A distinct clinicopathological variant of focal cortical dysplasia IIId characterized by loss of layer 4 in the occipital lobe in 12 children with remote hypoxic-ischemic injury.

OBJECTIVE: In 2011, the International League Against Epilepsy (ILAE) proposed a consensus classification system of focal cortical dysplasia (FCD) to distinguish clinicopathological subtypes, for example, "isolated" FCD type Ia-c and IIa-b, versus "associated" FCD type IIIa-d. The histopathological differentiation of FCD type I and III variants remains, however, a challenging issue in everyday practice. We present a unique histopathological pattern in patients with difficult-to-diagnose FCD, which highlights this dilemma, but also helps to refine the current ILAE classification scheme of FCD. METHODS: We present a retrospective series of 11 male and one female patient with early onset pharmacoresistant epilepsy of the posterior quadrant (mean age at seizure onset = 4.6 years). All surgical specimens were reviewed. Clinical histories were retrieved and extracted from archival patient files. RESULTS: Microscopic inspection revealed abnormalities in cortical architecture with complete loss of layer 4 in all surgical samples of the occipital lobe, as confirmed by semiquantitative measurements (p < 0.01). Clinical history reported early transient hypoxic condition in nine patients (75%). Magnetic resonance imaging (MRI) revealed abnormal signals in the occipital lobe in all patients, and signal changes suggestive of subcortical encephalomalacia were found in seven patients. Surgical treatment achieved favorable seizure control (Engel class I and II) in seven patients with an available follow-up period of 6.1 years. SIGNIFICANCE: Prominent disorganization of cortical layering and lack of any other microscopically visible principle lesion in the surgical specimen would result in this neuropathological pattern hitherto being classified as FCD ILAE type Ib. However, perinatal hypoxia with distinctive MRI changes suggested primarily a hypoxemic lesion and acquired pathomechanism of neuronal cell loss in the occipital lobe of our patient series. We propose, therefore, classifying this distinctive clinicopathological pattern as a separate variant of FCD ILAE type IIId.

Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation.

Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage beta-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet beta-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.

Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors.

Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone single-cell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1- or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia.

High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells.

Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes.

In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow.

Efforts toward routine islet cell transplantation as a means for reversing type 1 diabetes have been hampered by islet availability as well as allograft rejection. In vitro transdifferentiation of mouse bone marrow (BM)-derived stem (mBMDS) cells into insulin-producing cells could provide an abundant source of autologous cells for this procedure. For this study, we isolated and characterized single cell-derived stem cell lines obtained from mouse BM. In vitro differentiation of these mBMDS cells resulted in populations meeting a number of criteria set forth to define functional insulin-producing cells. Specifically, the mBMDS cells expressed multiple genes related to pancreatic beta-cell development and function (insulin I and II, Glut2, glucose kinase, islet amyloid polypeptide, nestin, pancreatic duodenal homeobox-1 [PDX-1], and Pax6). Insulin and C-peptide production was identified by immunocytochemistry and confirmed by electron microscopy. In vitro studies involving glucose stimulation identified glucose-stimulated insulin release. Finally, these mBMDS cells transplanted into streptozotocin-induced diabetic mice imparted reversal of hyperglycemia and improved metabolic profiles in response to intraperitoneal glucose tolerance testing. These results indicate that mouse BM harbors cells capable of in vitro transdifferentiating into functional insulin-producing cells and support efforts to derive such cells in humans as a means to alleviate limitations surrounding islet cell transplantation.

Comparison of the effects of polysaccharides from wood-cultured and bag-cultured Ganoderma lucidum on murine spleen lymphocyte proliferation in vitro.

AIM: To compare the influences of wood-cultured Ganoderma lucidum polysaccharides (Gl-PS-WC) and bag-cultured Ganoderma lucidum polysaccharides (Gl-PS-BC) on the proliferation activities of murine spleen lymphocytes in vitro, and investigate whether Gl-PS-BC can be substituted for Gl-PS-WC. METHODS: Mixed lymphocyte culture (MLC) reaction, lymphocyte proliferation induced by concanavalin A (Con A, 1 mg.L-1) or lipopolysaccharide (LPS, 5 mg.L-1), MLC reactions inhibited by immunosuppressive drugs, cyclosporine A (CsA, 0.1 mg.L-1), mitomycin (Mit C, 0.1 mg.L-1), or antitumor drug, etoposide (VP-16, 0.1 mg.L-1), were detected in the presence or absence of Gl-PS-WC and Gl-PS-BC in the concentration range of 0.2-12.8 mg.L-1. RESULTS: Two kinds of polysaccharides were shown to promote MLC in the range of 0.2-12.8 mg.L-1, increase lymphocyte proliferation induced by Con A or LPS and antagonize the inhibitory effects of CsA, Mit C or VP-16 on MLC. No significant difference was observed between these two kinds of polysaccharides in selected concentrations. CONCLUSION: Gl-PS-WC and Gl-PS-BC showed similar effects on the proliferation activities of murine spleen lymphocytes in vitro.

Regulatory effect of Ganoderma lucidum polysaccharides on cytotoxic T-lymphocytes induced by dendritic cells in vitro.

AIM: To study the regulatory effects of Ganoderma lucidum polysaccharides (Gl-PS) on cytotoxicity and mechanism of specific cytotoxic T-lymphocytes (CTL) induced by dendritic cells (DC) in vitro during the stage of antigen presentation. METHODS: Cultured murine bone marrow-derived DC were pulsed with P815 tumor cell lysates and co-incubated with or without various concentrations of Gl-PS (0.8, 3.2, or 12.8 mg/L) at the same time. P815 specific CTL were induced by spleen lymphocytes stimulated with mature DC. Non-adherent cells and culture supernatants were harvested on d 5 for analysis of specific cytotoxicity with lactate dehydrogenase (LDH) activity assay, mRNA expression of IFNgamma, granzyme B with RT-PCR assay, and protein expression of IFNgamma, granzyme B with ELISA or Western blot assay, respectively. RESULTS: Three concentrations of Gl-PS promoted LDH activities released into culture supernatants (P<0.01). It also increased mRNA expression of IFNgamma in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) and granzyme B in CTL (P<0.01). Protein production of IFNgamma in culture supernatants (P<0.05) and protein expression of granzyme B in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) were also augmented by Gl-PS. CONCLUSION: Gl-PS is shown to promote the cytotoxicity of specific CTL induced by DC which were pulsed with P815 tumor antigen during the stage of antigen presentation, and the mechanism of cytotoxicity is believed to be going through IFNgamma and granzyme B pathways.

Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides.

Ganoderma lucidum polysaccharides (Gl-PS) exhibits a variety of immunomodulatory activities, and dendritic cells (DC) are professional antigen presenting cells, which are pivotal for initiation of primary immune response. In this study, the regulatory effects of Gl-PS on maturation and function of cultured murine bone marrow derived DC were investigated in vitro. Gl-PS (0.8, 3.2, or 12.8 microg/ml) could increase the co-expression of CD11c and I-A/I-E molecules on DC surface, promote mRNA expression of cytokine IL-12 p40 in DC and augment protein production of IL-12 P40 in culture supernatants. The lymphocyte proliferation of mixed lymphocyte culture (MLC) induced by mature DC was enhanced by Gl-PS, either. Gl-PS was shown to promote not only the maturation of cultured murine bone marrow derived DC in vitro, but also the immune response initiation induced by DC.