NSCLC extracellular vesicles containing miR-374a-5p promote leptomeningeal metastasis by influencing blood‒brain barrier permeability.

Leptomeningeal metastasis (LM) is a devastating complication of advanced non-small cell lung cancer (NSCLC). Diagnosis and monitoring of LM can be challenging. Extracellular vesicles (EVs) microRNAs (miRNAs) have become a new noninvasive diagnostic biomarker. The purpose of this study was to examine the clinical value and role of EVs miRNAs in NSCLC-LM. According to next-generation sequencing (NGS), miRNAs with differential expression of EVs in serum of NSCLC patients with LM and non-LM were detected to find biological markers for the diagnosis of LM. Cellular and in vivo experiments were conducted to explore the pathogenesis of EVs miRNA promoting LM in NSCLC. In the present study, we first demonstrated the serum level of EV-associated miR-374a-5p in patients with LM of lung cancer was much higher than that in patients without LM and was correlated with the survival time of patients with LM. Further studies showed that EVs miR-374a-5p efficiently destroys tight junctions and the integrity of the cerebral microvascular endothelial cell barrier, resulting in increased blood-brain barrier (BBB) permeability. Mechanistically, miR-374a-5p regulates the distribution of ZO-1 and occludin in endothelial cells by targeting ADD3, increasing vascular permeability and promoting LM. Implications: These results suggest that serum NSCLC-derived EVs miR-374a-5p is involved in premetastatic niche formation by regulating the permeability of BBB to promote NSCLC-LM, and can be used as a blood biomarker for the diagnosis and prognosis of NSCLC-LM.

Cerebrospinal fluid circulating tumour DNA genotyping and survival analysis in lung adenocarcinoma with leptomeningeal metastases.

PURPOSE: The prognosis of patients with leptomeningeal metastasis (LM) remains poor. Circulating tumour DNA (ctDNA) has been proven to be abundantly present in cerebrospinal fluid (CSF); hence, its clinical implication as a biomarker needs to be further verified. METHODS: We conducted a retrospective study of 35 lung adenocarcinoma (LUAD) patients with LM, and matched CSF and plasma samples were collected from all patients. All paired samples underwent next-generation sequencing (NGS) of 139 lung cancer-associated genes. The clinical characteristics and genetic profiling of LM were analysed in association with survival prognosis. RESULTS: LM showed genetic heterogeneity, in which CSF had a higher detection rate of ctDNA (P = 0.003), a higher median mutation count (P < 0.0001), a higher frequency of driver mutations (P < 0.01), and more copy number variation (CNV) alterations (P < 0.001) than plasma. The mutation frequencies of the EGFR, TP53, CDKN2A, MYC and CDKN2B genes were easier to detect in CSF than in LUAD tissue (P < 0.05), possibly reflecting the underlying mechanism of LM metastasis. CSF ctDNA is helpful for analysing the mechanism of EGFR-TKI resistance. In cohort 1, which comprised patients who received 1/2 EGFR-TKIs before the diagnosis of LM, TP53 and CDKN2A were the most common EGFR-independent resistant mutations. In cohort 2, comprising those who progressed after osimertinib and developed LM, 7 patients (43.75%) had EGFR CNV detected in CSF but not plasma. Furthermore, patient characteristics and various genes were included for interactive survival analysis. Patients with EGFR-mutated LUAD (P = 0.042) had a higher median OS, and CSF ctDNA mutation with TERT (P = 0.013) indicated a lower median OS. Last, we reported an LM case in which CSF ctDNA dynamic changes were well correlated with clinical treatment. CONCLUSIONS: CSF ctDNA could provide a more comprehensive genetic landscape of LM, indicating the potential metastasis-related and EGFR-TKI resistance mechanisms of LM patients. In addition, genotyping of CSF combined with clinical outcomes can predict the prognosis of LUAD patients with LM.

Cerebrospinal fluid exosomal protein alterations via proteomic analysis of NSCLC with leptomeningeal carcinomatosis.

PURPOSE: Leptomeningeal carcinomatosis (LC) is a rare complication of non-small cell lung cancer (NSCLC) with highly mortality. Cerebrospinal fluid (CSF) as a special kind of tumor microenvironment (TME) better represents alterations than plasma. However, the clinical value of protein profiles of exosome in CSF as liquid biopsy remains unclear. METHODS: In this study, CSF samples of NSCLC patients with (LC group) or without (NSCLC group) LC were collected and compared to patients without tumors (normal group). CSF exosomes were isolated by ultracentrifugation and protein profiles were performed by label-free proteomics. Differentially expressed proteins (DEPs) were detected by bioinformatics tools and verified by parallel reaction monitoring (PRM). RESULTS: A total of 814 proteins were detected. Bioinformatics analysis revealed their shared function in the complement activation, extracellular region, and complement and coagulation cascades. Between LC and NSCLC group, 72 DEPs were found among which FN1 demonstrated the highest betweenness centrality (BC) after protein-protein interaction network analysis. CONCLUSION: We investigated the application of label free and PRM based proteomics to detect key proteins related to LC. FN1 may serve as potential indicator to classify LC and NSCLC. Extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) are important in the process of LC. These data is promising for early prediction and diagnosis of LC.

Development and validation of a machine learning model to predict prognosis in HIV-negative cryptococcal meningitis patients: a multicenter study.

PURPOSE: To predict prognosis in HIV-negative cryptococcal meningitis (CM) patients by developing and validating a machine learning (ML) model. METHODS: This study involved 523 HIV-negative CM patients diagnosed between January 1, 1998, and August 31, 2022, by neurologists from 3 tertiary Chinese centers. Prognosis was evaluated at 10 weeks after the initiation of antifungal therapy. RESULTS: The final prediction model for HIV-negative CM patients comprised 8 variables: Cerebrospinal fluid (CSF) cryptococcal count, CSF white blood cell (WBC), altered mental status, hearing impairment, CSF chloride levels, CSF opening pressure (OP), aspartate aminotransferase levels at admission, and decreased rate of CSF cryptococcal count within 2 weeks after admission. The areas under the curve (AUCs) in the internal, temporal, and external validation sets were 0.87 (95% CI 0.794-0.944), 0.92 (95% CI 0.795-1.000), and 0.86 (95% CI 0.744-0.975), respectively. An artificial intelligence (AI) model was trained to detect and count cryptococci, and the mean average precision (mAP) was 0.993. CONCLUSION: A ML model for predicting prognosis in HIV-negative CM patients was built and validated, and the model might provide a reference for personalized treatment of HIV-negative CM patients. The change in the CSF cryptococcal count in the early phase of HIV-negative CM treatment can reflect the prognosis of the disease. In addition, utilizing AI to detect and count CSF cryptococci in HIV-negative CM patients can eliminate the interference of human factors in detecting cryptococci in CSF samples and reduce the workload of the examiner.

Adult spinal cord diffuse midline glioma, H3 K27-altered mimics symptoms of central nervous system infection: a case report.

Diffuse midline gliomas, H3 K27-altered are infiltrative growth gliomas with histone H3K27M mutations. This glioma is more common in the pediatric population, and the prognosis is usually poor. We report a case of diffuse midline gliomas, H3 K27-altered in an adult patient that mimicked symptoms of central nervous system infection. The patient was admitted due to double vision for 2 months and paroxysmal unconsciousness for 6 days. Initially, lumbar puncture showed persistent high intracranial pressure, high protein, and low chlorine. Magnetic resonance imaging showed diffuse thickening and enhancement of meninges and spinal meninges, and later, fever occurred. The initial diagnosis was meningitis. We suspected central nervous system infection, so we started anti-infection treatment, but the treatment was ineffective. The patient's condition gradually worsened, with lower limb weakness and even the consciousness became unclear. A repeat magnetic resonance imaging and positron emission tomography-computed tomography scan showed space-occupying lesions in the spinal cord, which was considered a tumor. Following neurosurgery, pathological tests identified the tumor as diffuse midline gliomas, H3 K27-altered. The patient was recommended for radiotherapy and temozolomide chemotherapy. The patient's condition improved after chemotherapy treatment, and he survived for an additional 6 months. Our case shows that diagnosing diffuse midline gliomas, H3 K27-altered in the central nervous system is complex and can be confused with the clinical characteristics of central nervous system infection. Therefore, clinicians should pay attention to such diseases to avoid misdiagnosis.

Next-generation sequencing in the diagnosis of neurobrucellosis: a case series of eight consecutive patients.

BACKGROUND: Neurobrucellosis (NB) presents a challenge for rapid and specific diagnosis. Next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has showed power in detection of causative pathogens, even some infrequent and unexpected pathogens. In this study, we presented 8 cases of NB diagnosed by the NGS of CSF. METHODS: Between August 1, 2018 and September 30, 2020, NGS was used to detect causative pathogens in clinically suspected central nervous system (CNS) infections. Data on demographics, clinical features, and laboratory tests, imaging results and NGS results were collected and reviewed. RESULTS: Among the presented 8 patients, Brucella was rapidly detected using NGS of CSF within 1-4 days, despite those eight patients had variable medical history, disease course, clinical manifestations, laboratory tests and imaging findings. NGS showed the sequence reads corresponded to Brucella species were 8 to 448, with genomic coverage of 0.02 to 0.87%. The relative abundance was 0.13% to 82.40% and sequencing depth was 1.06 to 1.24. Consequently, patients were administered with 3 to 6 months of doxycycline, ceftriaxone and rifampicin, double or triple combination, supplemented with symptomatic therapy and were fully recovered except for case 1. CONCLUSION: NGS of CSF provides a powerful tool in detection of Brucella in a prompt and specific manner, and can be considered for first-line diagnostic use in practice.

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer.

BACKGROUND: Leptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was underinvestigated in GCLM. METHODS: We retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes. RESULTS: CSF had higher mutation allele frequency (P = 0.015), more somatic mutations (P = 0.032), and more copy-number variations (P < 0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients' overall survival (P = 0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation (P = 0.014), IGF1R mutation (P = 0.034), AR mutation (P = 0.038), SMARCB1 deletion (P < 0.001), SMAD4 deletion (P = 0.0034), and TGF-beta pathway aberration (P = 0.0038). Additionally, improvement in intracranial pressure (P < 0.001), improvement in CSF cytology (P = 0.0038), and relatively low levels of CSF ctDNA (P = 0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment. CONCLUSIONS: CSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Metal-Organic Frameworks-Based Nanoplatforms for the Theranostic Applications of Neurological Diseases.

Neurological diseases are the foremost cause of disability and the second leading cause of death worldwide. Owing to the special microenvironment of neural tissues and biological characteristics of neural cells, a considerable number of neurological disorders are currently incurable. In the past few years, the development of nanoplatforms based on metal-organic frameworks (MOFs) has broadened opportunities for offering sensitive diagnosis/monitoring and effective therapy of neurology-related diseases. In this article, the obstacles for neurotherapeutics, including delayed diagnosis and misdiagnosis, the existence of blood brain barrier (BBB), off-target treatment, irrepressible inflammatory storm/oxidative stress, and irreversible nerve cell death are summarized. Correspondingly, MOFs-based diagnostic/monitoring strategies such as neuroimaging and biosensors (electrochemistry, fluorometry, colorimetry, electrochemiluminescence, etc.) and MOFs-based therapeutic strategies including higher BBB permeability, targeting specific lesion sites, attenuation of neuroinflammation/oxidative stress as well as regeneration of nerve cells, are extensively highlighted for the management of neurological diseases. Finally, the challenges of the present research from perspective of clinical translation are discussed, hoping to facilitate interdisciplinary studies at the intersections between MOFs-based nanoplatforms and neurotheranostics.

Next-generation sequencing for the diagnosis of Listeria monocytogenes meningoencephalitis: a case series of five consecutive patients.

Introduction. The prompt and specific diagnosis of Listeria monocytogenes meningoencephalitis (LMM) is challenging. Next-generation sequencing (NGS) of cerebrospinal fluid (CSF) is an emerging technique for diagnosing infrequent causative pathogens.Hypothesis/Gap statement. We hypothesized that NGS of CSF is an effective approach for diagnosing LMM.Aim. To evaluate the effectiveness of NGS, we present five cases of LMM diagnosed using NGS of the CSF.Methodology. Between August 2017 and 30 September 2020, we used NGS of the CSF to detect pathogens in patients with clinically suspected central nervous system infections. The clinical characteristics, laboratory tests, imaging findings and NGS results are reviewed.Results. Five patients were diagnosed with LMM using NGS of the CSF within 2 to 4 days, although the clinical manifestations, medical history and imaging findings varied strikingly. NGS of CSF showed sequence reads corresponding to L. monocytogenes species ranging from 118 to 1997 bp, genomic coverage of 0.29-5.96 %, relative abundance of 14.83-32.16 % and sequencing depth of 1.12 to 1.35. The prompt diagnosis resulted in targeted and effective treatment with the appropriate antibiotics, although two patients with the most severe cerebral parenchymal lesions showed little improvement.Conclusion. Our results demonstrate the power of NGS of CSF for the prompt diagnosis of LMM. NGS of CSF is an important complementary tool for identifying L. monocytogenes.

Combined Effects of Programmed Cell Death-1 Blockade and Endostar on Brain Metastases of Lung Cancer.

BACKGROUND: The blockade of programmed cell death-1 (PD-1) and recombinant human endostatin can be used for the treatment of non-small cell lung cancer (NSCLC) and its metastasis. This study aims to explore the therapeutically potential of PD-1 blockade plus Endostar in brain metastasis of NSCLC. METHODS: The mouse brain metastases model was established using Lewis lung carcinoma luciferase (LLC-Luc) and PC-9-Luc cells. Tumor metastasis in the brain and tumor burden were analyzed by using bioluminescence imaging (BLI), qRT-PCR and ELISA which were used to determine the mRNA and protein levels of biomarkers in tumor tissues. Immunohistochemical staining was used to determine the expression and location of CD31 in tumor tissues in the brain. RESULTS: Treatment with anti-PD-1 and Endostar suppressed tumor metastasis in the brain and prolonged overall survival rate in LLC-Luc and PC-9-Luc brain metastases mouse model. In addition, treatment with anti-PD-1 and Endostar inhibited the expressions of CD31 and VEGF in tumor tissues in the brain. Furthermore, treatment with anti-PD- 1 and Endostar significantly suppressed the levels of IL1ß, IFNγ, and TGFß in the tumor tissues. CONCLUSION: The combination of PD-1 blockade and endostar suppressed brain metastases of NSCLC.

Destination Attributes of Memorable Chinese Rural Tourism Experiences: Impact on Positive Arousal, Memory and Behavioral Intention.

Purpose: There is a lack of research on the composition of destination attributes of memorable tourism experiences (MTEs) and their impact on tourist loyalty in Chinese rural tourism. Based on the extended SOR (stimulus-organism-response) theory, this study constructs a model of destination attributes (gastronomy, accommodation, physiography, and rural lifestyle) of MTEs on tourists' recommend intention and revisit intention under the chain mediating effect of positive arousal and memory in Chinese rural tourism. Methods: Through the judgment sampling method, this study distributed questionnaires to the subjects who met the sampling standard in all provincial administrative regions in China. Finally, 270 valid questionnaires from 29 provincial administrative regions were obtained and the proposed hypotheses were verified using a structural equation model. Results: The results show that gastronomy, accommodation, physiography and rural lifestyle are all destination attributes of MTEs in Chinese rural tourism, and all have a positive impact on positive arousal. In addition, they are positively correlated with recommend intention and revisit intention through the chain mediating effect of positive arousal and memory. Conclusion: This study explored the impact mechanism of destination attributes of MTEs on tourist destination loyalty in the field of Chinese rural tourism to enrich findings pertaining to the study of MTEs in different contexts. Four destination attributes of MTEs were proposed and verified, and this study also confirmed that destination attributes of MTEs vary with respect to the research context. The new destination attribute of MTEs was discovered. The research results showed that managers can deliver MTEs to tourists through optimizing gastronomy, accommodation, physiography and rural lifestyle, thus generating positive arousal, deepening their memory and then gaining their loyalty. In addition, the extended SOR theory proved that it could effectively and comprehensively explain the influencing mechanism of MTEs on tourist loyalty.

Metagenomic next-generation sequencing of cell-free and whole-cell DNA in diagnosing central nervous system infections.

Background: Central nervous system (CNS) infections pose a fatal risk to patients. However, the limited sample volumes of cerebrospinal fluid (CSF) and low detection efficiency seriously hinder the accurate detection of pathogens using conventional methods. Methods: We evaluated the performance of metagenomics next-generation sequencing (mNGS) in diagnosing CNS infections. CSF samples from 390 patients clinically diagnosed with CNS infections were used for the mNGS of cell-free DNA (cfDNA) (n =394) and whole-cell DNA (wcDNA) (n =150). Results: The sensitivity of mNGS using cfDNA was 60.2% (237/394, 95% confidence interval [CI] 55.1%-65.0%), higher than that of mNGS using wcDNA (32.0%, 95% [CI] 24.8%-40.2%, 48/150) and conventional methods (20.9%, 95% [CI] 16.2%-26.5%, 54/258) (P < 0.01, respectively). The accuracy of mNGS using cfDNA in positive samples was 82.6%. Most of viral (72.6%) and mycobacterial (68.8%) pathogens were only detected by the mNGS of cfDNA. Meningitis and encephalitis with Streptococcus pneumoniae infection might be more likely to result in critically ill diseases, while Human alphaherpesvirus 3 was prone to cause non-critically ill diseases. Conclusions: This is the first report on evaluating and emphasizing the importance of mNGS using CSF cfDNA in diagnosing CNS infections, and its extensive application in diagnosing CNS infections could be expected, especially for viral and mycobacterial CNS infections.

Fucoidan Alleviates Renal Fibrosis in Diabetic Kidney Disease via Inhibition of NLRP3 Inflammasome-Mediated Podocyte Pyroptosis.

Background: Fucoidan (FPS) has been widely used to treat renal fibrosis (RF) in patients with diabetic kidney disease (DKD); however, the precise therapeutic mechanisms remain unclear. Recently, research focusing on inflammation-derived podocyte pyroptosis in DKD has attracted increasing attention. This phenomenon is mediated by the activation of the nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, leading to RF during DKD progression. Therefore, we designed a series of experiments to investigate the ameliorative effects of FPS on RF in DKD and the mechanisms that are responsible for its effect on NLRP3 inflammasome-mediated podocyte pyroptosis in the diabetic kidney. Methods: The modified DKD rat models were subjected to uninephrectomy, intraperitoneal injection of streptozotocin, and a high-fat diet. Following induction of renal injury, the animals received either FPS, rapamycin (RAP), or a vehicle for 4 weeks. For in vitro research, we exposed murine podocytes to high glucose and MCC950, an NLRP3 inflammasome inhibitor, with or without FPS or RAP. Changes in the parameters related to RF and inflammatory podocyte injury were analyzed in vivo. Changes in podocyte pyroptosis, NLRP3 inflammasome activation, and activation of the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1)/NLRP3 signaling axis involved in these changes were analyzed in vivo and in vitro. Results: FPS and RAP ameliorated RF and inflammatory podocyte injury in the DKD model rats. Moreover, FPS and RAP attenuated podocyte pyroptosis, inhibited NLRP3 inflammasome activation, and regulated the AMPK/mTORC1/NLRP3 signaling axis in vivo and in vitro. Notably, our data showed that the regulative effects of FPS, both in vivo and in vitro, on the key signaling molecules, such as p-AMPK and p-raptor, in the AMPK/mTORC1/NLRP3 signaling axis were superior to those of RAP, but similar to those of metformin, an AMPK agonist, in vitro. Conclusion: We confirmed that FPS, similar to RAP, can alleviate RF in DKD by inhibiting NLRP3 inflammasome-mediated podocyte pyroptosis via regulation of the AMPK/mTORC1/NLRP3 signaling axis in the diabetic kidney. Our findings provide an in-depth understanding of the pathogenesis of RF, which will aid in identifying precise targets that can be used for DKD treatment.

Neurosyphilis with a rare magnetic resonance imaging pattern confirmed by metagenomic next-generation sequencing: a case report.

BACKGROUND: Neurosyphilis refers to infection of the central nervous system by Treponema pallidum. The clinical presentation is variable and nonspecific. Neuroimaging findings are complex and that the diagnosis is based on clinical presentation, cerebrospinal fluid (CSF) parameters, and serologic and CSF evidence of syphilis. To date, there is no case report describing Treponema pallidum detected by metagenomic next-generation sequencing (mNGS) in CSF. CASE PRESENTATION: In this report, we describe a case of neurosyphilis in a HIV-negative, 29-year-old man, who was admitted to our hospital with an epileptic seizure and progressive cognitive impairment. Brain magnetic resonance imaging (MRI) revealed fluid-attenuated inversion recovery (FLAIR) high signal intensities in bilateral medial and anterior temporal lobes, insula, right pulvinar of the thalami, precuneus, frontal and temporo-occipital lobes. Laboratory examination showed positive results by means of nontreponemal or specific treponemal test in serum and CSF. mNGS of the CSF was also performed to identify Treponema pallidum for the first time. CONCLUSIONS: This case underscores the importance of considering neurosyphilis as a potential cause of mesiotemporal abnormality. In addition, the rapid improvement and wide usability of mNGS technology will bring new breakthroughs in the clinical diagnosis of neurosyphilis.

Primary meningeal central nervous system lymphoma: A case report and literature review.

RATIONALE: Primary central nervous system lymphoma (PCNSL) is a rare extranodal non-Hodgkin lymphoma, and isolated meningeal PCNSL, without evidence of parenchymal involvement, is even less common, occurring in only 10% to 15% of cases. PATIENT CONCERNS: A 65-years-old female presented to our hospital with progressive lower extremity motor dysfunction and blurred vision. The initial neurological examination revealed decreased muscle strength in both lower extremities and sensory dysfunction of lower extremities, saddle area, and buttocks. Brain magnetic resonance imaging showed no abnormalities. Lumbar enhanced magnetic resonance imaging showed T11 to L3 horizontal meningeal enhancement. Cerebrospinal fluid (CSF) cytology revealed lymphoma cells. Immunohistochemistry and flow cytometry of the CSF were performed as auxiliary methods to establish the diagnosis of lymphoma. DIAGNOSES: The patient was diagnosed primary meningeal central nervous system lymphoma. INTERVENTIONS: During hospitalization, the patient was treated with 2 courses of high-dose intrathecal methotrexate and rituximab combined with intrathecal chemotherapy and supportive treatment. OUTCOMES: After 2 years of follow-up, the patient was able to walk and take care of herself. LESSONS: Cases of PCNSL involving only the meninges are rare. Multimodal analysis of the CSF comprises an important component of the diagnostic work-up for patients with primary meningeal central nervous system lymphoma.

Clinical efficacy and safety of different doses of intrathecal methotrexate in the treatment of leptomeningeal carcinomatosis: a prospective and single-arm study.

OBJECTIVE: To investigate the clinical efficacy and safety of different doses of intrathecal methotrexate in the treatment of leptomeningeal carcinomatosis. METHODS: 53 patients admitted to the Second Hospital of Hebei Medical University with leptomeningeal carcinomatosis were recruited. They were divided into two groups: 15-mg-group received 15 mg methotrexate intrathecally, while the other received 10 mg methotrexate. All patients were followed up to 31 December 2020 or until death. Primary endpoint was the response rate. Secondary endpoints were survival and safety. Treatment-related adverse events were recorded. RESULTS: The intrathecal chemotherapy was regularly maintained in 42 cases. Most primary cancers were lung (60.4%), stomach (18.9%) or breast (5.7%). The clinical response rate was higher in the 15 mg group than the 10 mg group (62.5 vs. 34.5%, P = 0.042). In the 15 mg group, two cases showed myelosuppression and one case showed seizures. In the 10 mg group, one patient appeared fever, three patients appeared myelosuppression and one showed leukoencephalopathy. However, there were no serious irreversible adverse reactions in neither of the two groups. In terms of survival, the median survival was 15.7 weeks in the 15 mg group and 27.1 weeks in the 10 mg group (P = 0.116). Multivariate analysis showed that only targeted therapy improved the survival (P < 0.0001, HR = 5.386). CONCLUSION: Increased dose of methotrexate did not prolong the overall survival, but it was more effective in relieving clinical symptoms with no increased adverse reactions. Targeted therapy might improve the survival.

[Effects and mechanisms of Supplemented Gegen Qinlian Decoction Formula against podocyte pyroptosis and insulin resistance in model rats with diabetic kidney disease].

This study explored the in vivo effects and mechanisms of the modern classical prescription Supplemented Gegen Qinlian Decoction Formula(SGDF) against diabetic kidney disease(DKD). Sixty rats were randomly divided into the normal group, model group, SGDF group, and rosiglitazone(ROS) group. The modified DKD rat model was established by employing the following three methods: exposure to high-fat diet, unilateral nephrectomy, and intraperitoneal injection of streptozotocin(STZ). After modeling, rats in the four groups were treated with double distilled water, SGDF suspension, and ROS suspension, respectively, by gavage every day. At the end of the 6 th week of drug administration, all the rats were sacrificed for collecting urine, blood, and kidney tissue, followed by the examination of rat general conditions, urine and blood biochemical indicators, glomerulosclerosis-related indicators, podocyte pyroptosis markers, insulin resistance(IR)-related indicators, and key molecules in the insulin receptor substrate(IRS) 1/phosphatidylinositol-3-kinase(PI3 K)/serine threonine kinase(Akt) signaling pathway. The results showed that SGDF and ROS improved the general conditions, some renal function indicators and glomerulosclerosis of DKD model rats without affecting the blood glucose(BG). Besides, they ameliorated the expression characteristics and levels of podocyte pyroptosis markers, alleviated IR, and up-regulated the protein expression levels of the key molecules in IRS1/PI3 K/Akt pathway to varying degrees. In conclusion, similar to ROS, SGDF relieves DKD by targeting multiple targets in vivo. Specifically, it exerts the therapeutic effects by alleviating podocyte pyroptosis and IR. This study has preliminarily provided the pharmacological evidence for the research and development of new drugs for the treatment of DKD based on SGDF.

Total Flavones of Abelmoschus manihot Ameliorates Podocyte Pyroptosis and Injury in High Glucose Conditions by Targeting METTL3-Dependent m6A Modification-Mediated NLRP3-Inflammasome Activation and PTEN/PI3K/Akt Signaling.

Background: The total flavones of Abelmoschus manihot (TFA), a compound that is extracted from Abelmoschus manihot, has been widely used in China to reduce podocyte injury in diabetic kidney disease (DKD). However, the mechanisms underlying the therapeutic action of this compound have yet to be elucidated. Podocyte pyroptosis is characterized by activation of the NLRP3 inflammasome and plays an important role in inflammation-mediated diabetic kidneys. Regulation of the PTEN/PI3K/Akt pathway is an effective strategy for improving podocyte damage in DKD. Previous research has also shown that N6-methyladenosine (m6A) modification is involved in DKD and that m6A-modified PTEN regulates the PI3K/Akt pathway. In this study, we investigated whether TFA alleviates podocyte pyroptosis and injury by targeting m6A modification-mediated NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. Methods: We used MPC-5 cells under high glucose (HG) conditions to investigate the key molecules that are involved in podocyte pyroptosis and injury, including activation of the NLRP3 inflammasome and the PTEN/PI3K/Akt pathway. We detected alterations in the levels of three methyltransferases that are involved in m6A modification. We also investigated changes in the levels of these key molecules in podocytes with the overexpression or knockdown of methyltransferase-like (METTL)3. Results: Analysis showed that TFA and MCC950 protected podocytes against HG-induced pyroptosis and injury by reducing the protein expression levels of gasdermin D, interleukin-1ß, and interleukin-18, and by increasing the protein expression levels of nephrin, ZO-1, WT1 and podocalyxin. TFA and 740Y-P inhibited activation of the NLRP3 inflammasome via the PI3K/Akt pathway by inhibiting the protein levels of NIMA-related kinase7, NLRP3, ASC, and caspase-1, and by increasing the protein expression levels of p-PI3K and p-Akt. TFA improved pyroptosis and injury in HG-stimulated podocytes by regulating METTL3-dependent m6A modification. Conclusion: Collectively, our data indicated that TFA could ameliorate pyroptosis and injury in podocytes under HG conditions by adjusting METTL3-dependent m6A modification and regulating NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. This study provides a better understanding of how TFA can protect podocytes in DKD.

Inhibition of Renal Tubular Epithelial Mesenchymal Transition and Endoplasmic Reticulum Stress-Induced Apoptosis with Shenkang Injection Attenuates Diabetic Tubulopathy.

Background: The proximal renal tubule plays a critical role in diabetic kidney disease (DKD) progression. Early glomerular disease in DKD triggers a cascade of injuries resulting in renal tubulointerstitial disease. These pathophysiological responses are collectively described as diabetic tubulopathy (DT). Thus, therapeutic strategies targeting DT hold significant promise for early DKD treatment. Shenkang injection (SKI) has been widely used to treat renal tubulointerstitial fibrosis in patients with chronic kidney disease in China. However, it is still unknown whether SKI can alleviate DT. We designed a series of experiments to investigate the beneficial effects of SKI in DT and the mechanisms that are responsible for its effect on epithelial-to-mesenchymal transition (EMT) and endoplasmic reticulum (ER) stress-induced apoptosis in DT. Methods: The modified DKD rat models were induced by uni-nephrectomy, streptozotocin intraperitoneal injection, and a high-fat diet. Following the induction of renal injury, these animals received either SKI, rosiglitazone (ROS), or vehicle, for 42 days. For in vitro research, we exposed NRK-52E cells to high glucose (HG) and 4-phenylbutyric acid (4-PBA) with or without SKI or ROS. Changes in parameters related to renal tubular injury and EMT were analyzed in vivo. Changes in the proportion of apoptotic renal tubular cells and ER stress, and the signaling pathways involved in these changes, were analyzed both in vivo and in vitro. Results: SKI and ROS improved the general condition, the renal morphological appearance and the key biochemical parameters, and attenuated renal injury and EMT in the rat model of DKD. In addition, SKI and ROS alleviated apoptosis, inhibited ER stress, and suppressed PERK-eIF2α-ATF4-CHOP signaling pathway activation both in vivo and in vitro. Notably, our data showed that the regulatory in vitro effects of SKI on PERK-eIF2α-ATF4-CHOP signaling were similar to those of 4-PBA, a specific inhibitor of ER stress. Conclusion: This study confirmed that SKI can alleviate DT in a similar manner as ROS, and SKI achieves this effect by inhibiting EMT and ER stress-induced apoptosis. Our findings thereby provide novel information relating to the clinical value of SKI in the treatment of DT.

A Diagnostic Scoring System for Distinguishing between Tuberculous and Bacterial Meningitis Based on Clinical and Laboratory Findings.

It is very difficult to diagnose and distinguish tuberculous meningitis, and the current laboratory methods are unsubstantial in developing countries. The study is aimed at creating a scoring system on the basis of basic laboratory and clinical achievements that could be used as diagnostic aid for tuberculous meningitis for Chinese patients. A retrospective study of cases was conducted for comparison between clinical characteristics and laboratory features of 241 patients on admission who conformed to inclusion criteria of tuberculous meningitis (n = 141) or bacterial meningitis (n = 100). Logistic regression was employed to establish a diagnostic formula to distinguish between tuberculous meningitis and bacterial meningitis. The receiver operating characteristic curve analysis was applied to determine the best diagnostic critical point of the diagnostic formula. It was found that five variables (disease course, white blood cell count, serum sodium, total white cell count of cerebrospinal fluid, and neutrophil proportion in cerebrospinal fluid) were independently associated with tuberculous meningitis. The 87% sensitivity and 94% specificity were included in the diagnostic scoring system derived from these variables. Especially in the case of limited microbial resources, doctors can use this diagnostic scoring system to distinguish tuberculous meningitis from bacterial meningitis.