Microbial infection promotes amyloid pathology in a mouse model of Alzheimer's disease via modulating γ-secretase.

Microbial infection as a type of environmental risk factors is considered to be associated with long-term increased risk of dementia, including Alzheimer's disease (AD). AD is characterized by two neuropathologically molecular hallmarks of hyperphosphorylated tau and amyloid-ß (Aß), the latter generated by several biochemically reactive enzymes, including γ-secretase. However, how infectious risk factors contribute to pathological development of the AD core molecules remains to be addressed. In this work, we utilized a modified herpes simplex virus type 1 (mHSV-1) and found that its hippocampal infection locally promotes Aß pathology in 5 × FAD mice, the commonly used amyloid model. Mechanistically, we identified HSV-1 membrane glycoprotein US7 (Envelope gI) that interacts with and modulates γ-secretase and consequently facilitates Aß production. Furthermore, we presented evidence that adenovirus-associated virus-mediated locally hippocampal overexpression of the US7 aggravates Aß pathology in 5 × FAD mice. Collectively, these findings identify a herpesviral factor regulating γ-secretase in the development and progression of AD and represent a causal molecular link between infectious pathogens and neurodegeneration.

Differential pathogenic and molecular features in neurological infection of SARS-CoV-2 Omicron BA.5.2 and BA.2.75 and Delta.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global threat, exacerbated by the emergence of viral variants. Two variants of SARS-CoV-2, Omicron BA.2.75 and BA.5, led to global infection peaks between May 2022 and May 2023, yet their precise characteristics in pathogenesis are not well understood. In this study, we compared these two Omicron sublineages with the previously dominant Delta variant using a human angiotensin-converting enzyme 2 knock-in mouse model. As expected, Delta exhibited higher viral replication in the lung and brain than both Omicron sublineages which induced less severe lung damage and immune activation. In contrast, the Omicron variants especially BA.5.2 showed a propensity for cellular proliferation and developmental pathways. Both Delta and BA.5.2 variants, but not BA.2.75, led to decreased pulmonary lymphocytes, indicating differential adaptive immune response. Neuroinvasiveness was shared with all strains, accompanied by vascular abnormalities, synaptic injury, and loss of astrocytes. However, Immunostaining assays and transcriptomic analysis showed that BA.5.2 displayed stronger immune suppression and neurodegeneration, while BA.2.75 exhibited more similar characteristics to Delta in the cortex. Such differentially infectious features could be partially attributed to the weakened interaction between Omicron Spike protein and host proteomes decoded via co-immunoprecipitation followed by mass spectrometry in neuronal cells. Our present study supports attenuated replication and pathogenicity of Omicron variants but also highlights their newly infectious characteristics in the lung and brain, especially with BA.5.2 demonstrating enhanced immune evasion and neural damage that could exacerbate neurological sequelae.

Triplet-drug chemotherapy combined with anti-EGFR antibody as an effective therapy for patients with initially unresectable metastatic colorectal cancer: a meta-analysis.

The meta-analysis aimed to assess the clinical efficacy of chemotherapeutic triplet-drug regimen combined with anti-EGFR antibody in patients with initially unresectable metastatic colorectal cancer (mCRC). A systematic literature search was performed in PubMed Publisher. Studies evaluating FOLFOXIRI combine with panitumumab or cetuximab as the therapy for initially unresectable mCRC were included. The primary outcome was objective response rate (ORR) and rate of R0 resections. The secondary outcomes included overall survival (OS), progression-free survival (PFS), and grades 3 or 4 adverse events. R software (version 4.0.2) and RevMan (version 5.3) were used to analyze the extracted data. The studies included were published between 2010 and 2021, involving four single-arm phase II trials and two randomized phase II trials. A total of 6 studies with 282 patients were included. The data showed a significant benefit for the FOLFOXIRI + anti-EGFR antibody arm compared with FOLFOXIRI arm (RR 1.33; 95% CI, 1.13-1.58; I2 = 0%, P < 0.05). The pooled ORR and pooled rate of R0 resection in patients who receiving FOLFOXIRI + anti-EGFR antibody were 85% (95% CI, 0.78-0.91; I2 = 58%) and 42% (95% CI, 0.32-0.53; I2 = 62%), respectively. The range of median PFS between all the six studies was 9.5-15.5 months, with weighted pooled median PFS mean 11.7 months. The range of median OS between all the four studies was 24.7-37 months, with weighted pooled median PFS mean 31.9 months. The common grades 3 and 4 adverse events were diarrhea and neutropenia. Our findings show that triplet-drug chemotherapy (FOLFOXIRI) combined with anti-EGFR antibody (panitumumab or cetuximab) represents a very effective therapeutic combination associated with a significant ORR and R0 rection rate for patients with molecularly unselected and surgically unresectable metastatic CRC.

Activation of innate immune cGAS-STING pathway contributes to Alzheimer's pathogenesis in 5×FAD mice.

cGAS senses microbial and host-derived double-stranded DNA in cytoplasm to trigger cellular innate immune response in a STING-dependent manner; however, it remains unknown whether the cGAS-STING pathway in innate immunity contributes to Alzheimer's disease (AD). Here we demonstrated the detectable binding of the cGAS double-stranded DNA in cytoplasm and the activation of the microglial cGAS-STING pathway in brains of human AD and aged mice. Cgas-/-;5×FAD mice were largely protected from cognitive impairment, amyloid-ß pathology, neuroinflammation and other sequelae associated with AD. Furthermore, Cgas deficiency in microglia inhibited a neurotoxic A1 astrocytic phenotype and thus alleviated oligomeric amyloid-ß peptide-induced neurotoxicity. Finally, administration of STING inhibitor H-151 potently suppressed the activation of the cGAS-STING pathway and ameliorated AD pathogenesis in 5×FAD mice. In conclusion, our present study has identified a critical molecular link between innate immunity and AD and suggests that therapeutic targeting of the cGAS-STING pathway activity might effectively interfere with the progression of AD.

Microglia innate immune response contributes to the antiviral defense and blood-CSF barrier function in human choroid plexus organoids during HSV-1 infection.

The choroid plexus (ChP) is the source of cerebrospinal fluid (CSF). The ChP-CSF system not only provides the necessary cushion for the brain but also works as a sink for waste clearance. During sepsis, pathogens and host immune cells can weaken the ChP barrier and enter the brain, causing cerebral dysfunctions known as sepsis-associated encephalophagy. Here, we used human ChP organoid (ChPO) to model herpes simplex virus type 1 (HSV-1) infection and found ChP epithelial cells were highly susceptible to HSV-1. Since the current ChPO model lacks a functional innate immune component, particularly microglia, we next developed a new microglia-containing ChPO model, and found microglia could effectively limit HSV-1 infection and protect epithelial barrier in ChPOs. Furthermore, we found the innate immune cyclic GMP-AMP synthase (cGAS)-STING pathway and its downstream interferon response were essential, as cGAS inhibitor RU.512 or STING inhibitor H-151 abolished microglia antiviral function and worsened ChP barrier in organoids. These results together indicated that cGAS-STING pathway coordinates antiviral response in ChP and contributes to treating sepsis or related neurological conditions.

Characteristics of replication and pathogenicity of SARS-CoV-2 Alpha and Delta isolates.

The continuously arising of SARS-CoV-2 variants has been posting a great threat to public health safety globally, from B.1.17 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) to B.1.1.529 (Omicron). The emerging or re-emerging of the SARS-CoV-2 variants of concern is calling for the constant monitoring of their epidemics, pathogenicity and immune escape. In this study, we aimed to characterize replication and pathogenicity of the Alpha and Delta variant strains isolated from patients infected in Laos. The amino acid mutations within the spike fragment of the isolates were determined via sequencing. The more efficient replication of the Alpha and Delta isolates was documented than the prototyped SARS-CoV-2 in Calu-3 and Caco-2 â€‹cells, while such features were not observed in Huh-7, Vero E6 and HPA-3 â€‹cells. We utilized both animal models of human ACE2 (hACE2) transgenic mice and hamsters to evaluate the pathogenesis of the isolates. The Alpha and Delta can replicate well in multiple organs and cause moderate to severe lung pathology in these animals. In conclusion, the spike protein of the isolated Alpha and Delta variant strains was characterized, and the replication and pathogenicity of the strains in the cells and animal models were also evaluated.

Simultaneous Detection of Herpes Simplex Virus Type 1 Latent and Lytic Transcripts in Brain Tissue.

Neurotrophic herpes simplex virus type 1 (HSV-1) establishes lifelong latent infection in humans. Accumulating studies indicate that HSV-1, a risk factor of neurodegenerative diseases, exacerbates the sporadic Alzheimer's disease (AD). The analysis of viral genetic materials via genomic sequencing and quantitative PCR (qPCR) is the current approach used for the detection of HSV-1; however, this approach is limited because of its difficulty in detecting both latent and lytic phases of the HSV-1 life cycle in infected hosts. RNAscope, a novel in situ RNA hybridization assay, enables visualized detection of multiple RNA targets on tissue sections. Here, we developed a fluorescent multiplex RNAscope assay in combination with immunofluorescence to detect neuronal HSV-1 transcripts in various types of mouse brain samples and human brain tissues. Specifically, the RNA probes were designed to separately recognize two transcripts in the same brain section: (1) the HSV-1 latency-associated transcript (LAT) and (2) the lytic-associated transcript, the tegument protein gene of the unique long region 37 (UL37). As a result, both LAT and UL37 signals were detectable in neurons in the hippocampus and trigeminal ganglia (TG). The quantifications of HSV-1 transcripts in the TG and CNS neurons are correlated with the viral loads during lytic and latent infection. Collectively, the development of combinational detection of neuronal HSV-1 transcripts in mouse brains can serve as a valuable tool to visualize HSV-1 infection phases in various types of samples from AD patients and facilitate our understanding of the infectious origin of neurodegeneration and dementia.

Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice.

BACKGROUND: The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. METHODS: We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. FINDINGS: We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3-/- mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. INTERPRETATION: Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease. FUNDING: This work was supported by grants from the Bureau of Frontier Sciences and Education, CAS (grant no. QYZDJ-SSW-SMC005 to Y.G.Y.), the key project of the CAS "Light of West China" Program (to D.Y.) and Yunnan Province (202001AS070023 to D.Y.).

Protocol for in utero injection to investigate Zika virus-related fetal microcephaly in mice.

Zika virus (ZIKV) is linked to congenital defects including microcephaly. An infection model that can recapitulate most microcephaly-related phenotypes is crucial for understanding ZIKV pathogenesis. Here, we present a protocol to generate ZIKV from an infectious clone through a reverse genetic system and subsequently perform embryonic brain infection with the rescued ZIKV in pregnant mice. We optimized several aspects of the procedures including virus rescue and in utero injection. This protocol facilitates reproducible investigation of virus-induced cortical development defects. For complete details on the use and execution of this protocol, please refer to Zeng et al. (2020).

Microglia and its Genetics in Alzheimer's Disease.

Alzheimer's Disease (AD) is the most prevalent form of dementia across the world. While its discovery and pathological manifestations are centered on protein aggregations of amyloid- beta (Aß) and hyperphosphorylated tau protein, neuroinflammation has emerged in the last decade as a main component of the disease in terms of both pathogenesis and progression. As the main innate immune cell type in the central nervous system (CNS), microglia play a very important role in regulating neuroinflammation, which occurs commonly in neurodegenerative conditions, including AD. Under inflammatory response, microglia undergo morphological changes and status transition from homeostatic to activated forms. Different microglia subtypes displaying distinct genetic profiles have been identified in AD, and these signatures often link to AD risk genes identified from the genome-wide association studies (GWAS), such as APOE and TREM2. Furthermore, many AD risk genes are highly enriched in microglia and specifically influence the functions of microglia in pathogenesis, e.g. releasing inflammatory cytokines and clearing Aß. Therefore, building up a landscape of these risk genes in microglia, based on current preclinical studies and in the context of their pathogenic or protective effects, would largely help us to understand the complex etiology of AD and provide new insight into the unmet need for effective treatment.

Neuroimmune Evasion of Zika Virus to Facilitate Viral Pathogenesis.

Zika virus (ZIKV), which preferentially targets neural stem and progenitor cells (NSCs) especially in developing brain, is causally associated with fetal microcephaly, intrauterine retardation, and other congenital malformations in humans. However, there are, so far, no effective drugs and vaccines against ZIKV epidemics, warranting an enhanced understanding of ZIKV biology. Immune response is essential for neuronal cells to combat viral invasion. In turn, neurotropic ZIKV has developed a complex strategy of neuroimmune evasion to facilitate viral pathogenesis, especially developmental impairment in embryonic brain. Here, we review not only overall knowledge of ZIKV-related immune responses, but also current advances in our understanding of immune evasion in ZIKV infection. We also review several specific mechanisms underlying ZIKV protein-mediated immune evasion for viral pathogenesis.

Mild traumatic brain injury induces microvascular injury and accelerates Alzheimer-like pathogenesis in mice.

INTRODUCTION: Traumatic brain injury (TBI) is considered as the most robust environmental risk factor for Alzheimer's disease (AD). Besides direct neuronal injury and neuroinflammation, vascular impairment is also a hallmark event of the pathological cascade after TBI. However, the vascular connection between TBI and subsequent AD pathogenesis remains underexplored. METHODS: In a closed-head mild TBI (mTBI) model in mice with controlled cortical impact, we examined the time courses of microvascular injury, blood-brain barrier (BBB) dysfunction, gliosis and motor function impairment in wild type C57BL/6 mice. We also evaluated the BBB integrity, amyloid pathology as well as cognitive functions after mTBI in the 5xFAD mouse model of AD. RESULTS: mTBI induced microvascular injury with BBB breakdown, pericyte loss, basement membrane alteration and cerebral blood flow reduction in mice, in which BBB breakdown preceded gliosis. More importantly, mTBI accelerated BBB leakage, amyloid pathology and cognitive impairment in the 5xFAD mice. DISCUSSION: Our data demonstrated that microvascular injury plays a key role in the pathogenesis of AD after mTBI. Therefore, restoring vascular functions might be beneficial for patients with mTBI, and potentially reduce the risk of developing AD.

Cancer-Associated Fibroblast-Mediated Cellular Crosstalk Supports Hepatocellular Carcinoma Progression.

BACKGROUND AND AIMS: Cancer-associated fibroblasts (CAFs) are key players in multicellular, stromal-dependent alterations leading to HCC pathogenesis. However, the intricate crosstalk between CAFs and other components in the tumor microenvironment (TME) remains unclear. This study aimed to investigate the cellular crosstalk among CAFs, tumor cells, and tumor-associated neutrophils (TANs) during different stages of HCC pathogenesis. APPROACH AND RESULTS: In the HCC-TME, CAF-derived cardiotrophin-like cytokine factor 1 (CLCF1) increased chemokine (C-X-C motif) ligand 6 (CXCL6) and TGF-ß secretion in tumor cells, which subsequently promoted tumor cell stemness in an autocrine manner and TAN infiltration and polarization in a paracrine manner. Moreover, CXCL6 and TGF-ß secreted by HCC cells activated extracellular signal-regulated kinase (ERK) 1/2 signaling of CAFs to produce more CLCF1, thus forming a positive feedback loop to accelerate HCC progression. Inhibition of ERK1/2 or CLCF1/ciliary neurotrophic factor receptor signaling efficiently impaired CLCF1-mediated crosstalk among CAFs, tumor cells, and TANs both in vitro and in vivo. In clinical samples, up-regulation of the CLCF1-CXCL6/TGF-ß axis exhibited a marked correlation with increased cancer stem cells, "N2"-polarized TANs, tumor stage, and poor prognosis. CONCLUSIONS: This study reveals a cytokine-mediated cellular crosstalk and clinical network involving the CLCF1-CXCL6/TGF-ß axis, which regulates the positive feedback loop among CAFs, tumor stemness, and TANs, HCC progression, and patient prognosis. These results may support the CLCF1 cascade as a potential prognostic biomarker and suggest that selective blockade of CLCF1/ciliary neurotrophic factor receptor or ERK1/2 signaling could provide an effective therapeutic target for patients with HCC.

The efficacy and safety of the combination of axitinib and pembrolizumab-activated autologous DC-CIK cell immunotherapy for patients with advanced renal cell carcinoma: a phase 2 study.

OBJECTIVES: Although axitinib has achieved a preferable response rate for advanced renal cell carcinoma (RCC), patient survival remains unsatisfactory. In this study, we evaluated the efficacy and safety of a combination treatment of axitinib and a low dose of pembrolizumab-activated autologous dendritic cells-co-cultured cytokine-induced killer cells in patients with advanced RCC. METHODS: All adult patients, including treatment-naive or pretreated with VEGF-targeted agents, were enrolled from May 2016 to March 2019. Patients received axitinib 5 mg twice daily and pembrolizumab-activated dendritic cells-co-cultured cytokine-induced killer cells intravenously weekly for the first four cycles, every 2 weeks for the next four cycles, and every month thereafter. RESULTS: The 43 patients (22 untreated and 21 previously treated) showed a median progression-free survival (mPFS) of 14.7 months (95% CI, 11.16-18.30). mPFS in treatment-naive patients was 18.2 months, as compared with 14.4 months in pretreated patients (log-rank P-value = 0.07). Overall response rates were 25.6% (95% CI, 13.5-41.2%). Grade 3 or higher adverse events occurred in 5% of patients included hypertension (11.6%) and palmar-plantar erythrodysesthesia (7.0%). Peripheral blood lymphocyte immunophenotype and serum cytokine profile analyses demonstrated increased antitumor immunity after combination treatment particularly in patients with a long-term survival benefit, while those with a minimal survival benefit demonstrated an elevated proportion of peripheral CD8+TIM3+ T cells and lower serum-level immunostimulatory cytokine profile. CONCLUSIONS: The combination therapy was active and well tolerated for treatment of advanced RCC, either as first- or second-line treatment following other targeted agents. Changes in immunophenotype and serum cytokine profile may be used as prognostic biomarkers.

Functional Mapping of AGO-Associated Zika Virus-Derived Small Interfering RNAs in Neural Stem Cells.

Viral interfering RNA (viRNA) has been identified from several viral genomes via directly deep RNA sequencing of the virus-infected cells, including zika virus (ZIKV). Once produced by endoribonuclease Dicer, viRNAs are loaded onto the Argonaute (AGO) family proteins of the RNA-induced silencing complexes (RISCs) to pair with their RNA targets and initiate the cleavage of target genes. However, the identities of functional ZIKV viRNAs and their viral RNA targets remain largely unknown. Our recent study has shown that ZIKV capsid protein interacted with Dicer and antagonized its endoribonuclease activity, which requires its histidine residue at the 41st amino acid. Accordingly, the engineered ZIKV-H41R loss-of-function (LOF) mutant virus no longer suppresses Dicer enzymatic activity nor inhibits miRNA biogenesis in NSCs. By combining AGO-associated RNA sequencing, deep sequencing analysis in ZIKV-infected human neural stem cells (NSCs), and miRanda target scanning, we defined 29 ZIKV derived viRNA profiles in NSCs, and established a complex interaction network between the viRNAs and their viral targets. More importantly, we found that viRNA production from the ZIKV mRNA is dependent on Dicer function and is a limiting factor for ZIKV virulence in NSCs. As a result, much higher levels of viRNAs generated from the ZIKV-H41R virus-infected NSCs. Therefore, our mapping of viRNAs to their RNA targets paves a way to further investigate how viRNAs play the role in anti-viral mechanisms, and perhaps other unknown biological functions.

Screening and identification of hub genes in bladder cancer by bioinformatics analysis and KIF11 is a potential prognostic biomarker.

Bladder cancer (BC) is the ninth most common lethal malignancy worldwide. Great efforts have been devoted to clarify the pathogenesis of BC, but the underlying molecular mechanisms remain unclear. To screen for the genes associated with the progression and carcinogenesis of BC, three datasets were obtained from the Gene Expression Omnibus. A total of 37 tumor and 16 non-cancerous samples were analyzed to identify differentially expressed genes (DEGs). Subsequently, 141 genes were identified, including 55 upregulated and 86 downregulated genes. The protein-protein interaction network was established using the Search Tool for Retrieval of Interacting Genes database. Hub gene identification and module analysis were performed using Cytoscape software. Hierarchical clustering of hub genes was conducted using the University of California, Santa Cruz Cancer Genomics Browser. Among the hub genes, kinesin family member 11 (KIF11) was identified as one of the most significant prognostic biomarkers among all the candidates. The Kaplan Meier Plotter database was used for survival analysis of KIF11. The expression profile of KIF11 was analyzed using the ONCOMINE database. The expression levels of KIF11 in BC samples and bladder cells were measured using reverse transcription-quantitative pCR, immunohistochemistry and western blotting. In summary, KIF11 was significantly upregulated in BC and might act as a potential prognostic biomarker. The present identification of DEGs and hub genes in BC may provide novel insight for investigating the molecular mechanisms of BC.