Antitumor CD8 T cell responses in glioma patients are effectively suppressed by T follicular regulatory cells.

Regulatory T (Treg) cells are thought to contribute to tumor pathogenesis by suppressing tumor immunosurveillance and antitumor immunity. T follicular regulatory (Tfr) cells are a recently characterized Treg subset that expresses both the Treg transcription factor (TF) Foxp3 and the T follicular helper (Tfh) TF Bcl-6. The role of Tfr cells in glioma patients remains unclear. In this study, we found that the level of Tfr cells, identified as Foxp3+Bcl-6+ CD4 T cells, was significantly elevated in tumor-infiltrating CD4 T cells from resected glioma tumors. Both Tfr cells and Treg cells significantly suppressed the proliferation and the cytotoxic capacity of CD8 T cells toward glioma tumor cells, and the suppression was positively associated with the proportion of Tfr cells and Treg cells, respectively. Tfr and Treg cells from glioma tumor samples demonstrated higher suppression potency than those from healthy blood samples and glioma blood samples. Interestingly, canonical CXCR5- Treg cells could suppress both CXCR5+ and CXCR5- CD8 T cells, albeit with stronger potency toward CXCR5- CD8 T cells. However, Tfr cells presented much higher suppression potency toward CXCR5+ CD8 T cells, whereas CXCR5+ CD8 T cells are a potent CD8 T cell subset previously described to have antiviral and antitumor roles. Overall, these data indicate that Tfr cells are enriched in glioma tumors and have suppressive capacity toward CD8 T cell-mediated effector functions.

Circular RNA circ_0001588 sponges miR-211-5p to facilitate the progression of glioblastoma via up-regulating YY1 expression.

BACKGROUND: As the most common and detrimental brain tumor with high invasiveness and poor prognosis, glioblastoma (GBM) has severely threatened people's health globally. Therefore, it is of great importance and necessary to identify the molecular mechanisms involved in tumorigenesis and development, thus contributing to potential therapeutic targets and strategies. METHODS: The level of circ_0001588 was detected in 68 pairs of GBM tissues and adjacent normal tissues and human glioma cell lines via a real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Then, the effect of circ_0001588 on the proliferation, migration and invasion of glioma cells was evaluated. In addition, potential downstream targets of circ_0001588 were forecasted by circBANK and Starbase. Their interaction was confirmed by introducing luciferase reporter assays. Moreover, sh-circ_0001588 transfected U251 cells were used to form tumors in vivo. Finally, the functional mechanism of circ_0001588 was identified by qRT-PCR, western blotting, xenograft and immunohistochemistry (IHC) assays. RESULTS: The expression of circ_0001588 is markedly up-regulated in GBM tissues and human gliomas cells. Additionally, increased expression of circ_0001588 is positively relevant with poor survival in GBM patients. The down-regulation of circ_0001588 distinctly inhibits the proliferation, migration and invasion of GBM in vitro, as well as tumor growth in vivo. Moreover, knockdown of circ_0001588 reduces the tumor volume and weight, enhances the relative IHC staining index of E-cadherin and decreases the relative IHC staining index of Ki-67, Yin Yang 1 (YY1) and vinmentin in vivo. Mechanistically, circ_0001588 locates in the cytoplasm, which is directly bound with miR-211-5p. Furthermore, circ_0001588 can positively regulate YY1 via sponging miR-211-5p. Moreover, circ_0001588 accelerates the proliferation, migration and invasion of GBM by modulating miR-211-5p/YY1 signaling. CONCLUSIONS: These results illustrate a new circ_0001588/miR-211-5p/YY1 regulatory signaling axis in GBM.

Intrahepatic CD206+ macrophages contribute to inflammation in advanced viral-related liver disease.

BACKGROUND & AIMS: Liver inflammation is key in the progression of chronic viral hepatitis to cirrhosis and hepatocellular carcinoma. The magnitude of viral replication and the specific anti-viral immune responses should govern the degree of inflammation, but a direct correlation is not consistently found in chronic viral hepatitis patients. We aim to better define the mechanisms that contribute to chronic liver inflammation. METHODS: Intrahepatic CD14+ myeloid cells from healthy donors (n=19) and patients with viral-related liver cirrhosis (HBV, HBV/HDV or HCV; n=15) were subjected to detailed phenotypic, molecular and functional characterisation. RESULTS: Unsupervised analysis of multi-parametric data showed that liver disease was associated with the intrahepatic expansion of activated myeloid cells mainly composed of pro-inflammatory CD14+HLA-DRhiCD206+ cells, which spontaneously produced TNFα and GM-CSF. These cells only showed heightened pro-inflammatory responses to bacterial TLR agonists and were more refractory to endotoxin-induced tolerance. A liver-specific enrichment of CD14+HLA-DRhiCD206+ cells was also detected in a humanised mouse model of liver inflammation. This accumulation was abrogated following oral antibiotic treatment, suggesting a direct involvement of translocated gut-derived microbial products in liver injury. CONCLUSIONS: Viral-related chronic liver inflammation is driven by the interplay between non-endotoxin-tolerant pro-inflammatory CD14+HLA-DRhiCD206+ myeloid cells and translocated bacterial products. Deciphering this mechanism paves the way for the development of therapeutic strategies specifically targeting CD206+ myeloid cells in viral-related liver disease patients. Lay summary: Viral-related chronic liver disease is driven by intrahepatic pro-inflammatory myeloid cells accumulating in a gut-derived bacterial product-dependent manner. Our findings support the use of oral antibiotics to ameliorate liver inflammation in these patients.

Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection.

OBJECTIVE: HCV infection affects millions of people worldwide, and many patients develop chronic infection leading to liver cancers. For decades, the lack of a small animal model that can recapitulate HCV infection, its immunopathogenesis and disease progression has impeded the development of an effective vaccine and therapeutics. We aim to provide a humanised mouse model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. DESIGN: Recently, we have established human liver cells with a matched human immune system in NOD-scid Il2rg(-/-) (NSG) mice (HIL mice). These mice are infected with HCV by intravenous injection, and the pathologies are investigated. RESULTS: In this study, we demonstrate that HIL mouse is capable of supporting HCV infection and can present some of the clinical symptoms found in HCV-infected patients including hepatitis, robust virus-specific human immune cell and cytokine responses as well as liver fibrosis and cirrhosis. Similar to results obtained from the analysis of patient samples, the human immune cells, particularly T cells and macrophages, play critical roles during the HCV-associated liver disease development in the HIL mice. Furthermore, our model is demonstrated to be able to reproduce the therapeutic effects of human interferon alpha 2a antiviral treatment. CONCLUSIONS: The HIL mouse provides a model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. It could also serve as a platform for antifibrosis and immune-modulatory drug testing.

Induction of functional human macrophages from bone marrow promonocytes by M-CSF in humanized mice.

Engraftment of human CD34⁺ hematopoietic stem/progenitor cells into immunodeficient mice leads to robust reconstitution of human T and B cells but not monocytes and macrophages. To identify the cause underlying the poor monocyte and macrophage reconstitution, we analyzed human myeloid cell development in humanized mice and found that it was blocked at the promonocyte stage in the bone marrow. Expression of human M-CSF or GM-CSF by hydrodynamic injection of cytokine-encoding plasmid completely abolished the accumulation of promonocytes in the bone marrow. M-CSF promoted the development of mature monocytes and tissue-resident macrophages whereas GM-CSF did not. Moreover, correlating with an increased human macrophages at the sites of infection, M-CSF-treated humanized mice exhibited an enhanced protection against influenza virus and Mycobacterium infection. Our study identifies the precise stage at which human monocyte/macrophage development is blocked in humanized mice and reveals overlapping and distinct functions of M-CSF and GM-CSF in human monocyte and macrophage development. The improved reconstitution and functionality of monocytes/macrophages in the humanized mice following M-CSF expression provide a superior in vivo system to investigate the role of macrophages in physiological and pathological processes.

Evidence for Scgb1a1(+) cells in the generation of p63(+) cells in the damaged lung parenchyma.

Transformation-related protein 63-expressing (p63(+)) basal cells are confined to the trachea in the mouse lung. However, after influenza virus infection or bleomycin treatment, patches of p63(+) cells were observed in the damaged lung parenchyma. To address whether the newly induced p63(+) cells are derived from the p63(+) basal cells, we performed lineage tracing. In a keratin 5 promoter-driven CreER system, although preexisting p63(+) basal cells were labeled by enhanced green fluorescent protein (EGFP) after tamoxifen treatment, none or only a small fraction (∼ 15%) of the p63(+) patches was labeled by EGFP after bleomycin treatment or influenza virus infection, respectively. In contrast, > 60% of p63(+) patches contained EGFP(+) cells in Scgb1a1-CreER transgenic system where club cells are labeled. Many p63(+) cells were found in bronchiole-like lumen structures with columnar cells at the lumen side. The columnar cells were positive for club cell marker Cyp2f2 and could be traced to the newly induced p63(+) cells. These results suggest that most of the newly induced p63(+) cells in the damaged parenchyma are likely derived from club cells rather than from p63(+) basal cells and that newly induced p63(+) cells may be involved in the regeneration of bronchioles.

Aging exacerbates damage and delays repair of alveolar epithelia following influenza viral pneumonia.

BACKGROUND: Influenza virus infection causes significantly higher levels of morbidity and mortality in the elderly. Studies have shown that impaired immunity in the elderly contributes to the increased susceptibility to influenza virus infection, however, how aging affects the lung tissue damage and repair has not been completely elucidated. METHODS: Aged (16-18 months old) and young (2-3 months old) mice were infected with influenza virus intratracheally. Body weight and mortality were monitored. Different days after infection, lung sections were stained to estimate the overall lung tissue damage and for club cells, pro-SPC+ bronchiolar epithelial cells, alveolar type I and II cells to quantify their frequencies using automated image analysis algorithms. RESULTS: Following influenza infection, aged mice lose more weight and die from otherwise sub-lethal influenza infection in young mice. Although there is no difference in damage and regeneration of club cells between the young and the aged mice, damage to alveolar type I and II cells (AT1s and AT2s) is exacerbated, and regeneration of AT2s and their precursors (pro-SPC-positive bronchiolar epithelial cells) is significantly delayed in the aged mice. We further show that oseltamivir treatment reduces virus load and lung damage, and promotes pulmonary recovery from infection in the aged mice. CONCLUSIONS: These findings show that aging increases susceptibility of the distal lung epithelium to influenza infection and delays the emergence of pro-SPC positive progenitor cells during the repair process. Our findings also shed light on possible approaches to enhance the clinical management of severe influenza pneumonia in the elderly.

CD166(pos) subpopulation from differentiated human ES and iPS cells support repair of acute lung injury.

Previous efforts to derive lung progenitor cells from human embryonic stem (hES) cells using embryoid body formation or stromal feeder cocultures had been limited by low efficiencies. Here, we report a step-wise differentiation method to drive both hES and induced pluripotent stem (iPS) cells toward the lung lineage. Our data demonstrated a 30% efficiency in generating lung epithelial cells (LECs) that expresses various distal lung markers. Further enrichment of lung progenitor cells using a stem cell marker, CD166 before transplantation into bleomycin-injured NOD/SCID mice resulted in enhanced survivability of mice and improved lung pulmonary functions. Immunohistochemistry of lung sections from surviving mice further confirmed the specific engraftment of transplanted cells in the damaged lung. These cells were shown to express surfactant protein C, a specific marker for distal lung progenitor in the alveoli. Our study has therefore demonstrated the proof-of-concept of using iPS cells for the repair of acute lung injury, demonstrating the potential usefulness of using patient's own iPS cells to prevent immune rejection which arise from allogenic transplantation.

Influences and mechanisms of pyrolytic conditions on recycling BTX products from passenger car waste tires.

Pyrolysis is an effective method for waste tire disposal. However, it has rarely been used to recycle specific highly valuable components (such as benzene, toluene, and xylene (BTX)) from tire rubbers, owing to complicated pyrolytic reactions. This study investigated the pyrolysis process of passenger-car-waste-tires (PCWT) with the help of TG-DTG and Py-GC/MS. Based on response surface methodology (RSM), the effect of pyrolytic parameters on the yields of pyrolytic oil and BTX is evaluated. Furthermore, the BTX generation mechanisms are discussed from the perspective of aliphatic and aromatic hydrocarbon transformations. Additionally, pyrolytic conditions including temperature, rubber particle size, pressure, and gas flow rate were systemically investigated and the optimum pyrolytic condition for yield of BTX (26.5 g per 100 g tire rubber) was obtained [765 K, 0.7 mm, 0.52 MPa and 2.5 mL (g min)-1]. Therein, yield of benzene, toluene and xylene were 1.07, 5.03 and 20.40 g per 100 g tire rubber, respectively. During PCWT pyrolysis, BTX is primarily obtained via the Diels-Alder reactions of small-chain alkenes and transformations of limonene and aromatics. This study elucidates the BTX generation mechanisms during PCWT pyrolysis and clarifies the effects of varying pyrolytic conditions on BTX generation.

Formation Mechanism of Monocyclic Aromatic Hydrocarbons during Pyrolysis of Styrene Butadiene Rubber in Waste Passenger Car Tires.

The production of aromatic hydrocarbons from the waste tire pyrolysis attracts more and more attention because of its tremendous potential. Based on styrene-butadiene rubber (SBR), which is the main rubber in the waste passenger car tires, this work studies the temperature influence on primary pyrolysis product distribution by experimental techniques (Py-GC/MS, TG-MS), and then, the formation mechanism of monocyclic aromatic hydrocarbons (MAHs) observed in the experiment was analyzed by first-principles calculations. The experimental results show that the MAHs during the pyrolysis mainly include styrene, toluene, and xylene, and subsequent calculations showed that these compounds were formed through a series of primary and secondary reactions. The formation pathways of these typical MAHs were studied via the reaction energy barrier analysis, respectively. It shows that the MAHs were not only derived from the benzene ring in the SBR chain but also generated from short-chain alkenes through the Diels-Alder reaction. The obtained pyrolysis reaction mechanism provides theoretical guidance for the regulation of the pyrolysis product distribution of MAHs.

Study of passenger-car-waste-tire pyrolysis: Behavior and mechanism under kinetical regime.

The pyrolysis of passenger-car-waste-tires (PCWT) has recently attracted widespread attention because it is a highly effective disposal method. However, a comprehensive understanding of real tire pyrolytic processes is limited owing to the complicated PCWT pyrolysis reaction system, particularly regarding the reaction mechanism. This study investigated the PCWT pyrolytic processes using a thermogravimetric analyzer coupled with mass spectrometry and analyzed all the pyrolytic products using pyrolysis-gas chromatography coupled with mass spectrometry. The composition and distribution of the PCWT pyrolytic products were investigated under a kinetic regime to eliminate other influences on the intrinsic reaction. The pyrolytic products mainly consisted of chain and cyclic alkenes, and monocylic aromatics. Importantly, an integral pyrolytic mechanism network for the PCWT was established based on the pyrolysis of single rubbers (natural, styrene butadiene, and butadiene rubbers). The reaction routes for the main products were determined according to the mechanism. Moreover, a kinetic study of the PCWT pyrolysis revealed the activation energy for this complicated reaction system.

LncRNA MACC1-AS1/MACC1 enhances the progression of glioma via regulating metabolic plasticity.

This study plans to investigate the effects of long-noncoding RNA MACC1-AS1 on glioma cells and its mechanism at metabolic plasticity angle. The MACC1-AS1 level was identified both in glioma tissues and in cells. Then the effects of MACC1-AS1 abnormal level on cell viability, apoptosis, the expression of apoptosis associated protein, glucose metabolism and redox status were measured in A172 and U251 cells by different methods. Furthermore, the interaction of MACC1-AS1 and MACC1 in glioma cells was investigated and the role of AMPK pathway was specifically examined. Our results demonstrated that MACC1-AS1 level was high in glioma tissues and cells, and MACC1-AS1 overexpression was closely associated with poor prognosis of glioma. Notably, under glucose deprivation, the MACC1-AS1 level was significantly increased, and overexpression of MACC1-AS1 increased cell viability but inhibited apoptosis. Also, MACC1-AS1 overexpression obviously increased the levels of GLUT1, HK2, G6PD, MCT1, ATP, lactate and NAPDH as well as promoted the activities of HK2 and LDHA, while reduced ROS level and the ratio of NADP+/NAPDH. In particular, the effects of proliferation, apoptosis and metabolic plasticity of glioma cells caused by MACC1-AS1 overexpression were achieved by positively regulating MACC1, and MACC1-AS1 promoted MACC1 expression via the AMPK pathway. In conclusions, the MACC1-AS1/MACC1 axis exertes the tumor-promoting effect by regulating glucose metabolism and redox homeostasis in glioma cells by activating the AMPK signals.

Expression of TRPC3 in cortical lesions from patients with focal cortical dysplasia.

Focal cortical dysplasia (FCD) is one of the main causes of medically intractable epilepsy. Some studies have reported that transient receptor potential canonical channel 3 (TRPC3) may play an important role in the occurrence of seizures. In this study, we investigated the expression patterns of TRPC3 in different types of FCD. Forty-five FCD specimens and 12 control samples from autopsies were used in our study. Western blotting, immunohistochemistry, and immunofluorescence staining were employed to detect protein expression and distribution. The amount of TRPC3 protein was markedly elevated in the FCD group. The immunohistochemistry results revealed that TRPC3 staining was strong in the malformed cells and microcolumns. Most of the TRPC3-positive cells were colabeled with glutamatergic and GABAergic markers. The overexpression and altered cellular distribution of TRPC3 in the FCD samples suggest that TRPC3 may be related to epileptogenesis in FCD.

LncRNA NNT-AS1 promote glioma cell proliferation and metastases through miR-494-3p/PRMT1 axis.

Long noncoding RNAs (lncRNAs) are key players in cancer progression. However, the function of lncRNA NNT-AS1 on glioma is unclear. In the present study, a total of 73 tumor tissues and matched adjacent non-tumor tissues were collected, and glioma cell lines were cultured in vitro. mRNA expression was tested using RT-qPCR. The protein expression level was determined using the western blot assay, cell proliferation was measured using the CCK-8 and BrdU proliferation assay, and the cell cycle, cell migration and invasion were determined using flow cytometry analysis, the wound healing assay and transwell, respectively. The results showed that lncNNT-AS1 is significantly up-regulated during the early stages of glioma. In particular, high levels of NNT-AS1 are observed in glioma cell lines compared to human astrocyte (HA) cells. Furthermore, the inhibition of lnc-NNT-AS1 by siRNA interfere attenuates the cell viability, proliferation, migration and invasion of glioma cell lines. Mechanistically, the inhibition of NNT-AS1 directly interacted with miRNA-494-3p, and positively regulated the downstream target PRMT1 in vitro. Further study proved that the overexpression of miRNA-494-3p and the inhibition of PRMT1 could attenuate both glioma cell proliferation and metastases. Collectively, our results indicated that the miR-494-3p-PRMT1 axis is involved the tumor-suppressive effects of NNT-AS1 inhibition, which sheds new light on lncRNA-directed diagnostics and the therapeutics of glioma.

WBSCR22 confers cell survival and predicts poor prognosis in glioma.

Human WBSCR22 is involved in cancer proliferation, invasion and metastasis; however, its function in glioma remains unexplored. In our research, we aimed to investigate the role of WBSCR22 in the development of glioma and its possible molecular mechanisms. Using bioinformatic analysis of public datasets, we determined that WBSCR22 overexpression in glioma specimens was correlated with an unfavorable patient prognosis. Our results revealed that WBSCR22 was highly expressed in glioma cell lines. The loss of WBSCR22 inhibited the growth, invasion and migration of glioma cells, while WBSCR22 overexpression produced the opposite effects. Moreover, we found that WBSCR22 downregulation reduced the phosphorylation of Akt and GSK3ß and decreased the levels of ß-catenin and CyclinD1 in glioma cells. The opposite effects were observed when WBSCR22 was overexpressed. Additionally, we verified with a dual-luciferase reporter assay that WBSCR22 was a direct target of miR-146b-5p. Furthermore, overexpression of miR-146b-5p suppressed WBSCR22 mRNA and protein expression. Notably, the restoration of WBSCR22 expression remarkably reversed the effects of miR-146b-5p overexpression on cell survival, apoptosis and the cell cycle in glioma cells. Collectively, our findings revealed a tumor-promoting role for WBSCR22 in glioma cells, thus providing molecular evidence for WBSCR22 as a novel therapeutic target in glioma.

Galangin inhibits epithelial-mesenchymal transition and angiogenesis by downregulating CD44 in glioma.

Galangin (3,5,7­trihydroxyflavone), a natural flavonoid present in plants, has been reported to possess anticancer properties in various types of cancers comprising glioma. The underlying mechanism, however, has not been fully elucidated. CD44, a hall marker in glioma, has been reported to be associated with epithelial-mesenchymal transition (EMT) and angiogenesis, which play important roles in glioma progression. In this study, we aimed to investigate whether galangin can inhibit EMT, angiogenesis and CD44 expression in glioma. We observed that galangin inhibited the proliferation, migration, invasion and angiogenesis of glioma cells in a dose-dependent manner, suppressed the expression of CD44 and inhibited angiogenesis of glioma cells through downregulating vascular endothelial growth factor (VEGF) in HUVECs. In addition, the overexpression of CD44 in U87 and U251 cells partly abolished the effects of galangin on glioma cells. Moreover, galangin suppressed tumor growth in an intracranial glioma mouse model. These results indicate that galangin is a potential novel drug for glioblastoma treatment due to its ability to suppress of CD44, EMT and angiogenesis.

Culture and Differentiation of Lung Bronchiolar Epithelial Cells In Vitro.

Club cells are a major bronchiolar epithelial cell type in the lung. Using genetic lineage tracing in mice and in vitro culture of purified cells, we have shown that club cells can differentiate into alveolar type I and II cells. Here we describe the detailed protocol for culturing and differentiating club cells in 3-dimensional culture.

Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro.

Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures. This differentiation was further confirmed by transcriptomic analysis of freshly isolated club cells and their cultured progeny. Freshly isolated club cells express Sca-1 and integrin α6, markers commonly used to characterize lung stem/progenitor cells. Together, current study for the first time isolated highly purified club cells for in vitro study and demonstrated club cells' capacity to differentiate into alveolar epithelial cells at the single-cell level.

Metabolomics Investigation Reveals Metabolite Mediators Associated with Acute Lung Injury and Repair in a Murine Model of Influenza Pneumonia.

Influenza virus infection (IVI) can cause primary viral pneumonia, which may progress to acute lung injury (ALI) and respiratory failure with a potentially fatal outcome. At present, the interactions between host and influenza virus at molecular levels and the underlying mechanisms that give rise to IVI-induced ALI are poorly understood. We conducted a comprehensive mass spectrometry-based metabolic profiling of serum, lung tissue and bronchoalveolar lavage fluid (BALF) from a non-lethal mouse model with influenza A virus at 0, 6, 10, 14, 21 and 28 days post infection (dpi), representing the major stages of IVI. Distinct metabolite signatures were observed in mice sera, lung tissues and BALF, indicating the molecular differences between systematic and localized host responses to IVI. More than 100 differential metabolites were captured in mice sera, lung tissues and BALF, including purines, pyrimidines, acylcarnitines, fatty acids, amino acids, glucocorticoids, sphingolipids, phospholipids, etc. Many of these metabolites belonged to pulmonary surfactants, indicating IVI-induced aberrations of the pulmonary surfactant system might play an important role in the etiology of respiratory failure and repair. Our findings revealed dynamic host responses to IVI and various metabolic pathways linked to disease progression, and provided mechanistic insights into IVI-induced ALI and repair process.

Expression of TRPC6 and BDNF in Cortical Lesions From Patients With Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) likely results from abnormal migration of neural progenitor cells originating from the subventricular zone. To elucidate the roles in molecules that are involved in neural migration pathway abnormalities in FCDs, we investigated the expression patterns of transient receptor potential canonical channel 6 (TRPC6) and brain-derived neurotrophic factor (BDNF) in cortical lesions from FCD patients and in samples of normal control cortex. TRPC6 and BDNF mRNA and protein levels were increased in FCD lesions. By immunohistochemistry, they were strongly expressed in microcolumns, heterotopic neurons, dysmorphic neurons, and balloon cells (BCs). Colocalization assays revealed that most of the misshapen TRPC6-positive or heterotopic cells had a neuronal lineage with the exception of TRPC6-positive FCDiib patient BCs, which had both neuronal and glial features. Most TRPC6-positive cells were glutamatergic neurons. There was also greater expression of calmodulin-dependent kinase IV (CaMKIV), the downstream factor of TRPC6, in FCD lesions, suggesting that TRPC6 expression promoted dendritic growth and the development of dendritic spines and excitatory synapses via the CaMKIV-CREB pathway in FCD. Thus, overexpression of BDNF and TRPC6 and activation of the TRPC6 signal transduction pathway in cortical lesions of FCD patients may contribute to FC pathogenesis and epileptogenesis.