A combination of LCPUFAs regulates the expression of miRNA-146a-5p in a murine asthma model and human alveolar cells.

BACKGROUND: LCPUFAs are suggestive of having beneficial effects on inflammatory diseases such as asthma. However, little is known about the modulative capacity of omega-(n)-3 and n-6 LCPUFAs within the epigenetic regulation of inflammatory processes. OBJECTIVE: The aim of this study was to investigate whether a specific combined LCPUFA supplementation restores disease-dysregulated miRNA-profiles in asthmatic mice. In addition, we determined the effect of the LCPUFA supplementation on the interaction of the most regulated miRNA expression and oxygenase activity in vitro. METHODS: Sequencing of miRNA was performed by NGS from lung tissue of asthmatic and control mice with normal diet, as well as of LCPUFA supplemented asthmatic mice. Network analysis and evaluation of the biological targets of the miRNAs were performed by DIANA- miRPath v.3 webserver software, TargetScanMouse 7.2, and tool String v.10, respectively. Expression of hsa-miRNA-146a-5p and activity of COX-2 and 5-LO in LCPUFA-treated A549 cells were assessed by qPCR and flow cytometry, respectively. RESULTS: In total, 62 miRNAs were dysregulated significantly in murine allergic asthma. The LCPUFA combination restored 21 of these dysregulated miRNAs, of which eight (mmu-miR-146a-5p, -30a-3p, -139-5p, -669p-5p, -145a-5p, -669a-5p, -342-3p and -15b-5p) were even normalized compared to the control levels. Interestingly, six of the eight rescued miRNAs are functionally implicated in TGF-ß signaling, ECM-receptor interaction and fatty acid biosynthesis. Furthermore, in vitro experiments demonstrated that upregulation of hsa-miRNA-146a-5p is accompanied by a reduction of COX-2 and 5-LO activity. Moreover, transfection experiments revealed that LCPUFAs inhibit 5-LO activity in the presence and absence of anti-miR-146a-5p. CONCLUSION: Our results demonstrate the modulative capacity of LCPUFAs on dysregulated miRNA expression in asthma. In addition, we pointed out the high regulative potential of LCPUFAs on 5-LO regulation and provided evidence that miR-146a partly controls the regulation of 5-LO.

Transcriptomic signatures of neuronal differentiation and their association with risk genes for autism spectrum and related neuropsychiatric disorders.

Genes for autism spectrum disorders (ASDs) are also implicated in fragile X syndrome (FXS), intellectual disabilities (ID) or schizophrenia (SCZ), and converge on neuronal function and differentiation. The SH-SY5Y neuroblastoma cell line, the most widely used system to study neurodevelopment, is currently discussed for its applicability to model cortical development. We implemented an optimal neuronal differentiation protocol of this system and evaluated neurodevelopment at the transcriptomic level using the CoNTeXT framework, a machine-learning algorithm based on human post-mortem brain data estimating developmental stage and regional identity of transcriptomic signatures. Our improved model in contrast to currently used SH-SY5Y models does capture early neurodevelopmental processes with high fidelity. We applied regression modelling, dynamic time warping analysis, parallel independent component analysis and weighted gene co-expression network analysis to identify activated gene sets and networks. Finally, we tested and compared these sets for enrichment of risk genes for neuropsychiatric disorders. We confirm a significant overlap of genes implicated in ASD with FXS, ID and SCZ. However, counterintuitive to this observation, we report that risk genes affect pathways specific for each disorder during early neurodevelopment. Genes implicated in ASD, ID, FXS and SCZ were enriched among the positive regulators, but only ID-implicated genes were also negative regulators of neuronal differentiation. ASD and ID genes were involved in dendritic branching modules, but only ASD risk genes were implicated in histone modification or axonal guidance. Only ID genes were over-represented among cell cycle modules. We conclude that the underlying signatures are disorder-specific and that the shared genetic architecture results in overlaps across disorders such as ID in ASD. Thus, adding developmental network context to genetic analyses will aid differentiating the pathophysiology of neuropsychiatric disorders.

Relationship Between Cortical Gyrification, White Matter Connectivity, and Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition, which is accompanied by differences in gray matter neuroanatomy and white matter connectivity. However, it is unknown whether these differences are linked or reflect independent aetiologies. Using a multimodal neuroimaging approach, we therefore examined 51 male adults with ASD and 48 neurotypical controls to investigate the relationship between gray matter local gyrification (lGI) and white matter diffusivity in associated fiber tracts. First, ASD individuals had a significant increase in gyrification around the left pre- and post-central gyrus. Second, white matter fiber tracts originating and/or terminating in the cluster of increased lGI had a significant increase in axial diffusivity. This increase in diffusivity was predominantly observed in tracts in close proximity to the cortical sheet. Last, we demonstrate that the increase in lGI was significantly correlated with increased diffusivity of short tracts. This relationship was not significantly modulated by a main effect of group (i.e., ASD), which was more closely associated with gray matter gyrification than white matter diffusivity. Our findings suggest that differences in gray matter neuroanatomy and white matter connectivity are closely linked, and may reflect common rather than distinct aetiological pathways.

A mutation in caspase-9 decreases the expression of BAFFR and ICOS in patients with immunodeficiency and lymphoproliferation.

Lymphocyte apoptosis is mainly induced by either death receptor-dependent activation of caspase-8 or mitochondria-dependent activation of caspase-9. Mutations in caspase-8 lead to autoimmunity/lymphoproliferation and immunodeficiency. This work describes a heterozygous H237P mutation in caspase-9 that can lead to similar disorders. H237P mutation was detected in two patients: Pt1 with autoimmunity/lymphoproliferation, severe hypogammaglobulinemia and Pt2 with mild hypogammaglobulinemia and Burkitt lymphoma. Their lymphocytes displayed defective caspase-9 activity and decreased apoptotic and activation responses. Transfection experiments showed that mutant caspase-9 display defective enzyme and proapoptotic activities and a dominant-negative effect on wild-type caspase-9. Ex vivo analysis of the patients' lymphocytes and in vitro transfection experiments showed that the expression of mutant caspase-9 correlated with a downregulation of BAFFR (B-cell-activating factor belonging to the TNF family (BAFF) receptor) in B cells and ICOS (inducible T-cell costimulator) in T cells. Both patients carried a second inherited heterozygous mutation missing in the relatives carrying H237P: Pt1 in the transmembrane activator and CAML interactor (TACI) gene (S144X) and Pt2 in the perforin (PRF1) gene (N252S). Both mutations have been previously associated with immunodeficiencies in homozygosis or compound heterozygosis. Taken together, these data suggest that caspase-9 mutations may predispose to immunodeficiency by cooperating with other genetic factors, possibly by downregulating the expression of BAFFR and ICOS.

Variants of the CNTNAP2 5' promoter as risk factors for autism spectrum disorders: a genetic and functional approach.

Contactin-associated protein-like 2 gene (CNTNAP2), a member of the Neurexin gene superfamily, is one of the best-replicated risk genes for autism spectrum disorders (ASD). ASD are predominately genetically determined neurodevelopmental disorders characterized by impairments of language development, social interaction and communication, as well as stereotyped behavior and interests. Although CNTNAP2 expression levels were proposed to alter ASD risk, no study to date has focused on its 5' promoter. Here, we directly sequenced the CNTNAP2 5' promoter region of 236 German families with one child with ASD and detected four novel variants. Furthermore, we genotyped the three most frequent variants (rs150447075, rs34712024, rs71781329) in an additional sample of 356 families and found nominal association of rs34712024G with ASD and rs71781329GCG[7] with language development. The four novel and the three known minor alleles of the identified variants were predicted to alter transcription factor binding sites (TFBS). At the functional level, the respective sequences spanning these seven variants were bound by nuclear factors. In a luciferase promoter assay, the respective minor alleles showed cell line-specific and differentiation stage-dependent effects at the level of promoter activation. The novel potential rare risk-variant M2, a G>A mutation -215 base pairs 5' of the transcriptional start site, significantly reduced promoter efficiency in HEK293T and in undifferentiated and differentiated neuroblastoid SH-SY5Y cells. This variant was transmitted to a patient with autistic disorder. The under-transmitted, protective minor G allele of the common variant rs34712024, in contrast, increased transcriptional activity. These results lead to the conclusion that the pathomechanism of CNTNAP2 promoter variants on ASD risk is mediated by their effect on TFBSs, and thus confirm the hypothesis that a reduced CNTNAP2 level during neuronal development increases liability for ASD.

Osteopontin induces soluble urokinase-type plasminogen activator receptor production and release.

BACKGROUND: Osteopontin (OPN) and soluble urokinase plasminogen activator receptor (suPAR) have been proposed as markers of disease severity and risk-stratification in infection and inflammation. In breast cancer, OPN and the membrane bound form of urokinase plasminogen activator receptor (uPAR) are functionally related, as OPN-induced cell migration depends on uPAR triggering by urokinase plasminogen activator (uPA). The aim of this study was to prospectively evaluate the kinetic of OPN and suPAR blood levels in patients developing septic shock (SS) compared to those not developing SS, and to investigate the relationships between these two biomarkers in immune cells in vitro. METHODS: We measured the levels of OPN and suPAR for 15 days in forty-three patients, defined a priory as at risk to develop septic shock. Moreover, we investigated in vitro the effect of recombinant OPN on uPAR and suPAR expression in monocytes. RESULTS: We found that OPN and suPAR levels were directly correlated to each other both at intensive care unit admission and on the day patients met SIRS/sepsis or septic shock criteria. In patients developing septic shock, OPN increased prior to suPAR and was already detectable up to 4 days before the shock development. In vitro, OPN induced suPAR production in monocytes by increasing both uPAR gene expression, and suPAR release from the cell surface. CONCLUSION: These data suggest that OPN is partly responsible for the increased plasma levels of suPAR and might be a valuable tool to predict the occurrence of septic shock.

Solid lipid nanoparticles of cholesteryl butyrate inhibit the proliferation of cancer cells in vitro and in vivo models.

BACKGROUND AND PURPOSE: Solid lipid nanoparticles containing cholesteryl butyrate (cholbut SLN) can be a delivery system for the anti-cancer drug butyrate. These nanoparticles inhibit adhesion of polymorphonuclear and tumour cells to endothelial cells and migration of tumour cells, suggesting that they may act as anti-inflammatory and anti-tumour agents. Here we have evaluated the effects of cholbut SLN on tumour cell growth using in vitro and in vivo models. EXPERIMENTAL APPROACH: Cholbut SLNs were incubated with cultures of four tumour cell lines, and cell growth was analysed by assessing viability, clonogenic capacity and cell cycle. Effects on intracellular signalling was assessed by Western blot analysis of Akt expression. The in vivo anti-tumour activity was measured in two models of PC-3 cell xenografts in SCID/Beige mice. KEY RESULTS: Cholbut SLN inhibited tumour cell line viability, clonogenic activity, Akt phosphorylation and cell cycle progression. In mice injected i.v. with PC3-Luc cells and treated with cholbut SLN, . in vivo optical imaging and histological analysis showed no metastases in the lungs of the treated mice. In another set of mice injected s.c. with PC-3 cells and treated with cholbut SLN when the tumour diameter reached 2 mm, analysis of the tumour dimensions showed that treatment with cholbut SLN substantially delayed tumour growth. CONCLUSION AND IMPLICATIONS: Cholbut SLN were effective in inhibiting tumour growth in vitro and in vivo. These effects may involve, in part, inhibition of Akt phosphorylation, which adds another mechanism to the activity of this multipotent drug.

[Genetics and animal modeling of autism spectrum disorders. New developments]. / Genetik und Tiermodelle von Autismusspektrumstörungen. Neue Entwicklungen.

Autism spectrum disorders (ASD) are pervasive developmental disorders with a complex phenotype in respect to communication, verbal development, and social behavior. Manifold molecular genetic analyses point towards a multifactorial genetic predisposition. For the identification of central key mechanisms large consortia have performed linkage analysis, genome-wide association, and copy number variation (CNV) studies, which led to the characterization of risk factors for ASD like CNV and single nucleotide polymorphisms but also single rare mutations. The so far associated genomic regions and candidate genes impact neuronal development especially the establishment of the synaptic cleft, secretion of surface proteins, or dendritic translation. These findings point towards deficits of translation-dependent cell-cell connectivity and synaptic plasticity for ASD. Animal models are relevant to analyze the pathomechanisms of single genetic risk variants at the cellular, tissue-specific, and behavioral levels.

The osteopontin gene +1239A/C single nucleotide polymorphism is associated with type 1 diabetes mellitus in the Italian population.

Secreted phosphoprotein 1, also known as Osteopontin (Opn), is a proinflammatory cytokine involved in the TH1 response and is highly expressed in the islets and pancreatic lymph nodes of non-obese diabetic mice before the onset of diabetes. In humans, typing of the +1239A/C single nucleotide polymorphism (SNP) in the 3UTR of the Opn gene (SPP1) showed that +1239C carriers displayed higher Opn serum levels than +1239A homozygotes and a higher risk of developing autoimmune/lymphoproliferative syndrome, multiple sclerosis, and systemic lupus erythematosus. The aim of this work is to evaluate whether +1239A/C is also associated with type 1 diabetes mellitus (T1DM). We typed +1239A/C in an initial cohort of 184 T1DM patients and 361 controls, and confirmed our data in a second cohort of 513 patients and 857 controls. In both cohorts, +1239C carriers displayed a significantly higher risk of T1DM than +1239A homozygotes (combined cohorts: OR=1.63, 95 percent CI: 1.34-1.97). Clinical analysis did not detect any differences between patients carrying or not +1239C in terms of gender distribution and age at T1DM diagnosis. These data suggest that SPP1 variants marked by +1239C are associated with T1DM development in the Italian population. The predisposing effect may depend on its effect on Opn levels.

Variations of the perforin gene in patients with multiple sclerosis.

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13-3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10-1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

Defective function of the Fas apoptotic pathway in type 1 diabetes mellitus correlates with age at onset.

The Fas death receptor triggers lymphocyte apoptosis through an extrinsic and an intrinsic pathway involving caspase-8 and -9 respectively. Inherited defects of Fas function are displayed by a proportion of patients with Type 1 diabetes mellitus (T1DM) especially those with a second autoimmunity (T1DM-p). This study assesses activation of both pathways in Fas-resistant (FasR) patients to localize the defect. 21/28 (75 percent) T1DM-p, 14/50 (38 percent) T1DM, and 7/150 (5 percent) controls were FasR. Analysis of the 35 FasR patients and 20 Fas-sensitive (FasS) controls showed that caspase-9 activity was lower in T1DM-p and T1DM than in controls, whereas caspase-8 activity was lower in T1DM-p than in T1DM and the controls. Single patient analysis showed that 16/35 patients displayed defective activity of one (FasR1), whereas 19 displayed normal activity of both caspases (FasR2). Ages at onset of diabetes mellitus in T1DM and the second autoimmune disease in T1DM-p were lower in FasR than in FasS patients. All FasR1 patients developed diabetes mellitus before the age of 9 years, whereas a later onset was displayed by 26% FasR2 and 53% FasS patients. These data show that defective Fas function may involve both the extrinsic and intrinsic pathway in T1DM and severity correlates with the precocity of the autoimmune attack and its tissue polyreactivity.

Mutations in the ribosomal protein gene RPL10 suggest a novel modulating disease mechanism for autism.

Autism has a strong genetic background with a higher frequency of affected males suggesting involvement of X-linked genes and possibly also other factors causing the unbalanced sex ratio in the etiology of the disorder. We have identified two missense mutations in the ribosomal protein gene RPL10 located in Xq28 in two independent families with autism. We have obtained evidence that the amino-acid substitutions L206M and H213Q at the C-terminal end of RPL10 confer hypomorphism with respect to the regulation of the translation process while keeping the basic translation functions intact. This suggests the contribution of a novel, possibly modulating aberrant cellular function operative in autism. Previously, we detected high expression of RPL10 by RNA in situ hybridization in mouse hippocampus, a constituent of the brain limbic system known to be afflicted in autism. Based on these findings, we present a model for autistic disorder where a change in translational function is suggested to impact on those cognitive functions that are mediated through the limbic system.

Two single-nucleotide polymorphisms in the 5' and 3' ends of the osteopontin gene contribute to susceptibility to systemic lupus erythematosus.

OBJECTIVE: To test the association of osteopontin (OPN) polymorphisms with systemic lupus erythematosus (SLE). METHODS: The coding 5' and 3' flanking regions of the OPN gene were scanned for polymorphisms by denaturing high-performance liquid chromatography. A case-control association study was performed in 394 Italian SLE patients and 479 matched controls. OPN serum levels were determined by enzyme-linked immunosorbent assay in 40 patients and 124 controls, and the mean levels were compared between the different OPN genotypes. RESULTS: Among the 13 detected single-nucleotide polymorphisms (SNPs), alleles -156G (frequency 0.714 versus 0.651; P = 0.006, corrected P [P(corr)] = 0.036) and +1239C (0.377 versus 0.297; P = 0.00094, P(corr) = 0.0056) were significantly increased in the SLE patients compared with the controls. The presence of the associated allele in single or double dose conferred an odds ratio (OR) of 2.35 (95% confidence interval [95% CI] 1.38-4.02) for SNP -156 and an OR of 1.57 (95% CI 1.16-2.13) for SNP +1239. These effects were independent of each other, i.e., not a consequence of linkage disequilibrium between the 2 alleles. The risk associated with a double dose of susceptibility alleles at both SNPs was 3.8-fold higher (95% CI 2.0-7.4) relative to the complete absence of susceptibility alleles. With regard to individual clinical and immunologic features, a significant association was seen between lymphadenopathy and -156 genotypes (overall P = 0.0011, P(corr) = 0.046). A significantly increased OPN serum level was detected in healthy individuals carrying +1239C (P = 0.002), which is indicative of an association between the SLE susceptibility allele and OPN levels. CONCLUSION: These data suggest the independent effect of a promoter (-156) and a 3'-untranslated region (+1239) SNP in SLE susceptibility. We can speculate that these sequence variants (or others in perfect linkage disequilibrium) create a predisposition to high production of OPN, and that this in turn may confer susceptibility to SLE.

Defective function of Fas in T cells from paediatric patients with autoimmune thyroid diseases.

Triggering of the Fas receptor induces T cell apoptosis and is involved in shutting-off the immune response. Inherited defects impairing Fas function cause the autoimmune lymphoproliferative syndrome, and may play a role in other autoimmune diseases. The aim of this work was to analyse the Fas function in paediatric patients with thyroid autoimmunities. We found that T cells from 24/28 patients with Graves' disease (GD) and 12/35 patients with Hashimoto's thyroiditis (HT) displayed defective Fas function. In HT, the defect was more frequent in patients requiring replacement therapy (11/20) than in those not requiring (1/15); moreover, in untreated HT the highest defect was displayed by patients with the highest levels of autoantibodies. Fas was always expressed at normal levels and no Fas mutations were detected. Analysis of the healthy parents of seven Fas-resistant patients showed that several of them were Fas-resistant, which suggests a genetic component. Fusion of Fas-resistant T cells with the Fas-sensitive HUT78 T cell line generated Fas-resistant hybrid cells, which suggests the presence of molecules exerting a dominant negative effect on Fas function. Analysis of Fas-induced activation of caspase-8 and -9 showed decreased activity of both caspases in HT, whereas activity of caspase-9 was increased and that of caspase-8 was decreased in GD. These data suggest that heterogeneous inherited defects impairing Fas function favour the development of thyroid autoimmunities.

Defective T cell fas function in patients with multiple sclerosis.

BACKGROUND: Fas (CD95) triggers programmed cell death and is involved in shutting off the immune response. Inherited deleterious mutations hitting Fas or its signaling pathway cause autoimmune/lymphoproliferative syndrome (ALPS). OBJECTIVE: To assess the possibility that decreased Fas function plays a role in development of MS. METHODS: The authors evaluated Fas function in long-term T cell lines (21 days of culture) from 32 patients with relapsing-remitting MS (RRMS), 15 with secondary progressive MS (SPMS), and 15 with primary progressive MS (PPMS) by assessing cell survival upon Fas triggering by monoclonal antibodies (Mab). RESULTS: Fas-induced cell death was significantly lower in all patient groups than in controls, and lower in SPMS than in RRMS. Moreover, 8/15 patients with PPMS, 10/15 with SPMS, and 8/32 with RRMS were frankly resistant to Fas. Frequency of resistance to Fas-induced cell death was significantly higher in all patient groups than in controls (2/75), and higher in SPMS than in RRMS. The findings that the parents of two Fas-resistant patients were Fas-resistant and that fusion of T cells from two Fas-resistant patients with Fas-sensitive HUT78 cells gave rise to Fas-resistant hybrid lines suggest that Fas-resistance is due to inherited alterations of the Fas signaling pathway, with production of molecules exerting a dominant negative effect on a normal Fas system. CONCLUSIONS: Defects of the immune response shutting-off system may be involved in the pathogenesis of MS, particularly in its progressive evolution.

Detection of Fusarium oxysporum f. sp. dianthi in Carnation Tissue by PCR Amplification of Transposon Insertions.

ABSTRACT Strains of the carnation wilt pathogen, Fusarium oxysporum f. sp. dianthi, can be distinguished by DNA fingerprint patterns, using the fungal transposable elements Fot1 and impala as probes for Southern hybridization. The DNA fingerprints correspond to three groups of F. oxysporum f. sp. dianthi strains: the first group includes isolates of races 1 and 8; the second group includes isolates of races 2, 5 and 6; and the third group includes isolates of race 4. Genomic DNAs flanking race-associated insertion sites of Fot1 (from races 1, 2, and 8) or impala (from race 4) were amplified by the inverse polymerase chain reaction (PCR) technique. These regions were cloned and sequenced, and three sets of primers overlapping the 3' or 5' end of the transposon and its genomic insertion were designed. Using fungal genomic DNA as template in PCR experiments, primer pairs generated amplification products of 295, 564 and 1,315 bp, corresponding to races 1 and 8; races 2, 5, and 6; and race 4, respectively. When multiplex PCR was performed with genomic DNA belonging to races 1 and 8, 2, or 4, single amplimers were generated, allowing clear race determination of the isolate tested. PCR was successfully performed on DNA extracted from susceptible carnation cv. Indios infected with isolates representative of races 1, 2, 4, and 8.

Green fluorescent protein as a reporter of gene expression in transgenic mice.

We used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a reporter of gene expression in transgenic mice. The GFP coding sequence was placed under the control of the human hemopexin and the mouse beta1 integrin promoter that were previously studied in transgenic mice using the lacZ reporter gene. We showed that GFP has a higher degree of sensitivity compared to the lacZ reporter gene allowing to identify cells with low and otherwise undetectable beta-galactosidase activity. Thus we showed the potentiality of GFP in replacing lacZ as a reporter gene to investigate promoter mapping and gene regulation in transgenic mice.

Analysis of regulatory regions of the ciliary neutrophic factor gene in transgenic mice.

In order to study the regulatory regions of the human ciliary neurotrophic factor (CNTF) gene we made constructs containing sequences upstream and downstream of CNTF coding regions and the lacZ gene and analysed their expression in transgenic mice. We show that 240 bp upstream of the translation start codon are sufficient for the transcription of the lacZ gene. A further 4 kb upstream sequence is required for the expression of the transgene in Schwann cells. These two upstream regions together with a 2 kb downstream fragment drive high level of expression of the lacZ gene in the sciatic nerve. Our results indicate that these three fragments contain regulatory regions able to mimic the CNTF expression pattern in the mouse peripheral nervous system.