Identification of Correlative Shifts in Indices of Brain Cholesterol Metabolism in the C57BL6/Mecp2tm1.1Bird Mouse,a Model for Rett Syndrome

Rett syndrome (RS) is a pervasive neurodevelopmental disorder resulting from loss‐of‐function mutations in the X‐linked gene methyl‐Cpg‐binding protein 2 (MECP2). Using a well‐defined model for RS, the C57BL6/Mecp2^tm1.1Bird mouse, we have previously found a moderate but persistently lower rate of cholesterol synthesis, measured in vivo, in the brains of Mecp2^?/y mice, starting from about the third week after birth. There was no genotypic difference in the total cholesterol concentration throughout the brain at any age. This raised the question of whether the lower rate of cholesterol synthesis in the mutants was balanced by a fall in the rate at which cholesterol was converted via cholesterol 24‐hydroxylase (Cyp46A1) to 24S‐hydroxycholesterol (24S‐OHC), the principal route through which cholesterol is ordinarily removed from the brain. Here, we show that while there were no genotypic differences in the concentrations in plasma and liver of three cholesterol precursors (lanosterol, lathosterol, and desmosterol), two plant sterols (sitosterol and campesterol), and two oxysterols (27‐hydroxycholesterol [27‐OHC] and 24S‐OHC), the brains of the Mecp2 ^?/y mice had significantly lower concentrations of all three cholesterol precursors, campesterol, and both oxysterols, with the level of 24S‐OHC being ~20% less than in their Mecp2 ^+/y controls. Together, these data suggest that coordinated regulation of cholesterol synthesis and catabolism in the central nervous system is maintained in this model for RS. Furthermore, we speculate that the adaptive changes in these two pathways conceivably resulted from a shift in the permeability of the blood–brain barrier as implied by the significantly lower campesterol and 27‐OHC concentrations in the brains of the Mecp2^?/y mice.     

Peptide Antibody Reactivity to Homologous Regions in Glutamate Decarboxylase Isoforms and Coxsackievirus B4 P2C

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.     

Dual-Function Semaphorin 4D Released by Platelets: Suppression of Osteoblastogenesis and Promotion of Osteoclastogenesis

Effects of the antiosteoblastogenesis factor Semaphorin 4D (Sema4D), expressed by thrombin-activated platelets (TPs), on osteoblastogenesis, as well as osteoclastogenesis, were investigated in vitro. Intact platelets released both Sema4D and IGF-1. However, in response to stimulation with thrombin, platelets upregulated the release of Sema4D, but not IGF-1. Anti-Sema4D-neutralizing monoclonal antibody (mAb) upregulated TP-mediated osteoblastogenesis in MC3T3-E1 osteoblast precursors. MC3T3-E1 cells exposed to TPs induced phosphorylation of Akt and ERK further upregulated by the addition of anti-sema4D-mAb, suggesting the suppressive effects of TP-expressing Sema4D on osteoblastogenesis. On the other hand, TPs promoted RANKL-mediated osteoclastogenesis in the primary culture of bone-marrow-derived mononuclear cells (BMMCs). Among the known three receptors of Sema4D, including Plexin B1, Plexin B2 and CD72, little Plexin B2 was detected, and no Plexin B1 was detected, but a high level of CD72 mRNA was detected in RANKL-stimulated BMMCs by qPCR. Both anti-Sema4D-mAb and anti-CD72-mAb suppressed RANKL-induced osteoclast formation and bone resorptive activity, suggesting that Sema4D released by TPs promotes osteoclastogenesis via ligation to a CD72 receptor. This study demonstrated that Sema4D released by TPs suppresses osteogenic activity and promotes osteoclastogenesis, suggesting the novel property of platelets in bone-remodeling processes.     

Semaphorin 6C Suppresses Proliferation of Pancreatic Cancer Cells via Inhibition of the AKT/GSK3/β-Catenin/Cyclin D1 Pathway

Semaphorins (SEMAs) are axon guidance factors that participate in axonal connections and nerve system development. However, the functional roles of SEMAs in tumorigenesis are still largely uncovered. By using in silico data analysis, we found that SEMA6C was downregulated in pancreatic cancer, and its reduction was correlated with worse survival rates. RNA sequencing revealed that cell cycle-related genes, especially cyclin D1, were significantly altered after blockage of SEMA6C by neutralizing antibodies or ectopic expressions of SEMA6C. Mechanistic investigation demonstrated that SEMA6C acts as a tumor suppressor in pancreatic cancer by inhibiting the AKT/GSK3 signaling axis, resulting in a decrease in cyclin D1 expression and cellular proliferation. The enhancement of cyclin D1 expression and cyclin-dependent kinase activation in SEMA6C-low cancer created a druggable target of CDK4/6 inhibitors. We also elucidated the mechanism underlying SEMA6C downregulation in pancreatic cancer and demonstrated a novel regulatory role of miR-124-3p in suppressing SEMA6C. This study provides new insights of SEMA6C-mediated anti-cancer action and suggests the treatment of SEMA6C-downregulated cancer by CDK4/6 inhibitors.     

Delayed Ventricular Repolarization and Sodium Channel Current Modification in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a severe developmental disorder that is strongly linked to mutations in the MECP2 gene. RTT has been associated with sudden unexplained death and ECG QT interval prolongation. There are mixed reports regarding QT prolongation in mouse models of RTT, with some evidence that loss of Mecp2 function enhances cardiac late Na current, I_Na,Late. The present study was undertaken in order to investigate both ECG and ventricular AP characteristics in the Mecp2^Null/Y male murine RTT model and to interrogate both fast I_Na and I_Na,Late in myocytes from the model. ECG recordings from 8–10-week-old Mecp2^Null/Y male mice revealed prolongation of the QT and rate corrected QT (QTc) intervals and QRS widening compared to wild-type (WT) controls. Action potentials (APs) from Mecp2^Null/Y myocytes exhibited longer APD₇₅ and APD₉₀ values, increased triangulation and instability. I_Na,Late was also significantly larger in Mecp2^Null/Y than WT myocytes and was insensitive to the Nav1.8 inhibitor A-803467. Selective recordings of fast I_Na revealed a decrease in peak current amplitude without significant voltage shifts in activation or inactivation V_0.5. Fast I_Na ‘window current’ was reduced in RTT myocytes; small but significant alterations of inactivation and reactivation time-courses were detected. Effects of two I_Na,Late inhibitors, ranolazine and GS-6615 (eleclazine), were investigated. Treatment with 30 µM ranolazine produced similar levels of inhibition of I_Na,Late in WT and Mecp2^Null/Y myocytes, but produced ventricular AP prolongation not abbreviation. In contrast, 10 µM GS-6615 both inhibited I_Na,Late and shortened ventricular AP duration. The observed changes in I_Na and I_Na,Late can account for the corresponding ECG changes in this RTT model. GS-6615 merits further investigation as a potential treatment for QT prolongation in RTT.     

Over-Expression of Semaphorin4D, Hypoxia-Inducible Factor-1�� and Vascular Endothelial Growth Factor Is Related to Poor Prognosis in Ovarian Epithelial Cancer

Semaphorin4D (SEMA4D) has been regarded as an important protein in tumor angiogenesis, though originally identified in neurodevelopment. SEMA4D is extensively expressed in several malignant solid tumors. Nevertheless, the function and expression of SEMA4D in epithelial ovarian cancer (EOC) is as yet not well understood. The aim of this study was to investigate SEMA4D expression in EOC and evaluate its clinical–pathological and prognostic significance. Immunohistochemistry was used to analyze SEMA4D expression and tumor angiogenesis-related proteins (HIF-1α and VEGF) in tissues from 40 patients with normal ovarian epithelia and 124 EOC patients. SEMA4D was found to be expressed in 61.3% of the 124 EOC tissues, which was significantly higher than in the normal ovarian epithelia (p < 0.001). SEMA4D expression correlated with HIF-1α and VEGF closely (ρ = 0.349 and 0.263, p < 0.001). Positive SEMA4D staining was significantly higher in tissues from patients with low histological grade, FIGO stage III-IV, lymph node metastasis and residual disease ≥1 cm (p < 0.05). In the Cox proportional hazard mode, SEMA4D expression and histologic grade were independent indicators of overall survival (OS) and progress-free survival (PFS) for EOC patients. These findings suggest that the cooperation of SEMA4D, HIF-1α, and VEGF may indicate poor prognosis for patients with EOC, thereby demonstrating that SEMA4D and its role in angiogenesis in EOC warrants further study.     

Identifying Function Determining Residues in Neuroimmune Semaphorin 4A

Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.     

抗旋毛虫硒蛋白T单克隆抗体的制备及其对裸鼠肝癌H7402细胞的抑制效果

目的制备抗旋毛虫与肝癌H7402细胞相关抗原硒蛋白T单克隆抗体,并观察其对裸鼠肝癌H7402细胞的抑制效果。方法用纯化的旋毛虫硒蛋白T重组蛋白常规免疫BALB/c小鼠,采用杂交瘤细胞技术制备其单克隆抗体;将肝癌H7402细胞经大腿内侧皮下接种BALB/c裸鼠,1×10^7个细胞/只,采用制备的高效价单抗,按20、50及100μg/只剂量接种裸鼠,隔日1次,共5次,评价单抗对裸鼠肝癌H7402细胞的抑制效果。结果成功获得了3株稳定分泌抗旋毛虫硒蛋白T的单抗的细胞株:4D4、4H2及4F1,效价均达1∶204800以上,其中4H2抗体效价最高;20、50及100μg/只剂量组的抑瘤率分别为51.6%、56.2%和80.3%。结论制备的抗旋毛虫与肝癌H7402细胞相关抗原硒蛋白T单克隆抗体对裸鼠体内肝癌H7402细胞具有明显的抑制效果。     

Platelet-Released Growth Factors Influence Wound Healing-Associated Genes in Human Keratinocytes and Ex Vivo Skin Explants

Platelet-released growth factors (PRGFs) or other thrombocyte concentrate products, e.g., Platelet-Rich Fibrin (PRF), have become efficient tools of regenerative medicine in many medical disciplines. In the context of wound healing, it has been demonstrated that treatment of chronic or complicated wounds with PRGF or PRF improves wound healing in the majority of treated patients. Nevertheless, the underlying cellular and molecular mechanism are still poorly understood. Therefore, we aimed to analyze if PRGF-treatment of human keratinocytes caused the induction of genes encoding paracrine factors associated with successful wound healing. The investigated genes were Semaphorin 7A (SEMA7A), Angiopoietin-like 4 (ANGPLT4), Fibroblast Growth Factor-2 (FGF-2), Interleukin-32 (IL-32), the CC-chemokine-ligand 20 (CCL20), the matrix-metalloproteinase-2 (MMP-2), the chemokine C-X-C motif chemokine ligand 10 (CXCL10) and the subunit B of the Platelet-Derived Growth Factor (PDGFB). We observed a significant gene induction of SEMA7A, ANGPLT4, FGF-2, IL-32, MMP-2 and PDGFB in human keratinocytes after PRGF treatment. The CCL20- and CXCL10 gene expressions were significantly inhibited by PRGF therapy. Signal transduction analyses revealed that the PRGF-mediated gene induction of SEMA7A, ANGPLT4, IL-32 and MMP-2 in human keratinocytes was transduced via the IL-6 receptor pathway. In contrast, EGF receptor signaling was not involved in the PRGF-mediated gene expression of analyzed genes in human keratinocytes. Additionally, treatment of ex vivo skin explants with PRGF confirmed a significant gene induction of SEMA7A, ANGPLT4, MMP-2 and PDGFB. Taken together, these results describe a new mechanism that could be responsible for the beneficial wound healing properties of PRGF or related thrombocytes concentrate products such as PRF.     

灵芝精粉与灵芝孢子粉对Lewis肺癌模型小鼠免疫功能的影响

目的研究灵芝精粉与灵芝孢子粉对Lewis肺癌模型小鼠免疫功能的影响,并比较二者在肿瘤治疗中的免疫调节作用。方法经C57BL/6小鼠右腋皮下接种1×106个Lewis肺癌细胞,建立小鼠Lewis肺癌模型。于接种肿瘤细胞次日至第20天,分别隔日灌胃灵芝孢子粉(D1)、灵芝精粉001(D2)及灵芝精粉002(D3),剂量均为0.5g/kg,同时设立对照组和肿瘤组(灌胃等体积生理盐水)。监测各组小鼠体重及瘤体积,第21日处死小鼠,称量瘤重,并无菌制备脾细胞悬液,检测小鼠NK细胞杀伤活性,流式细胞术检测小鼠脾细胞中CD4+、CD8+T细胞和B细胞数量,淋巴细胞转化试验检测脾细胞中T、B细胞增殖功能。结果与对照组比较,肿瘤组小鼠脾细胞中T、B细胞增殖功能、细胞数量及NK细胞杀伤活性均有不同程度的下降。与肿瘤组相比,3种灵芝制剂均可提高Lewis肺癌模型小鼠NK细胞的杀伤活性和T、B细胞增殖功能,其中,以D2作用最为明显(P<0.05);3种灵芝制剂均可不同程度地提高Lewis肺癌模型小鼠CD4+、CD8+T细胞数量,其中,以D3作用最为显著(P<0.05)。3种灵芝制剂对Lewis肺癌模型小鼠的体重、瘤重、瘤体积及B细胞数量均无明显影响。结论灵芝制剂对Lewis肺癌小鼠的免疫功能具有正向调节作用,其中灵芝精粉的作用优于灵芝孢子粉。     

东方马脑炎病毒E2蛋白的表达、纯化及免疫原性

目的表达、纯化东方马脑炎病毒(eastern equine encephalitis virus,EEEV)E2蛋白,并检测其对小鼠的免疫原性。方法利用IPTG诱导重组大肠埃希菌BL21-pET30-EEEV-E2,表达E2蛋白,用包涵体纯化试剂盒纯化重组E2蛋白,进行SDS-PAGE和Western blot分析。将BALB/c小鼠随机分为4组:PBS对照组、弗氏佐剂对照组(弗氏佐剂与PBS按体积比1∶1乳化)、E2蛋白组(E2蛋白与PBS按体积比1∶1混合)和E2蛋白+弗氏佐剂组(E2蛋白与弗氏佐剂按体积比1∶1乳化),每组10只,各组均经小鼠后肢肌肉免疫3次,两次免疫间隔时间均为14 d,免疫剂量均为100μl/只。第2次免疫后第7天,采用流式细胞术检测小鼠体内CD4+和CD8+T细胞比例;第2次免疫后第14天,采用细胞因子ELISA定量试剂盒检测小鼠血清中IL-2、IL-4和IFNγ的含量;第3次免疫后第7天,采用MTT法检测小鼠淋巴细胞增殖情况;每次免疫后第14天,采用ELISA法检测小鼠血清中IgG抗体效价。结果表达的重组E2蛋白相对分子质量约为53 000,表达量为菌体总蛋白的26.3%;纯化的重组E2蛋白纯度达95%以上;表达和纯化的重组E2蛋白均可与鼠抗His标签单克隆抗体结合。与PBS对照组、弗氏佐剂对照组和E2蛋白组相比,E2蛋白+弗氏佐剂组小鼠体内CD4+与CD8+T细胞比值、血清中IL-2、IL-4和IFNγ浓度、体内淋巴细胞增殖指数均明显升高(P﹤0.01);小鼠初次免疫后即可产生E2蛋白IgG抗体,且随着免疫时间的延长,抗体效价逐渐上升,第3次免疫后第14天,抗体效价可达1∶320。结论表达并纯化了重组E2蛋白,其能使小鼠产生较强的免疫反应,为新型EEEV疫苗的研制提供了参考。     

Alpha-Lipoic Acid Inhibits Spontaneous Diabetes and Autoimmune Recurrence in Non-Obese Diabetic Mice by Enhancing Differentiation of Regulatory T Cells and Showed Potential for Use in Cell Therapies for the Treatment of Type 1 Diabetes

Type 1 diabetes (T1D) is caused by the destruction of β cells in pancreatic islets by autoimmune T cells. Islet transplantation has been established as an effective treatment for T1D. However, the survival of islet grafts is often disrupted by recurrent autoimmunity. Alpha-lipoic acid (ALA) has been reported to have immunomodulatory effects and, therefore, may have therapeutic potential in the treatment of T1D. In this study, we investigated the therapeutic potential of ALA in autoimmunity inhibition. We treated non-obese diabetic (NOD) mice with spontaneous diabetes and islet-transplantation mice with ALA. The onset of diabetes was decreased and survival of the islet grafts was extended. The populations of Th1 cells decreased, and regulatory T cells (Tregs) increased in ALA-treated mice. The in vitro Treg differentiation was significantly increased by treatment with ALA. The adoptive transfer of ALA-differentiated Tregs into NOD recipients improved the outcome of the islet grafts. Our results showed that in vivo ALA treatment suppressed spontaneous diabetes and autoimmune recurrence in NOD mice by inhibiting the Th1 immune response and inducing the differentiation of Tregs. Our study also demonstrated the therapeutic potential of ALA in Treg-based cell therapies and islet transplantation used in the treatment of T1D.     

Long,Noncoding RNA SRA Induces Apoptosis of β-Cells by Promoting the IRAK1/LDHA/Lactate Pathway

Long non-coding RNA steroid receptor RNA activators (LncRNA SRAs) are implicated in the β-cell destruction of Type 1 diabetes mellitus (T1D), but functional association remains poorly understood. Here, we aimed to verify the role of LncRNA SRA regulation in β-cells. LncRNA SRAs were highly expressed in plasma samples and peripheral blood mononuclear cells (PBMCs) from T1D patients. LncRNA SRA was strongly upregulated by high-glucose treatment. LncRNA SRA acts as a microRNA (miR)-146b sponge through direct sequence–structure interactions. Silencing of lncRNA SRA increased the functional genes of Tregs, resulting in metabolic reprogramming, such as decreased lactate levels, repressed lactate dehydrogenase A (LDHA)/phosphorylated LDHA (pLDHA at Tyr10) expression, decreased reactive oxygen species (ROS) production, increased ATP production, and finally, decreased β-cell apoptosis in vitro. There was a positive association between lactate level and hemoglobin A1c (HbA1c) level in the plasma from patients with T1D. Recombinant human interleukin (IL)-2 treatment repressed lncRNA SRA expression and activity in β-cells. Higher levels of lncRNA-SRA/lactate in the plasma are associated with poor regulation in T1D patients. LncRNA SRA contributed to T1D pathogenesis through the inhibition of miR-146b in β-cells, with activating signaling transduction of interleukin-1 receptor-associated kinase 1 (IRAK1)/LDHA/pLDHA. Taken together, LncRNA SRA plays a critical role in the function of β-cells.     

Endothelial Semaphorin 7A Promotes Inflammation in Seawater Aspiration-Induced Acute Lung Injury

Inflammation is involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have shown that Semaphorin 7A (SEMA7A) promotes inflammation, there are limited reports regarding immunological function of SEMA7A in seawater aspiration-induced ALI. Therefore, we investigated the role of SEMA7A during seawater aspiration-induced ALI. Male Sprague–Dawley rats were underwent seawater instillation. Then, lung samples were collected at an indicated time for analysis. In addition, rat pulmonary microvascular endothelial cells (RPMVECs) were cultured and then stimulated with 25% seawater for indicated time point. After these treatments, cells samples were collected for analysis. In vivo, seawater instillation induced lung histopathologic changes, pro-inflammation cytokines release and increased expression of SEMA7A. In vitro, seawater stimulation led to pro-inflammation cytokine release, cytoskeleton remodeling and increased monolayer permeability in pulmonary microvascular endothelial cells. In addition, knockdown of hypoxia-inducible factor (HIF)-1α inhibited the seawater induced increase expression of SEMA7A. Meanwhile, knockdown of SEMA7A by specific siRNA inhibited the seawater induced aberrant inflammation, endothelial cytoskeleton remodeling and endothelial permeability. These results suggest that SEMA7A is critical in the development of lung inflammation and pulmonary edema in seawater aspiration-induced ALI, and may be a therapeutic target for this disease.     

Defining Phenotype,Tropism, and Retinal Gene Therapy Using Adeno-Associated Viral Vectors (AAVs) in New-Born Brown Norway Rats with a Spontaneous Mutation in Crb1

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10^Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10^Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10^Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.     

Expression and characterisation of recombinant human CD48 and isolation of a human anti‐CD48 monoclonal antibody by phage display

Human CD48, a membrane‐bound, glycosylphosphatidylinositol (GPI)‐linked glycoprotein, is a potential tumour target for the treatment of leukaemias and lymphomas. CD48 is expressed on T‐ and B‐cells, however <5% of CD34⁺ progenitor cells express CD48. A truncated, 45 kDa soluble form of the full length CD48 was expressed in Chinese hamster ovary (CHO) cells, and was shown to consist of a broad range of charge isoforms, with the most abundant isoforms between pI 4.5 and 5.0. The truncated form of CD48 was shown to bind to antibodies raised against native, GPI‐linked CD48 by surface plasmon resonance analysis. A synthetic, human, scFv immunoglobulin gene library was screened against recombinant CD48 by phage display, and an scFv antibody fragment, (designated N2A) was isolated after four rounds of biopanning. N2A was reassembled as a human IgG1 human monoclonal antibody, expressed in CHO cells and the binding of IgG1‐N2A to recombinant CD48 was confirmed by surface plasmon resonance. Flow cytometry studies of IgG1‐N2A binding to Raji cells showed the specificity of N2A for GPI‐linked CD48 was conserved, and presents the potential for IgG1‐N2A as a lead antibody candidate for the treatment of white blood cell malignancies. Copyright © 2005 Society of Chemical Industry     

Proton conducting crosslinked polymer electrolyte membranes based on SBS block copolymer

A series of crosslinked polymer electrolyte membranes with controlled structures were prepared based on poly(styrene‐b‐butadiene‐b‐styrene) (SBS) triblock copolymer and a sulfonated monomer, 2‐sulfoethyl methacrylate (SEMA). SBS membranes were thermally crosslinked with SEMA in the presence of a thermal‐initiator, 4,4′‐azobis(4‐cyanovaleric acid) (ACVA), as confirmed by FT‐IR spectroscopy. The water uptake and ion exchange capacity (IEC) of membranes increased almost linearly with SEMA concentrations due to the increase of SO groups. However, the proton conductivity of membranes increased linearly up to 33 wt % of SEMA, above which it abruptly jumped to 0.04 S/cm presumably due to the formation of well‐developed proton channels. Microphase‐separated morphology and amorphous structures of crosslinked SBS/SEMA membranes were observed using wide angle X‐ray scattering (WAXS), small angle X‐ray scattering (SAXS), and transmission electron microscopy (TEM). The membranes exhibited good mechanical properties and high thermal stability up to 250°C, as determined by a universal testing machine (UTM) and thermal gravimetric analysis (TGA), respectively. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

High-Throughput Gene and Protein Analysis Revealed the Response of Disc Cells to Vitamin D,Depending on the VDR FokI Variants

Vitamin D showed a protective effect on intervertebral disc degeneration (IDD) although conflicting evidence is reported. An explanation could be due to the presence of the FokI functional variant in the vitamin D receptor (VDR), observed as associated with spine pathologies. The present study was aimed at investigating—through high-throughput gene and protein analysis—the response of human disc cells to vitamin D, depending on the VDR FokI variants. The presence of FokI VDR polymorphism was determined in disc cells from patients with discopathy. 1,25(OH)₂D₃ was administered to the cells with or without interleukin 1 beta (IL-1β). Microarray, protein arrays, and multiplex protein analysis were performed. In both FokI genotypes (FF and Ff), vitamin D upregulated metabolic genes of collagen. In FF cells, the hormone promoted the matrix proteins synthesis and a downregulation of enzymes involved in matrix catabolism, whereas Ff cells behaved oppositely. In FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D. Angiogenic markers were upregulated in FF cells, along with hypertrophic markers, some of them upregulated also in Ff cells after vitamin D treatment. Higher inflammatory protein modulation after vitamin D treatment was observed in inflammatory condition. These findings would help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.     

Circulating 4-F4t-Neuroprostane and 10-F4t-Neuroprostane Are Related to MECP2 Gene Mutation and Natural History in Rett Syndrome

Neuroprostanes, a family of non-enzymatic metabolites of the docosahexaenoic acid, have been suggested as potential biomarkers for neurological diseases. Objective biological markers are strongly needed in Rett syndrome (RTT), which is a progressive X-linked neurodevelopmental disorder that is mainly caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene with a predominant multisystemic phenotype. The aim of the study is to assess a possible association between MECP2 mutations or RTT disease progression and plasma levels of 4(RS)-4-F_4t-neuroprostane (4-F_4t-NeuroP) and 10(RS)-10-F_4t-neuroprostane (10-F_4t-NeuroP) in typical RTT patients with proven MECP2 gene mutation. Clinical severity and disease progression were assessed using the Rett clinical severity scale (RCSS) in n = 77 RTT patients. The 4-F_4t-NeuroP and 10-F_4t-NeuroP molecules were totally synthesized and used to identify the contents of the plasma of the patients. Neuroprostane levels were related to MECP2 mutation category (i.e., early truncating, gene deletion, late truncating, and missense), specific hotspot mutations (i.e., R106W, R133C, R168X, R255X, R270X, R294X, R306C, and T158M), and disease stage (II through IV). Circulating 4-F_4t-NeuroP and 10-F_4t-NeuroP were significantly related to (i) the type of MECP2 mutations where higher levels were associated to gene deletions (p ≤ 0.001); (ii) severity of common hotspot MECP2 mutation (large deletions, R168X, R255X, and R270X); (iii) disease stage, where higher concentrations were observed at stage II (p ≤ 0.002); and (iv) deficiency in walking (p ≤ 0.0003). This study indicates the biological significance of 4-F_4t-NeuroP and 10-F_4t-NeuroP as promising molecules to mark the disease progression and potentially gauge genotype–phenotype associations in RTT.