Brain structure, cognition and negative symptoms in schizophrenia are associated with serum levels of polysialic acid-modified NCAM.

The neural cell adhesion molecule (NCAM) is a glycoprotein implicated in cell-cell adhesion, neurite outgrowth and synaptic plasticity. Polysialic acid (polySia) is mainly attached to NCAM (polySia-NCAM) and has an essential role in regulating NCAM-dependent developmental processes that require plasticity, that is, cell migration, axon guidance and synapse formation. Post-mortem and genetic evidence suggests that dysregulation of polySia-NCAM is involved in schizophrenia (SZ). We enrolled 45 patients diagnosed with SZ and 45 healthy individuals who were submitted to polySia-NCAM peripheral quantification, cognitive and psychopathological assessment and structural neuroimaging (brain volumes and diffusion tensor imaging). PolySia-NCAM serum levels were increased in SZ patients, independently of antipsychotic treatment, and were associated with negative symptoms, blunted affect and declarative memory impairment. The increased polySia-NCAM levels were associated with decreased volume in the left prefrontal cortex, namely Brodmann area 46, in patients and increased volume in the same brain area of healthy individuals. As this brain region is involved in the pathophysiology of SZ and its associated phenomenology, the data indicate that polySia-NCAM deserves further scrutiny because of its possible role in early neurodevelopmental mechanisms of the disorder.

Genome-wide haplotype association study identifies the FRMD4A gene as a risk locus for Alzheimer's disease.

Recently, several genome-wide association studies (GWASs) have led to the discovery of nine new loci of genetic susceptibility in Alzheimer's disease (AD). However, the landscape of the AD genetic susceptibility is far away to be complete and in addition to single-SNP (single-nucleotide polymorphism) analyses as performed in conventional GWAS, complementary strategies need to be applied to overcome limitations inherent to this type of approaches. We performed a genome-wide haplotype association (GWHA) study in the EADI1 study (n=2025 AD cases and 5328 controls) by applying a sliding-windows approach. After exclusion of loci already known to be involved in AD (APOE, BIN1 and CR1), 91 regions with suggestive haplotype effects were identified. In a second step, we attempted to replicate the best suggestive haplotype associations in the GERAD1 consortium (2820 AD cases and 6356 controls) and observed that 9 of them showed nominal association. In a third step, we tested relevant haplotype associations in a combined analysis of five additional case-control studies (5093 AD cases and 4061 controls). We consistently replicated the association of a haplotype within FRMD4A on Chr.10p13 in all the data set analyzed (OR: 1.68; 95% CI: (1.43-1.96); P=1.1 × 10(-10)). We finally searched for association between SNPs within the FRMD4A locus and Aß plasma concentrations in three independent non-demented populations (n=2579). We reported that polymorphisms were associated with plasma Aß42/Aß40 ratio (best signal, P=5.4 × 10(-7)). In conclusion, combining both GWHA study and a conservative three-stage replication approach, we characterised FRMD4A as a new genetic risk factor of AD.

APOE and Alzheimer disease: a major gene with semi-dominant inheritance.

Apolipoprotein E (APOE) dependent lifetime risks (LTRs) for Alzheimer Disease (AD) are currently not accurately known and odds ratios alone are insufficient to assess these risks. We calculated AD LTR in 7351 cases and 10 132 controls from Caucasian ancestry using Rochester (USA) incidence data. At the age of 85 the LTR of AD without reference to APOE genotype was 11% in males and 14% in females. At the same age, this risk ranged from 51% for APOE44 male carriers to 60% for APOE44 female carriers, and from 23% for APOE34 male carriers to 30% for APOE34 female carriers, consistent with semi-dominant inheritance of a moderately penetrant gene. Using PAQUID (France) incidence data, estimates were globally similar except that at age 85 the LTRs reached 68 and 35% for APOE 44 and APOE 34 female carriers, respectively. These risks are more similar to those of major genes in Mendelian diseases, such as BRCA1 in breast cancer, than those of low-risk common alleles identified by recent GWAS in complex diseases. In addition, stratification of our data by age groups clearly demonstrates that APOE4 is a risk factor not only for late-onset but for early-onset AD as well. Together, these results urge a reappraisal of the impact of APOE in Alzheimer disease.

Interleukin-18, from neuroinflammation to Alzheimer's disease.

A large body of evidence on brain development and ageing has revealed that inflammatory processes profoundly affect brain functions during life span of mammalians, including humans. Activation of innate immune mechanisms leading to pro-inflammatory cytokine up-regulation is involved in devastating and disabling human brain illnesses, as Alzheimer's disease (AD), a progressive neurodegenerative disease that causes dementia in the elderly. Emerging data indicates that the cytokine Interleukin (IL)-18, one of the key mediator of inflammation and immune response, has relevance in the physiopathological processes of the brain, by ultimately influencing the integrity of neurons and putatively contributing to AD. In this review, the relationship between specific IL-18-mediated processes and AD neurodegeneration is summarized and clinical studies pointing to a role of the cytokine in the pathology are discussed. Altogether, the presented data indicate that a more complete knowledge of the molecular mechanisms underlying IL-18 implication in neuroinflammatory and neurodegenerative pathways could contribute toward the development of new therapeutic strategies for AD.

BDNF Val66Met polymorphism is associated with aggressive behavior in schizophrenia.

Brain-derived neurotrophic factor (BDNF) gene variants may potentially influence behaviour. In order to test this hypothesis, we investigated the relationship between BDNF Val66Met polymorphism and aggressive behaviour in a population of schizophrenic patients. Our results showed that increased number of BDNF Met alleles was associated with increased aggressive behaviour.

The etiology of poststroke depression: a review of the literature and a new hypothesis involving inflammatory cytokines.

Although poststroke depression is unlikely to represent a single disorder and numerous etiologies for different kinds of poststroke depression will likely emerge as the result of future research, we believe that a number of poststroke depressive disorders are likely to be the result of specific changes in brain pathology and neurophysiology. Nevertheless, there are relatively few hypotheses about the pathophysiology of poststroke depression. This paper, therefore, proposes a new hypothesis for poststroke depression involving increased production of proinflammatory cytokines resulting from brain ischemia in cerebral areas linked to the pathogenesis of mood disorders. This paper reviews the evidence supporting the hypothesis that proinflammatory cytokines are involved in the occurrence of stroke as well as mood disorders linked to the brain damage. The increased production of proinflammatory cytokines such as IL-1beta, TNF-alpha or IL-18 resulting from stroke may lead to an amplification of the inflammatory process, particularly in limbic areas, and widespread activation of indoleamine 2,3-dioxygenase (IDO) and subsequently to depletion of serotonin in paralimbic regions such as the ventral lateral frontal cortex, polar temporal cortex and basal ganglia. The resultant physiological dysfunction may lead to poststroke depression. Future investigations may explore this hypothesis through more extensive studies on the role of proinflammatory cytokines, such as IL-1beta, TNF-alpha or even IL-18, in patients with poststroke depression.

Immunopurification of B lymphocytes from sea bass Dicentrarchus labrax (L.).

The monoclonal antibody DLIg3, specific for immunoglobulins and B cells of the teleost fish Dicentrarchus labrax (Mediterranean sea bass), was used to enrich immunoreactive cells from peripheral blood, spleen, and head kidney leukocytes. The purification was performed by immunomagnetic sorting of leukocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by immunofluorescence and cytofluorimetric analysis. Following a single immunopurification step, the percentages of DLlg3-purified cells were 61% +/- 6% from peripheral blood leukocytes, 66% +/- 5% from splenocytes, and 77% +/- 9% from head kidney cells. DLIg3-immunopurified cells, from the head kidney of antigen-primed fish displayed an enhanced proliferation index when incubated with the immunization antigen. DLIg3-purified cells from head kidney lymphoid tissue were employed for RNA extraction and complementary DNA synthesis, and in reverse transcriptase polymerase chain reaction experiments using specific primers corresponding to the sequences of the sea bass immunoglobulin light chain, and of T-cell receptor. DLIg3-purified cells displayed enhanced expression of the immunoglobulin gene, and lower expression of T-cell receptor.

Neutrophil apoptosis in autoimmune Fas-defective MRL lpr/lpr mice.

The apoptosis-defective lpr (fas) mutation in MRL mice causes the early onset of a lupus-like autoimmune disease with concomitant inflammation. In order to analyse the consequences of the impaired Fas-dependent apoptosis on inflammation, the susceptibility to apoptosis of polymorphonuclear leukocytes (PMN), obtained from MRL lpr/lpr mice, has been studied. Peritoneal PMN from lpr/lpr and control (+/+) mice were recruited with a mild inflammatory stimulus. The number of cells collected from the peritoneal cavity of young lpr/lpr mice was comparable to that obtained from age-matched control mice, indicating that PMN homeostasis is maintained regardless of the loss-of-function Fas mutation. Recruited neutrophils were exposed in culture to apoptosis-inducing stimuli. Treatment with agonist anti-Fas antibody increased apoptosis of +/+ PMN, but did not affect lpr/lpr PMN which do not express Fas on their surface. However, lpr/lpr PMN could undergo both spontaneous and stimulus-induced apoptosis in a fashion comparable to or higher than that of control +/+ mice. Analysis of mRNA expression revealed that lpr/lpr PMN have reduced expression of IL-18, whereas IL-1beta, IFNgamma, caspase 1 and caspase 3 are expressed at levels comparable to those of +/+ cells. However, caspase-3-like activity was higher in PMN from lpr/lpr mice than in +/+ cells, and correlated with enhanced apoptosis. It could be concluded that in young, uncompromised lpr/lpr mice, PMN homeostasis is still fully regulated through the involvement of Fas-independent, compensatory, apoptotic mechanisms. This could include an increased participation of caspase 3 in the apoptotic pathway, consequent to enhanced activation of the enzyme and to the decreased production of IL-18, which acts as a competitive caspase 3 substrate.

Cycle-dependent expression of interleukin-1 receptor type II in the human endometrium.

Cytokines such as interleukin-1 (IL-1) play a major role in the reparative and inflammatory-like processes that occur in human endometrium during every menstrual cycle, but they also seem to be implicated in critical reproductive events such as ovulation and implantation. Interleukin-1 is tightly regulated in the body by a complex network of control systems. In the present study, we examined the expression of IL-1RII, a natural specific inhibitor of IL-1, in the human endometrium and found an interesting distribution and temporal pattern of expression throughout the menstrual cycle. Immunoreactive IL-1RII was found in stromal as well as epithelial cells, but it was predominant within the lumen of the glands and the apical side of surface epithelium. In situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed higher levels of mRNA in epithelial than in stromal cells. The IL-1RII cellular and luminal secretion followed a regulated cycle phase-dependent pattern of expression. Although elevated in the late proliferative/early secretory phase of the menstrual cycle, IL-1RII luminal secretion significantly decreased in the midsecretory phase, reaching its lowest levels at Day 21, before augmenting markedly again during the late secretory phase. This pattern of expression was less obvious at the level of cellular staining, as examined by immunohistochemistry, but it was corroborated by Western blot analysis of IL-1RII protein and semiquantitative RT-PCR of IL-1RII mRNA in the whole endometrial tissue and separated glandular epithelial cells. The reduced expression of IL-1RII within the implantation window suggests the existence of accurate regulatory mechanisms that, by down-regulating IL-1RII expression, alleviate IL-1 inhibition during this crucial period and facilitate IL-1 proimplantation actions. The elevated expression of IL-1RII observed during the late secretory phase suggests an involvement of IL-1RII in control of the proinflammatory state that takes place in the endometrium during the premenstrual and menstrual periods.

Model of interaction of the IL-1 receptor accessory protein IL-1RAcP with the IL-1beta/IL-1R(I) complex.

A preliminary model has been calculated for the activating interaction of the interleukin 1 receptor (IL-1R) accessory protein IL-1RAcP with the ligand/receptor complex IL-1beta/IL-1R(I). First, IL-1RAcP was modeled on the crystal structure of IL-1R(I) bound to IL-1beta. Then, the IL-1RAcP model was docked using specific programs to the crystal structure of the IL-1beta/IL-1R(I) complex. Two types of models were predicted, with comparable probability. Experimental data obtained with the use of IL-1beta peptides and antibodies, and with mutated IL-1beta proteins, support the BACK model, in which IL-1RAcP establishes contacts with the back of IL-1R(I) wrapped around IL-1beta.

Balance between autocrine interleukin-1beta and caspases defines life versus death of polymorphonuclear cells.

The role of endogenous IL-1beta in regulating spontaneous and Fas-triggered apoptosis of human PMN has been studied in relation to the activity of the IL-1beta-generating enzyme ICE (caspase-1), an enzyme also involved in the mechanism of cell death. Upon in vitro culture, PMN undergo spontaneous apoptosis and express increasing levels of IL-1beta, caspase-1- and caspase-3-like enzymes. Endogenous IL-1beta protects PMN from apoptosis, since inhibition of either IL-1beta or caspase-1 activity can accelerate PMN apoptotic death. Thus, in spontaneous PMN apoptosis caspase-1 essentially plays an anti-apoptotic role by inducing maturation of protective IL-1beta, whereas other molecules are responsible of driving apoptosis. Upon Fas triggering, PMN apoptosis is greatly accelerated, in correlation with increased caspase activity, whereas IL-1beta production is not augmented. Inhibition of IL-1beta activity can increase Fas-induced apoptosis, whereas caspase-1 inhibitors are without significant effect. It is hypothesized that in Fas-induced PMN apoptosis caspase-1 has a double role: it can protect from apoptosis through generation of protective IL-1beta, as in spontaneous apoptosis, and it can also exert pro-apoptotic activity which counterbalances the protective effect and allows accelerated apoptosis.

Lymphocytes from autoimmune MRL lpr/lpr mice are hyperresponsive to IL-18 and overexpress the IL-18 receptor accessory chain.

MRL lpr/lpr mice spontaneously develop a severe autoimmune lupus syndrome characterized by strong autoantibody production and massive lymphoproliferation, in which IFN-gamma plays a major pathogenic effect. The role of the IFN-gamma-inducing cytokine IL-18 in the autoimmune syndrome of lpr/lpr mice has been investigated. In response to IL-18, lymph node cells of lpr/lpr mice produce significant amounts of IFN-gamma and proliferate more potently as compared with cells from +/+ mice. Cells likely responsible for such hyperresponsiveness to IL-18 include NK cells and the CD4(+)/CD8(+) self-reactive T lymphocytes characteristically present in lymph nodes of lpr/lpr mice. Analysis of the expression of IL-18R complex revealed that mRNA for the IL-18R alpha-chain is constitutively expressed at similar level both in +/+ and lpr/lpr lymphocytes. In contrast, the expression of the accessory receptor chain IL-18R beta is low in unstimulated +/+ cells but significantly high in lpr/lpr cells. Thus, the abnormally high expression of the IL-18R chain IL-18R beta could be one of the causes of the hyperresponsiveness of lpr/lpr cells to IL-18 at the basis of consequent enhancement of IFN-gamma production and development of IFN-gamma-dependent autoimmune pathology.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II.

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.

Purification of human recombinant interleukin 1 receptor antagonist proteins upon Bacillus subtilis sporulation.

Human interleukin 1 receptor antagonist (IL-1ra) and IL-1ra mutants were constitutively expressed in recombinant Bacillus subtilis in endocellular and active form. In order to optimize the purification of the recombinant proteins, a new method has been developed. After bacterial growth in fermenter, release of recombinant protein was achieved by starvation-induced sporulation. The sporulation supernatant was recovered by centrifugation, filtered, and subjected sequentially to cation- and anion-exchange chromatography. Alternatively, the fermenter's contents were directly subjected to expanded bed adsorption on a Streamline cation-exchange column, thus avoiding the centrifugation and filtration steps. Up to 88 mg of biological active purified recombinant protein per liter of culture was obtained, with a 72-79% recovery and 98% purity, depending on the molecule. By using the method described here, it is possible to achieve a spontaneous release of recombinant proteins expressed endocellularly at high levels in B. subtilis without need of a cell breakage step. Thus, this method could allow purification of the endocellular recombinant protein as if it were secreted. Furthermore, when using the expanded bed adsorption, highly purified protein was obtained in only two steps after sporulation. Among the advantages of the method, one of the most relevant is the possibility of keeping the system closed up to completion of the first purification step.

Interaction between interleukin-1 and ciliary neurotrophic factor in the regulation of neuroblastoma cell functions.

Human neuroblastoma cells SK-N-SH express significant numbers of IL-1R type I on their surface, as detected by saturation binding and RT-PCR, and are responsive to IL-1beta activation by producing inflammatory cytokines IL-6 and IL-8. IL-1beta can also have an indirect effect on nervous cell functions, since it is able to modulate the stimulus-induced increase of intracellular Ca++ levels, one of the first steps of the cell activation mechanism. In fact, on SK-N-SH neuroblastoma cells, IL-1beta can inhibit the Ca++ increase induced by stimulation of acetylcholine receptors with carbachol. In parallel to IL-1beta, the neurotrophic factor CNTF also shows an inhibitory effect on carbachol-stimulated Ca++ increase in CNTFRalpha-expressing SK-N-SH cells. However, when simultaneously present, the two cytokines cross-inhibit, thus allowing full cell activation in response to the cholinoceptor agonist. The inhibitory effect of CNTF on IL-1beta activities on nervous cells was confirmed in the IL-6 production assay. In fact, while CNTF could not induce IL-6 production, it could strongly inhibit cytokine production in response to IL-1beta in SK-N-SH cells. The down-modulation of IL-1 effects by CNTF could be one of the mechanisms controlling the extent of the inflammatory reaction at the nervous system level.

Structure-function relationship in the IL-1 family.

The interleukin 1 (IL-1) family is a group of related cytokines including two agonist proteins (IL-1alpha and IL-1beta), each derived by enzymatic cleavage of precursor proteins (pro-IL-1alpha and pro-IL-1beta), and three forms of an antagonist protein (IL-1ra, icIL-1raI, icIL-1raII). IL-1 plays a key role in the onset and development of the host reaction to invasion, being an important factor in the initiation of the inflammatory response and in the triggering of immune functions. Due to its pleiotropic activity and to the high potency of its inflammatory effects, IL-1 activity is tightly regulated in the body by a complex network of control systems. These include the presence of two types of inhibitors, the receptor antagonist IL-1ra and the second type of IL-1 receptor (IL-1RI), which is a natural scavenger of IL-1. Furthermore, regulation of IL-1 activity is attained by a strict hierarchy of binding affinity of the two receptors (the activating IL-1RI and the inhibitory IL-1RII) for the various members of the IL-1 family. Additional levels of control are represented by the presence of soluble forms of both receptors and of immature pro-IL-1 forms with different characteristics of activity and receptor binding capacity. To clarify the features of reciprocal interaction among ligands and receptors, in the attempt to understand the rules regulating the IL-1 system and its effectiveness, a deep analysis of the relationship between structure and function in the proteins of the IL-1 family becomes of key importance. Information on this line has been provided by several groups mainly with studies of mutagenesis of IL-1alpha, IL-1beta and IL-1ra in parallel with biological assays of activity. In this review, a survey of the available data is provided, in order to construct a hypothetical model of the functional structure of IL-1 proteins as a basis for future therapeutic interventions based on genetic and protein engineering.

Transfected type II interleukin-1 receptor impairs responsiveness of human keratinocytes to interleukin-1.

Of the two known types of specific receptors for interleukin (IL)-1, the function of the type II IL-1 receptor (IL-1RII) is still elusive. IL-1RII is allegedly devoid of signaling capacity and is therefore thought to act by trapping and inhibiting IL-1. To directly assess the functional role of IL-1RII, a human keratinocyte cell line has been stably transfected with a cDNA coding for IL-1RII, and its responsiveness to IL-1 has been compared with that of nontransfected cells. Parental cells express IL-1RI and are responsive to low doses of IL-1, whereas transfected cells overexpress IL-1RII, both in its membrane and soluble form, and show a dramatically impaired response to IL-1. Selective block of IL-1RII restores the ability of transfected keratinocytes to respond to IL-1, indicating that the overexpressed IL-1RII is in fact uniquely responsible for their refractoriness to IL-1. The main mechanism of unresponsiveness in transfected keratinocytes appears to be the capture and neutralization of IL-1 by the soluble form of IL-1RII.

Sporulation: an alternative way to recover recombinant proteins from Bacillus subtilis.

A recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin-1 receptor antagonist (IL-Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step.