inhibitor-1 complex (t-PA/PAI-1), α-2-Antiplasmin, Plasmin-α2-Antiplasmin Complex, Thrombin-activatable Fibrinolysis Inhibitor (TAFI)/TAFIa, Protease Nexin-1 (PN-1), and Neuroserpin (the main t-PA inhibitor of the central nervous system). Inhibitors of the common (Antithrombin, Thrombin-antithrombin complex, Protein Z [PZ]/PZ inhibitor, Heparin Cofactor II, and α2-Macroglobulin), Protein C ([PC], Protein C inhibitor, and Protein S), contact (Kallistatin, Protease Nexin-2/Amyloid Beta Precursor Protein, and α-1-Antitrypsin), and complement (C1-Inhibitor) pathways, in addition to Factor XIII, Histidine-rich glycoprotein (HRG) and Vaspin were also investigated by enzyme-linked immunosorbent assay. The association of these markers with disease severity was evaluated by logistic regression. Pulmonary expression of PAI-1 and Neuroserpin in the lungs from eight post-mortem cases was assessed by immunohistochemistry. Results show that six patients (10%) developed thrombotic events, and mortality was 11%. There was no significant reduction in plasma anticoagulants, in keeping with a compensated state. However, an increase in fibrinolysis inhibitors (PAI-1, Neuroserpin, PN-1, PAP, and t-PA/PAI-1) was consistently observed, while HRG was reduced. Furthermore, these markers were associated with moderate and/or severe disease. Notably, immunostains demonstrated overexpression of PAI-1 in epithelial cells, macrophages, and endothelial cells of fatal COVID-19, while Neuroserpin was found in intraalveolar macrophages only. These results imply that the lungs in SARS-CoV-2 infection provide anti-fibrinolytic activity resulting in a shift toward a local and systemic hypofibrinolytic state predisposing to (immuno)thrombosis, often in a background of compensated disseminated intravascular coagulation.
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Human temporal bones (HTBs) are invaluable resources for the study of otologic disorders and for evaluating novel treatment approaches. Given the high costs and technical expertise required to collect and process HTBs, there has been a decline in the number of otopathology laboratories. Our objective is to encourage ongoing study of HTBs by outlining the necessary steps to establish a pipeline for collection and processing of HTBs. In this methods manuscript, we: (1) provide the design of a temporal bone plug sawblade that can be used to collect specimens from autopsy donors; (2) establish that decalcification time can be dramatically reduced from 9 to 3 months if ethylenediaminetetraacetic acid is combined with microwave tissue processing and periodic bone trimming; (3) show that serial sections of relatively-rapidly decalcified HTBs can be successfully immunostained for key inner ear proteins; (4) demonstrate how to drill down a HTB to the otic capsule within a few hours so that subsequent decalcification time can be further reduced to only weeks. We include photographs and videos to facilitate rapid dissemination of the developed methods. Collected HTBs can be used for many purposes, including, but not limited to device testing, imaging studies, education, histopathology, and molecular studies. As new technology develops, it is imperative to continue studying HTBs to further our understanding of the cellular and molecular underpinnings of otologic disorders.
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Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, which regulates intermediate filament (IF) turnover. Previous neuropathological studies and examination of postmortem brain tissue in the current study revealed involvement of astrocytes in GAN. To develop a clinically-relevant model, we reprogrammed skin fibroblasts from seven GAN patients to pluripotent stem cells (iPSCs), which were used to generate neural progenitor cells (NPCs), astrocytes, and brain organoids. Multiple isogenic control clones were derived via CRISPR/Cas9 gene editing of one patient line carrying the G332R gigaxonin mutation. All GAN iPSCs were deficient for gigaxonin and displayed patient-specific increased vimentin expression. GAN NPCs had lower nestin expression and fewer nestin-positive cells compared to isogenic controls, but nestin morphology was unaffected. GAN brain organoids were marked by the presence of neurofilament and GFAP aggregates. GAN iPSC-astrocytes displayed striking dense perinuclear vimentin and GFAP accumulations and abnormal nuclear morphology. In over-expression systems, GFAP oligomerization and perinuclear aggregation were augmented in the presence of vimentin. GAN patient cells with large perinuclear vimentin aggregates accumulated significantly more nuclear KLHL16 mRNA compared to cells without vimentin aggregates. As an early effector of KLHL16 mutations, vimentin may be a potential target in GAN.
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Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, a regulator of intermediate filament (IF) protein turnover. Previous neuropathological studies and our own examination of postmortem GAN brain tissue in the current study revealed astrocyte involvement in GAN. To study the underlying mechanisms, we reprogrammed skin fibroblasts from seven GAN patients carrying different KLHL16 mutations to iPSCs. Isogenic controls with restored IF phenotypes were derived via CRISPR/Cas9 editing of one patient carrying a homozygous missense mutation (G332R). Neural progenitor cells (NPCs), astrocytes, and brain organoids were generated through directed differentiation. All GAN iPSC lines were deficient for gigaxonin, which was restored in the isogenic control. GAN iPSCs displayed patient-specific increased vimentin expression, while GAN NPCs had decreased nestin expression compared to isogenic control. The most striking phenotypes were observed in GAN iPSC-astrocytes and brain organoids, which exhibited dense perinuclear IF accumulations and abnormal nuclear morphology. GAN patient cells with large perinuclear vimentin aggregates accumulated nuclear KLHL16 mRNA. In over-expression studies, GFAP oligomerization and perinuclear aggregation were potentiated in the presence of vimentin. As an early effector of KLHL16 mutations, vimentin may serve as a potential therapeutic target in GAN.
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Chronic neurodegeneration and acute injuries lead to neuron losses via diverse processes. We compared retinal ganglion cell (RGC) responses between chronic glaucomatous conditions and the acute injury model. Among major RGC subclasses, αRGCs and intrinsically photosensitive RGCs (ipRGCs) preferentially survive glaucomatous conditions, similar to findings in the retina subject to axotomy. Focusing on an αRGC intrinsic factor, Osteopontin (secreted phosphoprotein 1 [Spp1]), we found an ectopic neuronal expression of Osteopontin (Spp1) in other RGCs subject to glaucomatous conditions. This contrasted with the Spp1 downregulation subject to axotomy. αRGC-specific Spp1 elimination led to significant αRGC loss, diminishing their resiliency. Spp1 overexpression led to robust neuroprotection of susceptible RGC subclasses under glaucomatous conditions. In contrast, Spp1 overexpression did not significantly protect RGCs subject to axotomy. Additionally, SPP1 marked adult human RGC subsets with large somata and SPP1 expression in the aqueous humor correlated with glaucoma severity. Our study reveals Spp1's role in mediating neuronal resiliency in glaucoma.
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Glaucoma , Enfermedades del Nervio Óptico , Humanos , Células Ganglionares de la Retina/metabolismo , Osteopontina , Nervio Óptico/metabolismo , Enfermedades del Nervio Óptico/metabolismoRESUMEN
The molecular and functional contributions of intratumoral nerves to disease remain largely unknown. We localized synaptic markers within tumors suggesting that these nerves form functional connections. Consistent with this, electrophysiological analysis shows that malignancies harbor significantly higher electrical activity than benign disease or normal tissues. We also demonstrate pharmacologic silencing of tumoral electrical activity. Tumors implanted in transgenic animals lacking nociceptor neurons show reduced electrical activity. These data suggest that intratumoral nerves remain functional at the tumor bed. Immunohistochemical staining demonstrates the presence of the neuropeptide, Substance P (SP), within the tumor space. We show that tumor cells express the SP receptor, NK1R, and that ligand/receptor engagement promotes cellular proliferation and migration. Our findings identify a mechanism whereby intratumoral nerves promote cancer progression.
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Neoplasias de la Mama , Neuronas , Neoplasias Ováricas , Carcinoma de Células Escamosas de Cabeza y Cuello , Animales , Ratones , Modelos Animales de Enfermedad , Humanos , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Sustancia P/metabolismo , Línea Celular Tumoral , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Neoplasias Ováricas/epidemiología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/secundario , Neuronas/patología , Ratones Endogámicos C57BL , Organismos Libres de Patógenos Específicos , Ovario/inervación , Virus del Papiloma Humano , Análisis de SupervivenciaRESUMEN
CONTEXT.: Organ weights are an essential part of autopsy analysis. Deviations from normal organ weights provide important clues to disease processes. The assessment of normal organ weights depends on reliable reference tables, but most widely available reference tables are based on data that are either decades old or derived from relatively small sample sizes. OBJECTIVE.: To provide an updated reference table of organ weights based on contemporary sources and a large sample size. DESIGN.: Organ weights from 4197 carefully screened autopsies performed on adults at the Palm Beach County Medical Examiner's Office in West Palm Beach, Florida, and the Mayo Clinic Hospital in Rochester, Minnesota. RESULTS.: Height and body weight data in this study reflect the well-recognized increases in both variables, but most particularly in body weight, seen during the last decades. The study data show a strong positive association between organ weight and body weight for the heart, liver, and spleen. There is a similar but weaker association between body weight and the weight of the lungs and kidneys. Brain weight is independent of body weight but shows a strong negative association with age. Even when controlling for body weight, men's organs are heavier, except for the weight of the liver, which is comparable in men and women. These associations are in agreement with the findings of previous studies. The current study suggests that, for some of the commonly weighed organs, there has been an increase in median organ weight when compared with existing references. CONCLUSIONS.: The tables presented here provide an updated reference that should prove useful to autopsy pathologists in the forensic and hospital settings.
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Estatura , Adulto , Masculino , Femenino , Humanos , Tamaño de los Órganos , Índice de Masa Corporal , Autopsia , Peso CorporalRESUMEN
BACKGROUND: Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19. METHODS: We obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy. FINDINGS: The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury. INTERPRETATION: Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2. FUNDING: This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.
Asunto(s)
Trastornos de la Coagulación Sanguínea/metabolismo , COVID-19/metabolismo , Regulación hacia Abajo , Endotelio Vascular/metabolismo , Hipoxia/metabolismo , Pulmón/metabolismo , SARS-CoV-2/metabolismo , Trombomodulina/biosíntesis , Anciano , Anciano de 80 o más Años , Trastornos de la Coagulación Sanguínea/patología , COVID-19/patología , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Endotelio Vascular/ultraestructura , Femenino , Humanos , Hipoxia/patología , Pulmón/ultraestructura , Masculino , Persona de Mediana EdadRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.