Lifen Yang

Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier

PURPOSEnThe aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of human intestinal epithelial cell induced by tumor necrosis factor-alpha (TNF-α).nnnMETHODSnTo confirm that TNF-α induces epithelial barrier hyperpermeability by disrupting tight junction, Caco-2 cells were exposed to TNF-α, and changes in epithelial permeability (via TER assay), F-actin dynamics (via Rhodamine-phalloidin staining) and tight junction protein expression (via western blot) were monitored. Moreover, to ensure that NF-κB participated in the regulatory mechanisms, Caco-2 cells were transfected with DNMu-IκBα or control plasmids, the above experiments were repeated and the activation effect of TNF-α on NF-κB was detected by luciferase reporter assays. Lastly, we took dominant negative plasmid and knockdown approaches to investigate the potential importance of the NF-κB/myosin light chain kinase (MLCK)/myosin light chain phosphorylation (pMLC) pathways in TNF-a-mediated damage.nnnRESULTnTNF-α could cause NF-κB activation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were alleviated by inhibiting NF-κB. TNF-α induced increase of MLCK transcription and MLC phosphorylation act later than NF-κB activation, which could be suppressed both by inactivating and deleting NF-κB.nnnCONCLUSIONSnTNF-α induces intestinal epithelial cell hyperpermeability by disrupting TJs, in part through MLCK upregulation, in which NF-κB is the positive upstream regulator for MLCK.

Interleukin-1β plays a role in the pathogenesis of mesial temporal lobe epilepsy through the PI3K/Akt/mTOR signaling pathway in hippocampal neurons

Recently, the role of inflammation in the pathogenesis of mesial temporal lobe epilepsy (MTLE) has garnered great attention. Increasing evidence indicated that interleukin-1β (IL-1β) plays a critical role in the pathogenesis of MTLE. In this study, we cultured primary hippocampal neurons, using IL-1β to mimic the process of inflammatory reaction in neurons, then inhibited the inflammation using inhibitors of the PI3K/Akt/mTOR signaling pathway. The expression of proteins related to the PI3K/Akt/mTOR signaling pathway in rat hippocampal neurons was detected by western blot, and similar methods were applied to the hippocampi obtained from children with MTLE and normal controls. Neuronal somatic size and dendritic length were measured by immunohistochemistry and digital imaging. We observed that stimulation with IL-1β in neuron led to the up-regulation of p-Akt and p70S6K and promoted the growth of cell somatic size and dendritic length via the PI3K/Akt/mTOR signaling pathway. Pre-treatment with inhibitors of the pathway, LY294002 and rapamycin, decreased the expression of p-Akt and p70S6K and alleviated the morphological changes induced by IL-1β in hippocampal neurons. We further verified the increasement of P-Akt and p70S6K in the hippocampi of children with MTLE. These data are the first to demonstrate that the inflammatory response induced by IL-1β promotes seizures and plays an important role in the pathogenesis of MTLE via the PI3K/Akt/mTOR signaling pathway. Therefore, modulation of the PI3K/Akt/mTOR signaling pathway may be a novel therapeutic target for the treatment of MTLE.

Myoloid-related protein 8, an endogenous ligand of Toll-like receptor 4, is involved in epileptogenesis of mesial temporal lobe epilepsy via activation of the nuclear factor-κB pathway in astrocytes.

The role of Toll-like receptor 4 (TLR4) in the activation of innate immunity has been extensively studied in the past several years. Here, we are the first to report that myeloid-related protein 8 (MRP8), an endogenous TLR4 ligand, is involved in the epileptogenesis of mesial temporal lobe epilepsy (MTLE). We find that the expression of MRP8, TLR4, and interleukin 1-β (IL-1β) was upregulated in a MTLE model during both acute and chronic disease stages. We next investigated the possible roles played by astrocytes, which have been shown to be the major source of IL-1β during epilepsy. Stimulation via MRP8 led to the induction of IL-1β in astrocytes in vitro, accompanied by the activation of Nuclear Factor-κB, while knockdown of TLR4 or inhibition of NF-κB in astrocytes prevented this IL-1β induction. Thus, MRP8 may potentiate the perpetuation of MTLE by activating the NF-κB pathway in astrocytes, and could be a new target for anticonvulsant therapies.

PKC and RhoA signals cross-talk in Escherichia coli endotoxin induced alterations in brain endothelial permeability.

Escherichia coli endotoxin LPS regulates blood-brain barrier permeability by disrupting the tight junction (TJ) complex between brain endothelial cells. This study used Bend.3 cells to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. The LPS-induced alterations in the brain endothelial barrier were associated with PKC (a, β, ζ) and RhoA, but were independent of PI3K and the tyrosine kinase pathway. Inhibition of PKC (a, β, ζ) and RhoA activity using shRNA and dominant negative mutants diminished the effects of LPS on the brains endothelial TJs. The interactions between the PKC and Rho pathways were therefore examined. PKC-a and PKC-ζ, but not PKC-β interacted with RhoA in Bend.3 cells stimulated by LPS. PKC-a acted as the upstream molecule for Rho and PKC-ζ acted as the downstream target for Rho. Comparing the effect of double inhibition of Rho and PKC and single inhibition of Rho or PKC confirmed that this interaction is critical for LPS-induced brain endothelial cell hyperpermeability. Collectively these data are the first to suggest that LPS affects the brains endothelial TJ barrier via PKC (a, β, ζ)- and RhoA, independent of the PI3K and tyrosine kinase pathways. In addition, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulator for RhoA in the process.

The use of targeted genomic capture and massively parallel sequencing in diagnosis of Chinese Leukoencephalopathies

Leukoencephalopathies are diseases with high clinical heterogeneity. In clinical work, it’s difficult for doctors to make a definite etiological diagnosis. Here, we designed a custom probe library which contains the known pathogenic genes reported to be associated with Leukoencephalopathies, and performed targeted gene capture and massively parallel sequencing (MPS) among 49 Chinese patients who has white matter damage as the main imaging changes, and made the validation by Sanger sequencing for the probands’ parents. As result, a total of 40.8% (20/49) of the patients identified pathogenic mutations, including four associated with metachromatic leukodystrophy, three associated with vanishing white matter leukoencephalopathy, three associated with mitochondrial complex I deficiency, one associated with Globoid cell leukodystrophy (or Krabbe diseases), three associated with megalencephalic leukoencephalopathy with subcortical cysts, two associated with Pelizaeus-Merzbacher disease, two associated with X-linked adrenoleukodystrophy, one associated with Zellweger syndrome and one associated with Alexander disease. Targeted capture and MPS enables to identify mutations of all classes causing leukoencephalopathy. Our study combines targeted capture and MPS technology with clinical and genetic diagnosis and highlights its usefulness for rapid and comprehensive genetic testing in the clinical setting. This method will also expand our knowledge of the genetic and clinical spectra of leukoencephalopathy.

Analysis copy number variation of Chinese children in early-onset epileptic encephalopathies with unknown cause.

Copy number variations (CNVs) play an important role in the genetic etiology of unknown cause early‐onset epileptic encephalopathies (EOEEs), but the genomic CNVs analysis of Chinese EOEEs children was rare. Here, we identified CNVs by single nucleotide polymorphism array in 116 patients with different subtypes of EOEEs. Of 116 patients 17 (14.66%) carried 19 large CNVs. A total of 14 CNVs in 12 patients were further validated: four of the CNVs were classified as de novo, seven were maternal, and three were paternal. Follow‐up of those 12 patients showed that 5 had been seizure‐free for at least 9 months, 5 had seizures several times per month or per year, and 2 had seizures everyday. But eight patients have profound developmental delay. In this study, we found at least 3.4% of patients had pathogenic CNVs. For the patients, our study laid the foundation for prenatal interventions for their families. Further, we identified potential candidate gene involved in EOEEs. The association of CNVs and clinical features will contribute to the understanding of EOEEs.

Genetic etiologies of the electrical status epilepticus during slow wave sleep: systematic review

BackgroundElectrical status epilepticus during slow-wave sleep (ESESS) which is also known as continuous spike-wave of slow sleep (CSWSS) is type of electroencephalographic (EEG) pattern which is seen in ESESS/CSWSS/epilepsy aphasia spectrum. This EEG pattern can occur alone or with other syndromes. Its etiology is not clear, however, brain malformations, immune disorders, and genetic etiologies are suspected to contribute. We aimed to perform a systematic review of all genetic etiologies which have been reported to associate with ESESS/CSWSS/epilepsy-aphasia spectrum. We further aimed to identify the common underlying pathway which can explain it. To our knowledge, there is no available systematic review of genetic etiologies of ESESS/CSWSS/epilepsy-aphasia spectrum. MEDLINE, EMBASE, PubMed and Cochrane review database were searched, using terms specific to electrical status epilepticus during sleep or continuous spike–wave discharges during slow sleep or epilepsy-aphasia spectrum and of studies of genetic etiologies. These included monogenic mutations and copy number variations (CNVs). For each suspected dosage-sensitive gene, further studies were performed through OMIM and PubMed database.ResultsTwenty-six studies out of the 136 identified studies satisfied our inclusion criteria. I51 cases were identified among those 26 studies. 16 studies reported 11 monogenic mutations: SCN2A (Nu2009=u20096), NHE6/SLC9A6 (Nu2009=u20091), DRPLA/ ATN1 (Nu2009=u20091), Neuroserpin/SRPX2 (Nu2009=u20091), OPA3 (Nu2009=u20091), KCNQ2 (Nu2009=u20092), KCNA2 (Nu2009=u20095), GRIN2A (Nu2009=u200934), CNKSR2 (Nu2009=u20092), SLC6A1 (Nu2009=u20092) and KCNB1 (Nu2009=u20095). 10 studies reported 89 CNVs including 9 recurrent ones: Xp22.12 deletion encompassing CNKSR2 (Nu2009=u20096), 16p13 deletion encompassing GRIN2A (Nu2009=u20094), 15q11.2–13.1 duplication (Nu2009=u200915), 3q29 duplication (Nu2009=u200911), 11p13 duplication (Nu2009=u20092), 10q21.3 deletion (Nu2009=u20092), 3q25 deletion (Nu2009=u20092), 8p23.3 deletion (Nu2009=u20092) and 9p24.2 (Nu2009=u20092). 68 of the reported genetic etiologies including monogenic mutations and CNVs were detected in patients with ESESS/CSWSS/epilepsy aphasia spectrum solely. The most common underlying pathway was channelopathy (Nu2009=u200956).ConclusionsOur review suggests that genetic etiologies have a role to play in the occurrence of ESESS/CSWSS/epilepsy-aphasia spectrum. The common underlying pathway is channelopathy. Therefore we propose more genetic studies to be done for more discoveries which can pave a way for proper drug identification. We also suggest development of common cut-off value for spike-wave index to ensure common language among clinicians and researchers.

Novel West syndrome candidate genes in a Chinese cohort

West syndrome (WS) is a classic form of early infantile epileptic encephalopathy (EIEE) characterized by tonic spasms with clustering, arrest of psychomotor development, and hypsarrhythmia on electroencephalography. Genetic defects play a critical role in the pathology of WS, and 54 EIEE genes have been identified till date. This study was designed to uncover new candidate genes for West syndrome.

Effectiveness and Safety of Different Once-Daily Doses of Adrenocorticotropic Hormone for Infantile Spasms

IntroductionAdrenocorticotropic hormone (ACTH) has been commonly used as a first-line treatment for infantile spasms (IS), but its optimal dose and duration are still unclear. This study is the largest retrospective cohort to document the therapeutic efficacy and tolerability for three gradient doses of ACTH in IU/kg/day units in Chinese patients.ObjectiveThe aim of our study was to elucidate the effectiveness and safety of three different low doses and duration of ACTH treatment for IS in China.MethodsWe conducted a retrospective, chart review of IS cases that were treated with biologic short-acting ACTH and followed up for at least 6xa0months at a single center in China between June 2010 and June 2016. In total, 200 children met the inclusion criteria. Cases were divided into three groups according to dosage (1, 1.1–1.9, and 2–4xa0IU/kg/day). Furthermore, we divided the 2–4xa0IU/kg/day group into 2–3 and 3.1–4xa0IU/kg/day subgroups. All groups were evaluated for response rates, relapse rates, and adverse effects.ResultsElectroclinical remission by day 14 occurred in 41.4% of infants given 2–4xa0IU/kg/day and 36.4% of infants given 1.1–1.9xa0IU/kg/day, compared with only 14.7% of patients given 1xa0IU/kg/day (pxa0=xa00.004 and 0.03, respectively). Prolonging ACTH treatment for up to 28xa0days improved response by 24% in all 200 infants. Overall, 73.9% of infants receiving 2–4xa0IU/kg/day responded, significantly higher than the 52.7% responding to 1.1–1.9xa0IU/kg/day and the 23.5% responding to 1xa0IU/kg/day (pxa0<xa00.01). There was no significant difference in the number of relapses or adverse effects in the three groups. Moreover, in the 2–4xa0IU/kg/day group, 74.7% of children receiving 2–3xa0IU/kg/day of ACTH responded, compared with 70% who responded to 3.1–4xa0IU/kg/day (pxa0=xa00.78).ConclusionsACTH at a dosage of 2–3xa0IU/kg/day is superior to 1.1–1.9 and 1xa0IU/kg/day dosages, is as good as a 3.1–4xa0IU/kg/day dosage in terms of response rate, and causes no more adverse effects or relapses than other dosages. In addition, prolonging the duration of ACTH treatment can improve response.