SARS-CoV-2 induces blood-brain barrier and choroid plexus barrier impairments and vascular inflammation in mice.

The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains an important question, which is also centered on the mystery of whether the virus is capable of breaching the barriers into the central nervous system. By studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment. In addition, the blood-cerebrospinal fluid barrier at the choroid plexus was also impaired after infection. Therefore, cerebrovascular and choroid plexus dysfunctions are important aspects of COVID-19 and may contribute to neurological complications both acutely and in long COVID.

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.

A nerve injury-specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression.

Maladaptive changes of nerve injury-associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury-specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury-induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury-induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

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.

Severe delayed pulmonary toxicity following PD-L1-specific CAR-T cell therapy for non-small cell lung cancer.

OBJECTIVES: This phase I study aimed to evaluate the antitumor effect and safety of programmed death-ligand-1 (PD-L1)-targeting autologous chimeric antigen receptor T (CAR-T) cells for patients with non-small cell lung cancer (NSCLC). METHODS: Programmed death-ligand-1-specific CAR-T cells were generated using lentiviral transduction. Four patients with NSCLC were recruited, but only one patient was finally involved. CAR-T cells were infused on three different days (total dose during therapy, 1 × 106 CAR-T cells kg-1 body weight). The date on which the patient received the first CAR-T cell infusion was designated as Day 0. RESULTS: Circulating CAR-T cells accounted for 3.30% of the patient's peripheral blood T cells detected by FACS analysis during the first follow-up (Day +29). The chest CT scan showed subtle tumor shrinkage (stable disease). On Day +43, the patient developed pyrexia without any known causes and dyspnoea that rapidly deteriorated to respiratory failure in 3 days. The chest X-ray and CT scan showed bilateral extensive pulmonary infiltration in addition to the tumor silhouette on the left upper lung. The interleukin (IL)-6 levels in serum dramatically increased (> 100-fold). The patient was immediately transferred to the ICU where he received oxygen and intravenous infusions of tocilizumab and methylprednisolone. His symptoms rapidly improved and the pulmonary inflammation gradually resolved. CONCLUSION: The clinical manifestations and test findings for this patient with NSCLC might represent unique clinical manifestations of solitary organ damage secondary to PD-L1-specific CAR-T cell therapy. The differential diagnosis, underlying mechanism and prevention and treatment strategies for such complications have also been discussed.

Fgr contributes to hemorrhage-induced thalamic pain by activating NF-κB/ERK1/2 pathways.

Thalamic pain, a type of central poststroke pain, frequently occurs following ischemia/hemorrhage in the thalamus. Current treatment of this disorder is often ineffective, at least in part due to largely unknown mechanisms that underlie thalamic pain genesis. Here, we report that hemorrhage caused by microinjection of type IV collagenase or autologous whole blood into unilateral ventral posterior lateral nucleus and ventral posterior medial nucleus of the thalamus increased the expression of Fgr, a member of the Src family nonreceptor tyrosine kinases, at both mRNA and protein levels in thalamic microglia. Pharmacological inhibition or genetic knockdown of thalamic Fgr attenuated the hemorrhage-induced thalamic injury on the ipsilateral side and the development and maintenance of mechanical, heat, and cold pain hypersensitivities on the contralateral side. Mechanistically, the increased Fgr participated in hemorrhage-induced microglial activation and subsequent production of TNF-α likely through activation of both NF-κB and ERK1/2 pathways in thalamic microglia. Our findings suggest that Fgr is a key player in thalamic pain and a potential target for the therapeutic management of this disorder.

N6-Methyladenosine Demethylase FTO Contributes to Neuropathic Pain by Stabilizing G9a Expression in Primary Sensory Neurons.

Nerve injury-induced change in gene expression in primary sensory neurons of dorsal root ganglion (DRG) is critical for neuropathic pain genesis. N6-methyladenosine (m6A) modification of RNA represents an additional layer of gene regulation. Here, it is reported that peripheral nerve injury increases the expression of the m6A demethylase fat-mass and obesity-associated proteins (FTO) in the injured DRG via the activation of Runx1, a transcription factor that binds to the Fto gene promoter. Mimicking this increase erases m6A in euchromatic histone lysine methyltransferase 2 (Ehmt2) mRNA (encoding the histone methyltransferase G9a) and elevates the level of G9a in DRG and leads to neuropathic pain symptoms. Conversely, blocking this increase reverses a loss of m6A sites in Ehmt2 mRNA and destabilizes the nerve injury-induced G9a upregulation in the injured DRG and alleviates nerve injury-associated pain hypersensitivities. FTO contributes to neuropathic pain likely through stabilizing nerve injury-induced upregulation of G9a, a neuropathic pain initiator, in primary sensory neurons.

Identification of Medium-Length Antineurofilament Autoantibodies in Patients with Anti-N-Methyl-D-Aspartate Receptor Encephalitis.

BACKGROUND AND PURPOSE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe central nervous system disorder mediated by NMDAR antibodies that damages neurons. We investigated the correlation between cytoskeletal autoantibodies and the clinical severity in patients with anti-NMDAR encephalitis. METHODS: Non-NMDAR autoantibodies were identified by screening matched cerebrospinal fluid (CSF) and the serum samples of 45 consecutive patients with anti-NMDAR encephalitis and 60 healthy individuals against N-methyl-D-aspartate receptor 1-transfected and nontransfected human embryonic kidney 293T cells. Immunocytochemistry was performed to assess antibody binding in rat brain sections and primary cortical neurons. Cell-based assays and Western blotting were applied to identify autoantibodies targeting medium neurofilaments (NFMs). We compared clinical characteristics between patients with NMDAR encephalitis who were positive and negative for anti-NFM-autoantibodies. RESULTS: Anti-NFM autoantibodies were detected in both the serum and CSF in one patient (2%) and in the serum only in six patients (13%). No antibodies were detected in the serum of healthy controls (7/45 vs. 0/60, p=0.0016). Four of the seven patients with anti-NFM autoantibodies in serum were children (57%), and three (43%) had abnormalities in brain magnetic resonance imaging. These patients responded well to immunotherapy, and either no significant or only mild disability was observed at the last follow-up. Anti-NMDAR encephalitis did not differ with the presence of anti-NFM autoantibodies. CONCLUSIONS: Anti-NFM autoantibodies may be present in patients with anti-NMDAR encephalitis, indicating underlying neuronal damage. A large cohort study is warranted to investigate the clinical differences between patients with NMDAR encephalitis according to their anti-NFM antibody status.

Toll-like receptor 7 contributes to neuropathic pain by activating NF-κB in primary sensory neurons.

Toll like receptor 7 (TLR7) is expressed in neurons of the dorsal root ganglion (DRG), but whether it contributes to neuropathic pain is elusive. We found that peripheral nerve injury caused by ligation of the fourth lumbar (L4) spinal nerve (SNL) or chronic constriction injury of sciatic nerve led to a significant increase in the expression of TLR7 at mRNA and protein levels in mouse injured DRG. Blocking this increase through microinjection of the adeno-associated virus (AAV) 5 expressing TLR7 shRNA into the ipsilateral L4 DRG alleviated the SNL-induced mechanical, thermal and cold pain hypersensitivities in both male and female mice. This microinjection also attenuated the SNL-induced increases in the levels of phosphorylated extracellular signal-regulated kinase ½ (p-ERK1/2) and glial fibrillary acidic protein (GFAP) in L4 dorsal horn on the ipsilateral side during both development and maintenance periods. Conversely, mimicking this increase through microinjection of AAV5 expressing full-length TLR7 into unilateral L3/4 DRGs led to elevations in the amounts of p-ERK1/2 and GFAP in the dorsal horn, augmented responses to mechanical, thermal and cold stimuli, and induced the spontaneous pain on the ipsilateral side in the absence of SNL. Mechanistically, the increased TLR7 activated the NF-κB signaling pathway through promoting the translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from the injured DRG neurons. Our findings suggest that DRG TLR7 contributes to neuropathic pain by activating NF-κB in primary sensory neurons. TLR7 may be a potential target for therapeutic treatment of this disorder.

Influence of obesity on in-hospital and postoperative outcomes of hepatic resection for malignancy: a 10-year retrospective analysis from the US National Inpatient Sample.

OBJECTIVES: The influence of obesity on the outcomes of curative liver resection for malignancies remains controversial. We aimed to compare the in-hospital outcomes of liver resection for malignancy between obese and non-obese patients. DESIGN: This was a population-based, retrospective, observational study using data from the Nationwide Inpatient Sample (NIS), the largest all-payer US inpatient care database. SETTING: Hospitalisations of adults ≥18 years old with diagnoses of primary hepatobiliary malignancy or secondary malignant neoplasms of liver in the USA were identified from the NIS database between 2005 and 2014. PARTICIPANTS: Data of 18 398 patients ≥18 years old and underwent liver resection without pancreatic resection in the NIS were extracted. All included subjects had primary hepatobiliary malignancy or secondary malignant neoplasms of the liver. Patients were divided into obese and non-obese groups. These groups were compared with respect to postoperative complications, length of hospital stay and hospital cost according to surgical extent and approach. INTERVENTIONS: Patients were undergoing lobectomy of liver or partial hepatectomy. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoints of this study were postoperative complications, length of hospital stay and hospital cost. RESULTS: After adjustment, obese patients were significantly more likely to experience postoperative complications than were non-obese patients (adjusted OR 1.25, 95% CI 1.10 to 1.42), regardless of whether lobectomy or partial hepatectomy was performed. Furthermore, obesity was significantly associated with increased risk of postoperative complications in patients who underwent open liver resection, but not laparoscopic resection. No significant difference was observed in length of hospital stay or total hospital costs between obese and non-obese patients. CONCLUSIONS: After adjustment for preoperative comorbidities and other potential confounders, obesity is significantly associated with greater risk of complications in patients undergoing open liver resection for malignancy, but not laparoscopic resection.

BCL2L10/BECN1 modulates hepatoma cells autophagy by regulating PI3K/AKT signaling pathway.

The aim of this study was to investigate BCL2L10 and BECN1 expression and their effect on autophagy in hepatocellular carcinoma (HCC). We found that BCL2L10 expression was low in hepatoma tissues and cells. Overexpression of BCL2L10 decreased the activity of hepatoma cells. To analyze autophagic flux, we monitored the formation of autophagic vesicles by fluorescence protein method. Autophagy-related protein LC3B-II was accumulated and P62 was decreased, which indicated that autophagy was induced by BECN1, while BCL2L10 could suppress this trend. Immunofluorescence assay showed that BCL2L10 and Beclin 1 were co-located in hepatoma cells. Immunoprecipitation showed that BCL2L10 could inhibit the autophagy of hepatoma cells by combining with Beclin 1. ELISA and co-immunoprecipitation suggested that the combination between BCL2L10 and Beclin 1 reduced the bond between Beclin 1 and PI3KC3. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the PI3K/AKT signaling pathway was significantly upregulated in HCC. In conclusions, BCL2L10 had a low expression in HCC tissues and cells, which could release BECN1 to induce autophagy of hepatoma cells by downregulating PI3K/AKT signaling pathway.

Protective effects of lycopene on kainic acid-induced seizures.

Lycopene (LCP) is a carotenoid that protects against many diseases by alleviating oxidative stress. However, the effect of LCP on epileptic seizures has not been examined well in previous studies. In the current work, we employed kainic acid (KA) to induce experimental epileptic seizures in mice, and investigated the function of LCP during this process. We found that the onset and extent of KA-induced seizures were alleviated in LCP-pretreated mice. Nissl staining of hippocampus showed that the granule cell dispersion lesion induced by KA was improved by the LCP treatment. Additionally, we analyzed the oxidative stress levels in mice and found that LCP elevated SOD activity and suppressed MDA level in KA-induced seizures. Moreover, the expression of GABA receptors was influenced by LCP treatment. LCP suppressed the upregulation of gabrb2 and gabrb3 induced by KA, whereas it enhanced the expression of gabrb1. Results suggested that LCP plays a protective function in KA-induced seizures. Hence, it may be a potential functional food alternative for controlling and treating epileptic seizures.