Kang Xiao’s research while affiliated with National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention and other places

Prion diseases are fatal neurodegenerative disorders characterized by neuron damage and loss. Growth-associated protein 43 (GAP43) functions in neuronal plasticity and synaptic function, but its role in prion diseases is not fully elucidated. In this study, we investigated the changes of GAP43 in the central nerve system (CNS) of several prion-infected rodent models and explored the potential relationship of GAP43 with PrPSc deposit and neuron loss using various methods. We found that GAP43 levels were significantly decreased in the brain tissues of scrapie-infected rodent models at the terminal stage of the disease. Immunohistochemical analysis showed that GAP43 colocalized with NeuN-positive cells morphologically, indicating the presence of GAP43 in mature neurons. On contrary, the levels of GAP43 and p-GAP43 increased in a prion-infected cell line SMB-S15 in vitro, accompanying with the increase of intracellular calcium. Stimulation of lipopolysaccharide (LPS) upregulated while removal of PrPSc propagation downregulated the level of GAP43 in SMB-S15 cells. Morphological colocalization and molecular interaction between GAP43 and PrPSc have been addressed in the brains of prion-infected rodents and prion-infected cell line. Histological assays of the serial sections of the whole brains of prion-infected mice proposed that the reduced GAP43 level correlated with large amount of PrPSc deposits and notable neuron damage and loss showing cell crumpled and nuclear pyknosis. The impairment of GAP43 signaling and disturbance of calcium homeostasis by aberrance of brain GAP43/p-GAP43 not only reflect but also likely contribute to the pathology of severe neuron loss at the end of prion disease.

Importance Recent studies have revealed that autopsy skin samples from cadavers with prion diseases (PRDs) exhibited a positive prion seeding activity similar to cerebrospinal fluid (CSF). It is worthwhile to validate the findings with a large number of biopsy skin samples and compare the clinical value of prion seeding activity between skin biopsies and concurrent CSF specimens. Objective To compare the prion seeding activity of skin biopsies and CSF samples and to determine the effectiveness of combination of the skin biopsies from multiple sites and numerous dilutions on the diagnosis for various types of PRDs. Design, Setting, and Participants In the exploratory cohort, patients were enrolled from September 15, 2021, to December 15, 2023, and were followed up every 3 months until April 2024. The confirmatory cohort enrolled patients from December 16, 2023, to June 31, 2024. The exploratory cohort was conducted at a single center, the neurology department at Xuanwu Hospital. The confirmatory cohort was a multicenter study involving 4 hospitals in China. Participants included those diagnosed with probable sporadic Creutzfeldt-Jakob disease or genetically confirmed PRDs. Patients with uncertain diagnoses or those lost to follow-up were excluded. All patients with PRDs underwent skin sampling at 3 sites (the near-ear area, upper arm, lower back, and inner thigh), and a portion of them had CSF samples taken simultaneously. In the confirmatory cohort, a single skin biopsy site and CSF samples were simultaneously collected from a portion of patients with PRDs. Exposures The skin and CSF prion seeding activity was assessed using the real-time quaking-induced conversion (RT-QUIC) assay, with rHaPrP90-231, a Syrian hamster recombinant prion protein, as the substrate. In the exploratory cohort, skin samples were tested at dilutions of 10 ⁻² through 10 ⁻⁴ . In the confirmatory cohort, skin samples were tested at a dilution of 10 ⁻² . A total of four 15-μL wells of CSF were used in the RT-QUIC assay. Main Outcomes and Measures Correlations between RT-QUIC results from the skin and CSF and the final diagnosis of enrolled patients. Results In the exploratory cohort, the study included 101 patients (mean [SD] age, 60.9 [10.2] years; 63 female [62.4%]) with PRD and 23 patients (mean [SD] age, 63.4 [9.1] years; 13 female [56.5%]) without PRD. A total of 94 patients had CSF samples taken simultaneously with the skin biopsy samples. In the confirmatory cohort, a single skin biopsy site and CSF sample were taken simultaneously in 43 patients with PRDs. Using an experimental condition of 10 ⁻² dilution, the RT-QUIC positive rates of skin samples from different sites were comparable with those of the CSF (skin: 18 of 26 [69.2%] to 74 of 93 [79.6%] vs CSF: 71 of 94 [75.5%]). When tested at 3 different dilutions, all skin sample positivity rates increased to over 80.0% (79 of 93 for the near-ear area, 21 of 26 for the upper arm, 77 of 92 for the lower back, and 78 of 92 for the inner thigh). Combining samples from skin sites near the ear, inner thigh, and lower back in pairs yielded positivity rates exceeding 92.1% (93 of 101), significantly higher than CSF alone (71 of 94 [75.5%]; P =.002). When all skin sample sites were combined and tested at 3 dilution concentrations for RT-QUIC, the sensitivity reached 95.0% (96 of 101). In the confirmatory cohort, the RT-QUIC positive rate of a single skin biopsy sample was slightly higher than that of the CSF (34 of 43 [79.1%] vs 31 of 43 [72.1%]; P = .45). Conclusions and Relevance Results of this diagnostic study suggest that the sensitivity of an RT-QUIC analysis of a combination of 2 or more skin sites was superior to that of CSF in diagnosing PRDs.

Background The COVID-19 pandemic has underscored the critical role of sequencing technology in disease control and outbreak response. However, resource limitations and challenging environments often impede such efforts in low and middle-income countries. This study aimed to investigate the spectrum of viral co-infections, particularly with human viral pathogens, in SARS-CoV-2 positive individuals in Sierra Leone using metagenomic sequencing, evaluating the feasibility of utilizing this technology for epidemiological and evolutionary surveillance of pathogens related to public health in low-income environments. Methods We retrospectively collected and analyzed 98 nasopharyngeal swab specimens from SARS-CoV-2 positive individuals in Sierra Leone. Samples were pre-processed locally and transferred to China via FTA cards for metagenomic sequencing, which was performed using the Novaseq platform. The study focused on the identification of nasopharyngeal viruses co-infecting with SARS-CoV-2, with a deeper analysis of significant human viral pathogens such as HPV. Results The study identified 22 viral taxa from 20 families, including 4 human viruses. Notably, 19.4% of samples showed HPV co-infection with 34 distinct types, predominantly beta and gamma HPVs. Multiple HPV types were found in individual samples, indicating a high complexity of viral co-infections. Conclusions The identification of a wide range of co-infecting viruses, particularly multiple HPV genotypes, highlights the complexity of viral interactions and their potential implications for public health. These findings enhance our understanding of viral co-infections and provide valuable insights for public health interventions in Sierra Leone. Further research is needed to explore the clinical significance of these findings and their impact on disease outcomes.

Introduction Human prion disease (PrD), a group of fatal and transmissible neurodegenerative diseases, consists of Creutzfeldt–Jakob disease (CJD), kuru, fatal familial insomnia (FFI), Gerstmann-Sträussler-Scheinker disease (GSS), and variably protease-sensitive prionopathy (VPSPr). The emergence of bovine spongiform encephalopathy (BSE) in cattle and variant CJD (vCJD) has greatly threatened public health, both in humans and animals. Since the 1990's, dozens of countries and territories have conducted PrD surveillance programs. Methods In this study, the case numbers and alternative trends of different types of PrD globally and in various countries or territories from 1993 to 2020 were collected and analyzed based on the data from the websites of the international and national PrD surveillance programs, as well as from relevant publications. Results The total numbers of the reported PrD and sporadic CJD (sCJD) cases in 34 countries with accessible annual case numbers were 27,872 and 24,623, respectively. The top seven countries in PrD cases were the USA (n = 5,156), France (n = 3,276), Germany (n = 3,212), Italy (n = 2,995), China (n = 2,662), the UK (n = 2,521), Spain (n = 1,657), and Canada (n = 1,311). The annual PrD case numbers and mortalities, either globally or in the countries, showed an increased trend in the past 27 years. Genetic PrD cases accounted for 10.83% of all reported PrD cases; however, the trend varied largely among the different countries and territories. There have been 485 iatrogenic CJD (iCJD) cases and 232 vCJD cases reported worldwide. Discussion The majority of the countries with PrD surveillance programs were high- and upper-middle-income countries. However, most low- and lower-middle-income countries in the world did not conduct PrD surveillance or even report PrD cases, indicating that the number of human PrD cases worldwide is markedly undervalued. Active international PrD surveillance for both humans and animals is still vital to eliminate the threat of prion disease from a public health perspective.

Neuroinflammation is a common pathological feature in a number of neurodegenerative diseases, which is mediated primarily by the activated glial cells. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome-associated neuroinflammatory response is mostly considered. To investigate the situation of the NLRP3-related inflammation in prion disease, we assessed the levels of the main components of NLRP3 inflammasome and its downstream biomarkers in the scrapie-infected rodent brain tissues. The results showed that the transcriptional and expressional levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein (ASC) in the brains of scrapie-infected rodents were significantly increased at terminal stage. The increased NLPR3 overlapped morphologically well with the proliferated GFAP-positive astrocytes, but little with microglia and neurons. Using the brain samples collected at the different time-points after infection, we found the NLRP3 signals increased in a time-dependent manner, which were coincidental with the increase of GFAP. Two main downstream cytokines, IL-1β and IL-18, were also upregulated in the brains of prion-infected mice. Moreover, the gasdermin D (GSDMD) levels, particularly the levels of GSDMD-NT, in the prion-infected brain tissues were remarkably increased, indicating activation of cell pyroptosis. The GSDMD not only co-localized well with the astrocytes but also with neurons at terminal stage, also showing a time-dependent increase after infection. Those data indicate that NLRP3 inflammasomes were remarkably activated in the infected brains, which is largely mediated by the proliferated astrocytes. Both astrocytes and neurons probably undergo a pyroptosis process, which may help the astrocytes to release inflammatory factors and contribute to neuron death during prion infection.

Prion diseases are fatal neurodegenerative disorders characterized by neuron damage and loss. Growth-associated protein 43 (GAP43) functions in neuronal plasticity and synaptic function, but its role in prion diseases is not fully elucidated. In this study, we investigated the changes of GAP43 in the central nerve system (CNS) of several prion infected rodent models and explored the potential relationship of GAP43 with PrP Sc deposit and neuron loss using various methods. We found that GAP43 levels were significantly decreased in the brain tissues of scrapie-infected rodent models at the terminal stage of the disease. Immunohistochemical analysis showed that GAP43 colocalized with NeuN-positive cells morphologically, indicating the presence of GAP43 in mature neurons. On contrary, the levels of GAP43 and p-GAP43 increased in a prion-infected cell line SMB-S15 in vitro , accompanying with the increase of intracellular calcium. Stimulation of lipopolysaccharide (LPS) upregulated whilst removal of PrP Sc propagation downregulated the level of GAP43 in SMB-S15 cells. Morphological colocalization and molecular interaction between GAP43 and PrP Sc has been addressed in the brains of prion infected rodents and prion infected cell line. Histological assays of the serial sections of the whole brains of prion infected mice proposed that the reduced GAP43 level correlated with large amount of PrP Sc deposits and notable neuron damage and loss showing cell crumpled and nuclear pyknosis. The impairment of GAP43 signaling and disturbance of calcium homeostasis by aberrance of brain GAP43/p-GAP43 not only reflect but also likely contribute to the pathology of severe neuron loss at the end of prion disease.

Background and purpose Calmodulin (CaM) levels exhibit significant elevation in the brain tissue of rodent and cell line models infected with prion, as well as in the cerebrospinal fluid (CSF) samples from patients diagnosed with sporadic Creutzfeldt-Jakob disease (sCJD). However, the status of CSF CaM in patients with genetic prion diseases (gPrDs) remains unclear. This study aims to assess the characteristics of CSF CaM in Chinese patients presenting four subtypes of gPrDs. Methods A total of 103 CSF samples from patients diagnosed with T188K-gCJD, E200K-gCJD, D178N-FFI, P102L-GSS were included in this study, along with 40 CSF samples from patients with non-prion diseases (non-PrDs). The presence of CSF CaM and 14-3-3 proteins was assessed using Western blots analysis, while levels of CSF 14-3-3 and total tau were measured using enzyme-linked immunosorbent assays (ELISAs). Statistical methods including multivariate logistic regression were employed to evaluate the association between CSF CaM positivity and relevant clinical, laboratory, and genetic factors. Results The positive rates of CSF CaM were significantly higher in cases of T188K-gCJD (77.1%), E200K-gCJD (86.0%), and P102-GSS (90.9%) compared to non-PrD cases (22.5%). In contrast, CSF CaM positivity was slightly elevated in D178N-FFI (34.3%). CSF CaM positivity was remarkably high in patients who tested positive for CSF 14-3-3 by Western blot and exhibited high levels of total tau (≥1400 pg/ml) as measures by ELISA. Multivariate logistic regression analysis confirmed a significant association between CSF CaM positivity and specific mutations in PRNP , as well as with CSF 14-3-3 positivity. Furthermore, the diagnostic performance of CaM surpassed that of 14-3-3 and tau when analyzing CSF samples from T188K-gCJD and E200K-gCJD patients. Conclusion Western blot analysis reveals significant variations in the positivity of CSF CaM among the four genotypes of gPrD cases, demonstrating a positive correlation with 14-3-3 positivity and elevated tau levels in CSF.

Neuroinflammation is a common pathological feature in a number of neurodegenerative diseases, which is mediated primarily by the activated glial cells. NLRP3 inflammasomes associated neuroinflammatory response is mostly considered. To investigate the situation of the NLRP3 related inflammation in prion disease, we assessed the levels of the main components of NLRP3 inflammasome and its downstream biomarkers in the scrapie infected rodent brain tissues. The results showed that the transcriptional and expressional levels of NLRP3, caspase1, ASC in the brains of scrapie infected rodents were significantly increased at terminal stage. The increased NLPR3 overlapped morphologically well with the proliferated GFAP-positive astrocytes, but little with microglia and neurons. Using the brain samples collected at the different time-points after infection, we found the NLRP3 signals increased in a time-dependent manner, which were coincidental with the increase of GFAP. Two main downstream cytokines, IL-1β and IL-18, were also upregulated in the brains of prion infected mice. Moreover, the GSDMD levels, particularly the levels of GSDMD-NT, in the prion infected brain tissues were remarkably increased, indicating activation of cell pyroptosis. The GSDMD not only co-localized well with the astrocytes but also with neurons at terminal stage, also showing a time-dependent increase after infection. Those data indicate that NLRP3 inflammasomes were remarkably activated in the infected brains, which is largely mediated by the proliferated astrocytes. Both astrocytes and neurons probably undergo a pyroptosis process, which may help the astrocytes to release inflammatory factors and contribute to neuron death during prion infection.

Worldwide, 10–15% human prion disease are genetic and inherited, due to the special mutations or insertions in PRNP gene. Herein, we reported two Chinese patients with rapidly progressive dementia who were referred to the national Creutzfeldt–Jacob disease (CJD) surveillance as suspected CJD. Those two patients displayed sporadic CJD (sCJD)-like clinical phenotype, e.g. rapidly progressive dementia, visional and mental problems, sCJD-associated abnormalities in MRI. A missense mutation was identified in one PRNP allele of these two patients, resulting in a change from serine to asparagine at codon 97 (S97N). RT-QuIC of the cerebrospinal fluid samples from those two cases were positive. It indicates that they are very likely to be prion disease.

Introduction Human prion diseases (PrDs) are rare, fatal encephalopathies requiring comprehensive diagnostic analysis. This study examines hospital referral patterns to the Chinese National Surveillance for Creutzfeldt-Jakob Disease (CNS-CJD) from 2006 to 2019. Methods We assessed 1,970 PrD cases referred by various hospitals to CNS-CJD. Referral distributions were analyzed based on provincial-level administrative divisions (PLADs). Differences in referral numbers and confirmed cases between monitored and non-monitored PLADs were statistically evaluated. Results The study included cases from 344 hospitals across 29 Chinese PLADs. Hospital referrals increased over the surveillance years: from 28.2 hospitals annually during 2006–2010, to 64 in 2011–2015, and 107 in 2016–2019. Of these, 12.2% (42/344) of hospitals reported ≥10 PrD cases, accounting for 70.0% (1,379/1,970) of total cases. Referral numbers varied across PLADs, with the top 5 of Beijing (41), Henan (26), Shanghai (21), Guangdong (21), and Jiangsu (21) leading. Additionally, 12 CJD-surveillance PLADs had more referring hospitals and PrD cases than the other 17 non-surveillance PLADs. Conclusions Geographical variations in PrD recognition exist across Chinese PLADs, with certain regions and major cities reporting notably higher case numbers.