VDR), most genetic studies have primarily focused on variation within this gene. Therefore, analysis of genetic variation in VDR and other vitamin D pathway genes may provide insight into the role of vitamin D in renal cell carcinoma (RCC) etiology. RCC cases (N = 777) and controls  were genotyped to investigate the relationship between RCC risk and variation in eight target genes. Minimum-p-value permutation (Min-P) tests were used to identify genes associated with risk. A three single nucleotide polymorphism (SNP) sliding window was used to identify chromosomal regions with a False Discovery Rate of <10%, where subsequently, haplotype relative risks were computed in Haplostats. Min-P values showed that VDR  and retinoid-X-receptor-alpha (RXRA)  were associated with RCC risk. Within VDR, three haplotypes across two chromosomal regions of interest were identified. The first region, located within intron 2, contained two haplotypes that increased RCC risk by approximately 25%. The second region included a haplotype  across intron 4 that increased risk among participants with the TC  haplotype compared to participants with the common haplotype, TT. Across RXRA, one haplotype located 3′ of the coding sequence , increased RCC risk 35% among individuals with the variant haplotype compared to those with the most common haplotype. This study comprehensively evaluated genetic variation across eight vitamin D pathway genes in relation to RCC risk. We found increased risk associated with VDR and RXRA. Replication studies are warranted to confirm these findings.In the kidney vitamin D is converted to its active form. Since vitamin D exerts its activity through binding to the nuclear vitamin D receptor ( Given the importance of vitamin D metabolism in the kidney and the lack of studies that have evaluated the relationship between vitamin D, the VDR gene, and RCC, we previously evaluated how three common VDR gene polymorphisms  modified RCC risk f alleles in the FokI single-nucleotide polymorphism (SNP) compared with subjects with the FF genotype VDR in our previous candidate SNP investigation, and the numerous genes that interact directly with VDR in the vitamin D pathway, we expanded upon these findings to investigate variation in candidate genes that may interact directly with VDR to modify RCC risk and improved our analysis by increasing regional coverage of variation across each gene to 80–90%.Since 1,25(OH)VDR, RXRA, RXRB, CYP24A1, GC, STAT1, THRAP4, and TRAP5) of the vitamin D pathway among cases and controls from Central Europe, an area with one of the highest rates of RCC worldwide Therefore, to determine whether variation in vitamin D pathway genes modified RCC risk, we comprehensively investigated the relationship between renal cancer risk and 139 tagging SNPs across eight target genes (2) and hypertension, and to have a first degree relative with cancer. The association with smoking was no longer observed after adjustment for age, BMI, hypertension, study center, and sex A description of study participants and known RCC risk factors is provided in VDR  and RXR-alpha (RXRA)  genes were most strongly associated with RCC risk. Both the minimum p-value for all tagging SNPs within the gene and minimum false discover rate (FDR) adjusted p-values for the three SNP haplowalk sliding window for VDR and RXRA were significantly associated with RCC risk.VDR haplotypes  were identified in two regions that were associated with increased RCC risk. The first region  or GAC  haplotype were at a significantly increased risk compared to patients with the most common referent haplotype, GAT. Increased risk appeared to be driven by the T to C change at the third loci. The second region  haplotype compared to participants with most common referent haplotype, TT. The R adjusted global p-values for both of these regions were 0.04 . Across the RXRA gene , a single region located downstream, 3′ of the coding sequence, was shown to be associated with increased RCC risk among individuals with the CG  haplotype compared to subjects with the most common haplotype, GA. The R adjusted global p-value for this region was 0.03.Subsequently, HaploWalk based methods were used to identify regions of interest in each gene that remained significant at an FDR of less than 10%. Three significant plotypes Table 3Table S1. Thirteen SNPs in five vitamin D pathway genes were significantly associated with RCC risk using an additive model (number of significant tag SNPs): VDR(5), RXRA(5), CYP24A1(1), GC(1), and STAT1(1). No statistically significant interactions between these SNPs and potential confounders  were detected (data not shown). No difference in association between individual SNPs and RCC risk was observed when analyses were restricted to RCC cases histologically diagnosed with clear cell subtype (data not shown) nor when analyses were restricted to cases with only high grades tumors (data not shown).VDR and RXRA, for which we hypothesized a priori would be associated with risk, contained regions with significant associations. Across the VDR gene, three haplotypes within two regions (intron 2 and intron 4) were significantly associated with increased risk. Across the RXRA gene, RCC risk was higher among those with one particular haplotype located 3′ of the coding region.In this analysis of 139 tagging SNPs across eight vitamin D pathway genes, we identified three regions within two genes that were significantly associated with RCC risk. Both genes, VDR gene in relation to cancer VDR gene; twenty-two SNPs were genotyped to capture a high percentage of variation in the VDR gene VDR loci (rs2107301 and rs2238135), which were located within introns 2 and 4, respectively VDR variants and RCC risk, genetic susceptibility studies for common VDR polymorphisms, such as BsmI, FokI, TaqI, and ApaI, have been shown to modify RCC risk AA genotype at the ApaI site had a significant increase in RCC risk ; cases with the AA genotype also were found to have lower survival rates  compared to cases with the Aa or aa genotypes TT genotype at the TaqI site in a second case-control study of 102 RCC patients compared to subjects with the Tt or tt genotype  Only a handful of studies have comprehensively analyzed genetic variation in the VDR gene, such as poly(A), TaqI, and BsmI, have been speculated to result in variation of VDR expression and are hypothesized to subsequently result in changes to circulating levels of active vitamin D VDR protein expression has been reported in a number of tumor types including breast, malignant gliomas, prostate, and colon cancer VDR is associated with decreased RCC risk, and that active levels of vitamin D3 in serum are significantly lower in RCC patients compared to population controls RXR gene and cancer risk in vitro animal and human studies have shown that elevated levels of RXRA increases the antiproliferative effects of 1,25(OH)2D3Similarly, few previous studies of RCC risk and variation across vitamin D pathway genes have been conducted. A small number of polymorphisms in the In vitro and animal studies suggest that vitamin D and its metabolites may impede carcinogenesis by stimulating cell differentiation, inhibiting cell proliferation typically characterized by G0/G1 cell cycle arrest, apoptosis, and suppression of invassion, angiogenesis, and metastasis VDR and RXRA gene variants in our study may be small, some of these variants are relatively common in the population and therefore may be associated with a much higher attributable risk as a whole compared to rare high penetrance genes. Furthermore, the VDR variants associated with RCC risk in this study were intronic and were not located near intron-exon boundaries that may produce splicing errors. These alleles seem to have no effect on the expression levels or activity of translated VDR protein and it is hypothesized that they are correlated with other variants with functional relevance. Synonymous SNPs are often disregarded in many genetic susceptibility studies based on the assumption that they do not alter amino acid sequence, which would subsequently affect protein structure and function, nor protein expression Although the magnitude of association between RCC risk and VDR and seven other vitamin D pathway genes in relation to RCC risk, an additional strength of our study includes the use of HapMap data to tag genes of interest using high (80–90%) genomic coverage by tagging genomic regions, both upstream (20 kb) and downstream (10 kb) to reduce chance of false negative findings. In this study, we also observed high participation rates, used newly diagnosed cases, included only histologically confirmed cancers, and collected biologic materials from a high proportion of subjects. The large sample size of this study provided sufficient statistical power to detect relatively small associations between genotypes and risk. Analysis of genes with significant global p-values reduced the risk of Type I errors in our study, while the use of two different Haplotype-based methods  reduced the possibility of finding significant results based on chance. Furthermore, while hospital-based case-control studies have potential limitations due to the lack of population controls, these studies can improve response rates for the intense collection of biological specimens and therefore reduce the chances of bias in the assessment of gene-environment interactions In addition to being one of the first genetic susceptibility studies to comprehensively examine the association between a priori hypothesis was supported and we observed that genetic variations in VDR and RXRA were significantly associated with RCC risk. However, since our results are observed within introns rather than exons and because we are only beginning to understand the role of genetic variation in intronic regions and their effect on disease development, additional studies of genetic variations of these two genes and RCC risk are warranted.In conclusion, our Details of the Central and Eastern European Renal Cell Carcinoma (CEERCC) study have been previously reported Cases between 20 and 79 years of age were ascertained through a rapid reporting system. Participating physician and other professional staff members visiting the urology, surgery, radiology, or pathology departments of participating hospitals regularly identified patients admitted for kidney cancer work-up. All patients suspected of having kidney cancer were approached for interview, but only histologically confirmed cases were included in the final analysis. All tumors were centrally reviewed to confirm diagnosis and histology. The majority of renal tumors were clear cell carcinomas (83.4%), while other subtypes included papillary (7%), chromophobe (2.4%), oncocytoma (2.3%), oncocytic neoplasms (0.2%), transitional cell carcinomas (1.1%), and unclassified (3.6%). Controls were frequency-matched to cases on age (±3 years), sex, and study center. A total of 1,097 confirmed incident RCC cases and 1,476 controls were included in this study. Genomic DNA was obtained from 987 of 1,097 (90%) of cases and 1,298 of 1,476 (88%) of controls. The response rates across study centers among eligible subjects who were requested to participate ranged from 90.0–98.6% for cases and 90.3–96.1% for controls.Cases and controls were interviewed with the same questionnaire www.illumina.com) at the National Cancer Institute's (NCI's) Core Genotyping Facility (CGF) where lab staff was blinded to cases/control status. Suitable quantity and quality of genomic DNA was obtained from a subset of 777 (70.8%) RCC cases and 1,035 (70.1%) controls, due to strict quality and quantity criteria for analysis using the Illumina® OPA panel.Genomic DNA was extracted from whole blood buffy coat using a standard phenol chloroform method. Genotyping was conducted with a GoldenGate® Oligo Pool All (OPA) assay by Illumina®  that were ultimately selected for their involvement in vitamin D metabolism, transport, binding, function and/or expression, which may affect the mechanisms through which vitamin D may influence cancer risk. Since it is well known that VDR elicits a transcriptional response by forming a heterodimer complex with the retinoid-X-receptor, in this study both VDR and RXR were the primary genes suspected a priori to modify renal cancer risk. The group specific component (GC) vitamin D binding protein was selected for its involvement as the major carrier of vitamin D in plasma CYP24A1) was selected for its involvement in degrading and regulating 1,25(OH)2D3 levels STAT1) and the thyroid hormone receptor associated protein 4 and 5 (THRAP4 and THRAP5) genes were selected since studies indicate that these genes may induce or suppress transcription by interacting with the VDR-RXR complex A literature review of vitamin D associated genes helped identify eight candidate genes (http://www.hapmap.org) among SNPs with minor allele frequencies of at least 5% and an r2≥80% were selected. Additionally, non-synonymous SNPs or those correlated with polymorphisms with potential functional significance were included in analysis. In total, 139 SNPs in eight vitamin D-related pathway target genes were selected for analysis. The total length of sequence analyzed across all genes was 495,664 bases . Replicate quality control samples (5% samples) were interspersed among genotyping plates. The genotyping failure rate for the selected SNPs was 4.8% (7/146). The concordance rate between duplicate DNA samples ranged from 93–100% and completion rates ranged from 98–100%. The genotype frequencies among controls did not differ from the expected Hardy-Weinberg equilibrium proportions (p>0.05). All SNPs and assay information are reported in NCI's SNP500Cancer Database (http://snp500cancer.nci.nih.gov) Tag SNPs were selected to provide 80–90% genomic coverage across the genomic regions of interest. To ensure thorough coverage of the targeted region, additional coverage of regions both upstream (20 kb) of the start of transcription and downstream (10 kb) of the last exon using HapMap CEU data  were compared between genotyped cases and controls using the Chi-square test. These characteristics were further evaluated to determine if associations between SNPs and RCC were modified; however, no significant interactions were observed between cancer risk, RCC risk factors, and SNPs (data not shown).2>0.85). Of the 139 SNPs genotyped, two pairs of SNPs in the VDR gene were found to be highly correlated (r2 = 0.95 for rs1544410 (BsmI) and rs731236 (TaqI); r2 = 0.89 for rs2248098 and rs2239185) and therefore only one SNP was used per pair (rs1544410 (BsmI) and rs2248098).The associations between individual SNPs and RCC risk were estimated by calculating odds ratios (ORs) and 95% confidence intervals (95% CI) using unconditional logistic regression. Interactions were tested comparing regression models with and without interaction terms using a likelihood ratio test (LRT). All regression models were adjusted for age (continuous), sex, study center, and smoking habit . Additional risk factors  were also considered, however, these variables did not affect risk estimates by at least 10%. To avoid redundant analyses, only one SNP was evaluated when high correlations were observed between two SNPs . Similarly, analyses restricted to cases with only high grades tumors did not affect risk estimates (data not shown).S6.Risk estimates were calculated for the heterozygous and homozygous variant genotypes relative to the common homozygous genotype assuming an additive model. When the frequency of the homozygous variant allele was less than 5% among controls, a dominant model was used to determine risk estimates for the presence and absence of the variant allele. Associations between SNP variants and RCC were assessed using an additive model ) or a dominant model ). To assess global significance of association with genes, a minimum p-value permutation (Min-P) test was used for analysis because it corrects for multiple testing while also accounting for correlations between SNPs within a gene K SNPs. The HaploWalk procedure considered a three SNP window for each SNP from SNP 2 through SNP K-1. For each window, haplotype frequencies in cases and controls were reconstructed using the EM algorithm K SNPs, the K-2 p-values (one for each window) were adjusted for multiple comparisons using the False Discovery Rate (FDR)-controlling procedure of Benjamini and Hochberg HaploWalk, a haplotype-based sliding window analytic method, was used to evaluate candidate genes with N = http://www.r-project.org) 2 using Haploview among population controls In stage two, haplotype blocks identified with HaploWalk were analyzed in relation to RCC risk using Haplostats Click here for additional data file.Figure S1RXRB HaploWalk and Haploview analysis. Top: HaploWalk analysis identified no chromosomal regions of interest in the retinoid-X-receptor-beta (RXRB) gene that were significant at an FDR of 10%. The Min-P test for this gene was not statistically significant . Bottom: Haploview analysis identified two haplotype blocks; none were associated with RCC risk when analyzed in Haplostats.(0.24 MB TIF)Click here for additional data file.Figure S2CYP24A1 HaploWalk and Haploview analysis. Top: HaploWalk analysis identified no chromosomal regions of interest in the cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) gene that were significant at an FDR of 10%. The Min-P test for this gene was not statistically significant . Bottom: Haploview analysis identified several haplotype blocks; none were associated with RCC risk when analyzed in Haplostats.(0.76 MB TIF)Click here for additional data file.Figure S3GC HaploWalk and Haploview analysis. Top: HaploWalk analysis identified no chromosomal regions of interest in the group specific component (GC) vitamin D binding protein that were significant at an FDR of 10%. The Min-P test for this gene was not statistically significant . Bottom: Haploview analysis identified one haplotype block which was not associated with RCC risk when analyzed in Haplostats.(2.86 MB TIF)Click here for additional data file.Figure S4STAT1 HaploWalk and Haploview analysis. Top: HaploWalk analysis identified a single chromosomal region of interest in the signal transducer and activator of transcription 1 (STAT1) gene that was significant at an FDR of 10%. This region included SNPs 3′- rs3755821, rs6751855, rs1467199, rs13029532 -5′. The Min-P test for this gene was not statistically significant . Bottom: Haploview analysis also identified one haplotype block within the same chromosomal region of interest. Haplostats analysis adjusted for age, sex, study center, and smoking habit showed that subjects with the CGGA haplotype had an increased risk of RCC  compared to subjects with the referent haplotype, GACA.(1.76 MB TIF)Click here for additional data file.Figure S5THRAP4 HaploWalk and Haploview analysis. Top: HaploWalk analysis identified no chromosomal regions of interest in the thyroid hormone receptor associated protein 4 (THRAP4) gene that were significant at an FDR of 10%. The Min-P test for this gene was not statistically significant . Bottom: Haploview analysis identified one haplotype block which was not associated with RCC risk when analyzed in Haplostats.(1.42 MB TIF)Click here for additional data file.Figure S6THRAP5 HaploWalk and Haploview analysis. Top: HaploWalk analysis identified no chromosomal regions of interest in the thyroid hormone receptor associated protein 5 (THRAP5) gene that were significant at an FDR of 10%. The Min-P test for this gene was not statistically significant . Bottom: Haploview analysis identified several haplotype blocks; none were associated with RCC risk when analyzed in Haplostats.(0.64 MB TIF)Click here for additional data file.


LAMB2).We describe a severe form of congenital myasthenic syndrome (CMS) associated with congenital nephrosis and ocular malformations caused by two truncating mutations in the gene encoding the laminin β2 subunit (1478delG and a paternally inherited 4804delC. An anconeus muscle biopsy demonstrated a profound distortion of the architecture and function of the neuromuscular junction, which was strikingly similar to that seen in mice lacking laminin β2 subunit. The findings included: pronounced reduction of the axon terminal size with encasement of the nerve endings by Schwann cells, severe widening of the primary synaptic cleft and invasion of the synaptic space by the processes of Schwann cells, and moderate simplification of postsynaptic folds and intact expression of the endplate acetylcholinesterase. The endplate potential quantal content was notably reduced, while the frequencies and amplitudes of miniature endplate potentials were only moderately diminished and the decay phases of miniature endplate potentials were normal. Western blot analysis of muscle and kidney tissue and immunohistochemistry of kidney tissue showed no laminin β2 expression.Mutational analysis in the affected patient, who has a history of a serious untoward reaction to treatment with acetylcholinesterase inhibition, revealed two frame-shifting heteroallelic mutations, a maternally inherited LAMB2 mutations and underscores the fundamental role that laminin β2 plays in the development of the human neuromuscular junction.This case, which represents a new type of synaptic CMS, exemplifies the wide variability of phenotypes associated with  CHRNG (MIM 100730), CHRNA1 (100690), CHRNB1 (100710), CHRND (100720), and CHRNE (100725)), rapsyn (RAPSN (601592)), MuSK (MUSK (601296)), Dok-7 (DOK7 (610285)), and the skeletal muscle sodium channel (SCN4A (603967)).–CHAT (118490)), and the collagenic tail subunit of the acetylcholinesterase (COLQ (603033)) have been found to associate with presynaptic CMS and synaptic basal-lamina-associated CMS, respectively.Congenital myasthenic syndromes (CMS) represent a diverse group of neuromuscular disorders characterised by varying degrees of muscle weakness and fatigability resulting from impaired neuromuscular transmission. These syndromes are classified into presynaptic, synaptic basal-lamina-associated, and postsynaptic subgroups depending on which compartment of the neuromuscular junction (NMJ) is primarily targeted by the disease.HRNG MIM 00730, CHDOK7 61025), and tRNA1 10060, CHRNB1LAMB2 (150325)) have been found to be responsible for Pierson syndrome (MIM 609049), which is characterised by congenital nephrosis and ocular defects.LAMB2 defects are truncation mutations, which result in a complete lack of laminin β2 immunoreactivity in the glomerular basal membrane as well as a severe phenotype that leads to death within days or months after birth.10Mutations in the gene encoding the laminin β2 subunit (LAMB2.−/−) were negative, and a brain magnetic resonance image (MRI) was normal. A trial with a cholinesterase inhibitor resulted in profound weakness requiring hospitalisation and ventilatory support; however, she responded well to treatment with ephedrine. When she was 9 years old she required corrective surgery for severe ptosis. At that time an ophthalmologic examination revealed pronounced miosis, myopia and impaired visual acuity in both eyes. Fundoscopic examination revealed hypoplastic macular areas and poor foveal reflex, but her electroretinogram was normal. A neurologic examination revealed normal cognition, impaired visual acuity and reactive pinpoint pupils. Her external ocular movements were limited to 15–20° in the horizontal axis, and she had bilateral ptosis. She showed no bulbar deficit, but the motor examination revealed severe proximal limb weakness. She had spine surgery for scoliosis at the age of 12, and since then she has used bi-level positive airway pressure during night sleep and intermittently during the day.To elucidate the nature of the CMS in this patient, we performed an anconeus muscle biopsy at age 10, which included in vitro microelectrode recordings as previously described.2 (17) in the patient was reduced in comparison with the mean endplate area of 227.03 (11.9) μm2 (40) in controls.Except for occasional small angular fibres and type I fibre predominance there were no other histological abnormalities. The acetylcholinesterase reaction performed in teased muscle fibre bundles revealed, as in control muscles, only one endplate per muscle fibre; however, the mean endplate area of 169.9 (16.6) μmThe most consistent structural abnormality of the NMJ shown by electron microscopy were: (1) small axon terminal size and encasement of nerve endings by the Schwann cell; (2) severe widening of the primary synaptic clefts with invasion of the synaptic space by processes of Schwann cells; and (3) moderate simplification of the postsynaptic membranes . The smaThe morphometric analysis of the NMJ was performed as previously describedLAMB2 mutations . This has allowed the clinical manifestations of the failure of neuromuscular transmission to emerge.−/−Lamb2 mice. Thus, the extensive analysis performed in −/−Lamb2 and the conclusions drawn from this animal model provide an invaluable key to understanding the pathogenesis of the human neuromuscular disease reported here. First, as in −/−Lamb2 mice, our patient showed immature hypoplastic nerve terminals, which in the animal model have been attributed to the fact that laminin β2 acts as a potent “stop signal” for motor neurite growth and promotes presynaptic differentiation in vitro.v) subunit of the voltage gated calcium channel.−/−Lamb2 mice has been interpreted in the animal model as resulting from the loss of the inhibitory effect that the β2 chain containing laminin-11 exerts on Schwann cell propagation.−/−Lamb2, there was a simplification of the postsynaptic folding pattern. This simplification of postsynaptic folds may result from the lack of an interaction between β2 chain containing laminins and cell surface receptors for laminins such as integrins and dystroglycan that participate in the development and maintenance of the synaptic scaffold.13In terms of the pathogenesis of this CMS, the structural changes and the functional abnormalities of the NMJ encountered in our patient are strikingly similar to those reported in The URLs for data presented herein are:http://www.ncbi.nlm.nih.gov/National Center for Biotechnology Information (NCBI): http://www.ncbi.nlm.nih.gov/Omim/Online Mendelian Inheritance in Man (OMIM): http://blast.ncbi.nlm.nih.gov/Blast.cgiBasic Local Alignment Search Tool (BLAST): http://www.ncbi.nlm.nih.gov/sites/entrezEntrez Single Nucleotide Polymorphism Database: −/−Lamb2 mutant, while rescuing the structure of the NMJ with a muscle specific β2 transgene (MCK-B2) extends life by 50%.Using tissue specific laminin β2 transgenes, it was recently demonstrated that salvaging the glomerular defects with a podocyte specific β2 transgene (NEPH- β2) does not significantly improve the phenotype of the LAMB2 and long survival due to successful renal replacement therapy. From a therapeutic perspective, drugs that increase the release of neurotransmitter from the nerve terminal, such as 3,4-diaminopyridine, may be beneficial, but should be given with caution to patients who, like ours, have profound distortion of the function and architecture of the NMJ.Since we see no obvious central nervous system defects in humans, and since the ocular abnormalities are not lethal, the NMJ disease may become the most serious complication in patients with biallelic truncation mutations in


Understanding the pathogenesis of infection by neurotropic viruses represents a major challenge and may improve our knowledge of many human neurological diseases for which viruses are thought to play a role. Borna disease virus (BDV) represents an attractive model system to analyze the molecular mechanisms whereby a virus can persist in the central nervous system (CNS) and lead to altered brain function, in the absence of overt cytolysis or inflammation. Recently, we showed that BDV selectively impairs neuronal plasticity through interfering with protein kinase C (PKC)–dependent signaling in neurons. Here, we tested the hypothesis that BDV phosphoprotein (P) may serve as a PKC decoy substrate when expressed in neurons, resulting in an interference with PKC-dependent signaling and impaired neuronal activity. By using a recombinant BDV with mutated PKC phosphorylation site on P, we demonstrate the central role of this protein in BDV pathogenesis. We first showed that the kinetics of dissemination of this recombinant virus was strongly delayed, suggesting that phosphorylation of P by PKC is required for optimal viral spread in neurons. Moreover, neurons infected with this mutant virus exhibited a normal pattern of phosphorylation of the PKC endogenous substrates MARCKS and SNAP-25. Finally, activity-dependent modulation of synaptic activity was restored, as assessed by measuring calcium dynamics in response to depolarization and the electrical properties of neuronal networks grown on microelectrode arrays. Therefore, preventing P phosphorylation by PKC abolishes viral interference with neuronal activity in response to stimulation. Our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons. Neurotropic viruses have evolved diverse strategies to persist in their host, with variable consequences for brain function. The investigation of these mechanisms of persistence and associated disease represent a major issue in viral pathogenesis, as it may also improve our understanding of human neurological diseases of unclear etiology for which viruses are thought to play a role. In this study, we have examined the mechanisms whereby the neurotropic Borna disease virus (BDV) can selectively interfere with synaptic plasticity upon infection of neurons. Using genetically engineered recombinant viruses, we show that the phosphorylation of BDV phosphoprotein (P) by the cellular protein kinase C (PKC) is the main determinant for this interference, mainly by competing with the phosphorylation of the natural PKC substrates in neurons. A mutant virus in which the PKC phosphorylation site of P has been destroyed no longer interferes with this signaling pathway. As a result, the calcium dynamics and electrical activity in response to stimulation of neurons infected with this mutant virus are completely corrected and become similar to that of non-infected neurons. Thus, our findings uncover a previously undescribed mechanism whereby a viral protein interferes with neuronal response to stimulation. The finding that persistent viruses could selectively affect differentiated functions of their target cell without causing cell lysis or widespread inflammation was first demonstrated more than 25 years ago Mononegavirales, BDV replicates in the nucleus of infected cells Borna disease virus (BDV) is a highly neurotropic, non-cytolytic virus that provides an ideal paradigm for studying the behavioral correlates of CNS viral infections. BDV is an enveloped virus with a non-segmented, negative strand RNA genome Mononegavirales, BDV P is a component of the viral polymerase complex, which serves several functions in the viral life cycle. These functions are thought to be regulated, at least in part, by its phosphorylation by cellular kinases In an effort to better characterize the impact of BDV persistence on neuronal function, we recently analyzed the neuronal activity of primary cultures of neurons infected with BDV, using both functional imaging and electrophysiological approaches Mononegavirales. Here, we infected primary cultures of rat hippocampal and cortical neurons with these different recombinant viruses and analyzed their phenotype and responses to stimulation. Using a rBDV where P can no longer be phosphorylated by PKC, we demonstrate a complete suppression of viral interference with neuronal activity. This was shown not only at the molecular level in terms of PKC-dependent signaling, but also at the functional level by studying calcium responsiveness to depolarization and the electrophysiological network properties of cultured neurons.The newly established reverse genetics technique allowing the generation of recombinant BDV (rBDV) entirely from cDNA has provided means to test this hypothesis directly Thus, our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons.To study the impact of BDV P phosphorylation on viral neuropathogenesis, we used a recombinant BDV in which the two serine (S) residues in position 26 and 28 of P have been replaced by alanine (A) residues (rBDV-AASS). We have previously shown that these mutations, when introduced using the recently established technique for generating BDV from cDNA We first analyzed the impact of these mutations on the kinetics of viral spread in primary hippocampal rat neuronal cultures. To this end, we infected neuron-rich cultures (>80% neurons) one day after their plating with 300 focus forming units (FFU) per well of either rBDV-wt or rBDV-AASS cell-released virus (prepared from persistently infected Vero cells). We studied viral dissemination occurring after primary infection by assessing expression of the BDV nucleoprotein at different times post-infection, using immunofluorescence microscopy to quantify viral spread. Consistent with our previous reports using wild-type BDV Importantly, despite the delayed kinetics for rBDV-AASS, infection ultimately disseminated to the whole neuronal cultures. By 20–21 days post-infection, the large majority of neurons was positive for BDV antigens, with no significant differences between cultures infected with either rBDV-wt or rBDV-AASS, as assessed by immunofluorescence analysis . At thisIn conclusion, both recombinant viruses were able to infect the totality of our neuronal cultures, albeit with a delayed kinetics for rBDV-AASS. For our subsequent signaling and functional studies, we therefore used neurons that had been infected for at least 21 days and verified for each experiment that infection was indeed complete prior to any subsequent analysis.Recently, we have shown that BDV-induced impairment of potentiation of synaptic activity was due to an interference with the PKC-dependent phosphorylation of synaptic proteins that modulate neuronal activity SNAP25 is a synaptic protein that plays an essential role in neurotransmitter release through regulation of synaptic vesicle exocytosis Since P is expressed from a bicistronic mRNA encoding P and X, the introduced mutation into the open reading frame of P in the rBDV-AASS mutant also resulted in two amino acid substitutions in the X protein . We prevA receptor antagonist. Treatment with this antagonist leads to the removal of the tonic inhibition imposed by GABAergic interneurons on the network The S26/S28A mutation of BDV P corrects the defects in electrical activity observed in rBDV-wt infected neurons. Given the dramatic consequences of the S26/28A mutation on neuronal signaling and calcium responsiveness, we next studied its impact on the electrophysiological properties of infected neurons. Recently, we described a cell culture system using microelectrode arrays (MEA), which allows to monitor the firing pattern of a neuronal network grown on a grid of sixty electrodes embedded in a culture dish  [36],i was calculated using the equation [Ca2+]i = Kd (F−Fmin)/(Fmax−F), where Kd is the dissociation constant of the Ca2+-fluo-3 complex (390 nM), and F represents the fluorescence intensity of the cells expressed as the ratio between the highest fluorescence measurement between 10 and 90 s and the baseline. Fmin corresponds to the minimum fluorescence between 90 and 177 s. Fmax represents the maximum fluorescence between 177 and 250 s (see Neurons grown on flat-bottom 96-well plates were incubated for 30 min at 37°C in HBSS (Invitrogen) –BSA (Sigma) solution containing 2.5 mM probenecid (Sigma), pH 7.45 supplemented with fluo-3 acetoxymethyl (AM) 1 mM (Invitrogen) and 20% pluronic F-17 (Sigma). After this incubation period, neurons were washed twice with HBSS-BSA-probenecid solution and placed into a 37°C incubator in the dark for 30 min. Fluorescence was measured at 460–490 nm excitation and 515 nm emission in each well, using a Novostar plate reader . For each well, a series of 250 recordings (one per second) was performed; neurons were first exposed to 50 mM KCl (Sigma) from 10 to 90 s, then to HBSS-BSA-probenecid solution containing 50 µM thapsigargin , 5 µM ionomycin  and 10 µM EGTA (Sigma) from 90 to 177 s and finally to HBSS-BSA-probenecid solution containing 120 mM CaCl50 s see .5 cells per MEA and half of the MEA dishes were infected with BDV on day 1. All experiments were made on day 21, to allow spreading of the different recombinant viruses to the totality of the MEA dishes. Signals corresponding to the electrical activity from the 60 electrodes of the MEA were recorded using the MC Rack Software  for online visualization and raw data storage. The signal corresponding to the firing of a single action potential by a neuron in the vicinity of an electrode was identified as a spike. We also detected high frequency grouped spikes trains, known as bursts, which represent an important parameter of the analysis of neuronal network activity Neuronal cortical cultures were prepared from embryonic Sprague Dawley rats at gestational day 18, according to a previously described protocol t-test.Data are presented as mean±standard error of the mean (s.e.m). Statistical significance was determined using Student's unpaired http://www.expasy.org/sprot/) accession numbers for proteins mentioned in the text are BDV Nucleoprotein (Q01552), BDV Phosphoprotein (P26668), BDV X protein (Q912Z9), BDV matrix protein (P52637), BDV polymerase (P52639), MARCKS (P30009), SNAP-25 (P60881), PKC epsilon (P09216), CKII (P19139), TBK-1 (Q9WUN2). The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession number for the strain discussed in this paper is BDV strain He/80/FR (AJ311522).The SwissProt (


Simple and effective vaccine administration is particularly important for annually recommended influenza vaccination. We hypothesized that vaccine delivery to the skin using a patch containing vaccine-coated microneedles could be an attractive approach to improve influenza vaccination compliance and efficacy.Solid microneedle arrays coated with inactivated influenza vaccine were prepared for simple vaccine delivery to the skin. However, the stability of the influenza vaccine, as measured by hemagglutination activity, was found to be significantly damaged during microneedle coating. The addition of trehalose to the microneedle coating formulation retained hemagglutination activity, indicating stabilization of the coated influenza vaccine. For both intramuscular and microneedle skin immunization, delivery of un-stabilized vaccine yielded weaker protective immune responses including viral neutralizing antibodies, protective efficacies, and recall immune responses to influenza virus. Immunization using un-stabilized vaccine also shifted the pattern of antibody isotypes compared to the stabilized vaccine. Importantly, a single microneedle-based vaccination using stabilized influenza vaccine was found to be superior to intramuscular immunization in controlling virus replication as well as in inducing rapid recall immune responses post challenge.The functional integrity of hemagglutinin is associated with inducing improved protective immunity against influenza. Simple microneedle influenza vaccination in the skin produced superior protection compared to conventional intramuscular immunization. This approach is likely to be applicable to other vaccines too. Influenza virus causes serious respiratory disease, affecting 5–15% of the world population annually. The dose of currently used inactivated viral or detergent split vaccines is standardized based on the content of hemagglutinin of each vaccine strain. The efficacies of the vaccines in humans are also usually evaluated by immune responses to the hemagglutinin protein Vaccination is the most cost effective measure to prevent infectious diseases Previous intradermal vaccinations were performed using liquid injection devices  in vitro on inducing protective immunity in vivo are not well investigated.Recently, it was demonstrated that microneedle vaccination with inactivated influenza virus in the skin could induce similar protective immunity as intramuscular vaccination In this study, we hypothesized that vaccine integrity as represented by hemagglutination (HA) activity is a critical factor in inducing protective immune responses. We investigated the relationship between vaccine integrity and its immunogenicity, as well as the immunological differences between microneedle delivery of solid vaccine to the skin and intramuscular immunization with influenza vaccine in solution. We found that maintenance of HA activity in the vaccine was critically important in inducing isotype-switched antibodies and high levels of protective immune responses. In addition, results from this study suggest that microneedle vaccination in the skin using stabilized antigen provides insights into superior immunity against influenza infection, which might be applicable to vaccination with other antigens too.Most vaccines including influenza are administered in liquid form using a hypodermic needle. In contrast, microneedle-based vaccination involves coating vaccine onto microneedles using a drying process, and thus represents the delivery of vaccine in a solid state . This drWe next investigated the relationship between retention of HA activity of influenza vaccine and its immunogenicity after microneedle vaccination using both solid state formulations administered using MN or liquid state formulations intramuscularly. Groups of mice (n = 12) were immunized with a single dose of vaccine in the skin after coating the vaccine in the absence (MN) or presence of trehalose (MN+T) in the coating formulation . Also, to compare with the conventional intramuscular route of immunization (IM), two more groups (n = 12) were immunized with inactivated viral vaccine that was coated onto microneedles in the absence (IM) or presence of trehalose (IM+T), dissolved off the microneedles and then injected intramuscularly. A mock immunized control group with microneedle vaccination of coating buffer only in the skin was also included (n = 12).We determined vaccine (A/PR8 virus) specific total IgG and isotypes IgG1, IgG2a and IgG2b antibodies in sera at weeks 1, 2 and 4 after a single IM or microneedle vaccination in the skin. Trehalose-stabilized microneedle vaccination induced the highest levels of antibodies among the groups . TrehaloThe pattern of antibody isotypes indicates either T helper type 1 (Th1) or type 2 (Th2) like immune response corresponding to dominance of the IgG2a or IgG1 isotype antibody, respectively Levels of functional antibodies measured as hemagglutination inhibition (HAI) and/or virus neutralizing activities in immune sera are known to be better immune correlates for protection 50) at 5 weeks after a single microneedle vaccination in the skin or intramuscular immunization  affects protective immunity, vaccinated mice including a mock control were challenged with a lethal dose of A/PR8 virus  had lower lung viral titers by 20- and 75-fold compared to those in the mock-immunized control (p<0.01), but still showed lung viral titers in a range of from 2.9×10g tissue . The IM  control , p<0.005Acute infection by pathogenic influenza virus causes severe lung inflammation To better understand the significantly improved viral clearance in the hemagglutinin-stabilized microneedle group (MN+T), we determined virus-specific antibody levels in lungs and sera at day 4 post challenge infection . InteresHigher levels of both recall serum IgG1 and IgG2a antibodies were also observed in the group immunized by microneedle delivery with trehalose-formulated vaccine (MN+T) than those in the corresponding inramuscular immunization groups (p<0.05 between MN+T and IM+T for both IgG1 and IgG2a) . Increasin vitro stimulation of splenocytes with A/PR/8/34 hemagglutinin-specific MHC I or MHC II peptides, cytokine secreting cells were quantified , resulting in the induction of Th1 type immune responses Our data suggest that the functional integrity of hemagglutinin in the influenza vaccine may influence the types of antigen presenting cells. Th1 type immune responses are known to be induced most likely by pathogen interactions with receptors on host innate immune cells Intradermal influenza vaccination has attracted widespread interest. However, detailed immunologic studies have not been carried out. Vaccines delivered intradermally were effective against rabies, BCG, hepatitis B, and influenza antigens Microneedle-based delivery differs from intramuscular injection not only in the route of administration, but also in the preparation of the vaccine. To isolate out the effects of potential antigenic changes during microneedle vaccine preparation independent of the route of administration, we reconstituted vaccines from coated microneedles and injected them intramuscularly for comparison of immunogenicity and protective efficacies with microneedle vaccination to the skin. Using a trehalose formulation, microneedle vaccination in the skin was significantly more immunogenic than intramuscular immunization as evidenced by a range of different immunologic assays: binding and functional antibodies, post-challenge lung viral titers and inflammatory cytokine levels, and recall mucosal and systemic responses to influenza. Among the range of immunologic data, the recall immune responses to influenza in the microneedle group (MN+T) with a single vaccination to the skin were significantly stronger than intramuscular immunization (IM+T). After taking up intradermally delivered antigens, skin-derived dendritic cells are known to migrate to the systemic and mucosal compartments In summary, this study demonstrates that the integrity of influenza vaccine as represented by HA activities is a critically important factor for determining antibody isotypes, inducing functional antibodies, and providing effective protective immunity as well as recall immune responses to influenza. Also, our study provides insight into formulating a vaccine maintaining structural and conformational integrity through the use of trehalose as a stabilizer. Immunologic data from this study offers a partial explanation for improved protection by delivering vaccines to the skin. Finally, the results show that a solid formulation of an enveloped virus antigen with biologically active glycoproteins can be at least as effective as a liquid form of vaccine if an optimized stabilizer is used. In addition to possible immunologic advantages, solid microneedle vaccination may offer logistic advantages such as possible self-administration, less dependence on a cold-chain and highly trained medical personnel, and less pain compared to the conventional intradermal or intramuscular delivery of liquid form vaccines.Influenza virus, A/PR/8/1934 , was grown in 10-day old embryonated hen's eggs and purified from allantoic fluid by using a discontinuous sucrose gradient  layers. The purified virus was inactivated by mixing the virus with formalin at a final concentration of 1∶4000 (v/v) as previously described Stainless steel microneedles of approximately 700 µm in length  were fabTo determine the amount of inactivated virus vaccine coated on microneedles, vaccine-coated microneedles were incubated in PBS for 12 h at 4°C and the total protein content of the reconstituted vaccine from an array of five microneedles was measured by a DC protein assay kit . The retained HA activities were also determined in the reconstituted vaccines. Dissolved vaccines in PBS (50 µl) was serially diluted in 50 µl of PBS mixed with an equal volume of a fresh 0.5% suspension of chicken red blood cells  and incubated for 1 h at 25°C. The titers were determined as the endpoint dilutions inhibiting the precipitation of red blood cells.Female inbred BALB/c mice  aged 6 to 8 weeks were used. Groups of mice (12 mice per group) were immunized with coated microneedle vaccine  for delivery to the skin or reconstituted vaccines (0.4 µg protein/100 µl) for intramuscular immunization in the upper quadriceps muscles of mice (both legs each with 50 µl). The four groups of immunized mice were designated as trehalose-negative formulated microneedle vaccine (MN), trehalose-positive formulated microneedle vaccine (MN+T), trehalose-negative reconstituted vaccine (IM) and trehalose-positive reconstituted vaccine (IM+T) for intramuscular injection. For microneedle delivery, mice were anesthetized with ketamine  mixed with xylaxine . Hair on the dorsal surface of mice was removed by a hair-removing cream  with a moisturized cotton stick. After cleaning with a soaked cotton ball (70% ethanol) and drying with a hair dryer, an array of vaccine-coated microneedles was inserted into the skin and left in place for 10 min for release of the vaccine antigen from the microneedle.For challenge infections, mice lightly anesthetized with isoflurane were intranasally infected with a lethal dose of A/PR8 virus (20× LD50) in 50 µl of PBS at 5 weeks after a single dose immunization. Mice were observed daily to monitor changes in body weight and to record mortality. Mice with >25% loss of body weight were sacrificed to prevent undue suffering. These studies were approved by Emory University IACUC.Influenza virus-specific antibodies of different isotypes  were determined by enzyme-linked immunosorbent assay (ELISA) plates coated with A/PR8 viral antigen and by using anti-mouse IgG isotype specific secondary antibodies as described previously Virus neutralization assay was performed using MDCK cells  following a previously described procedure 6 cells per well of 96 well plates) were stimulated in vitro with A/PR8 hemagglutinin specific peptides Recall immune responses were determined from serum, lung, and spleen at day 4 post-challenge. To determine T cell responses, spleen cells were harvested from immunized and mock control mice at day 4 post-challenge and were used to determine cytokine producing T cell responses by ELISPOT as previously described P value less than 0.05 was considered to be significant.All parameters were recorded for individuals within all groups. A two-tailed Student's t-test was performed when comparing two different conditions. Statistical comparisons of three or more conditions were carried out using the correlation and regression test of the PC-SAS system . A


Prior structural studies of posterior subcapsular cataract (PSC) development in Royal College of Surgeons (RCS) rats suggest that migration of basal fiber ends was disrupted, ultimately resulting in a PSC. Therefore the goal of this study was to assess the overall migration patterns as well as changes to the structure and cytoskeleton of basal fiber ends during PSC development.+/Lav) from 2 to 8 weeks old were examined. Equatorial diameters were measured and suture patterns were photographed immediately following enucleation/dissection. Right eye lenses were fixed and processed to visualize the actin cytoskeleton via laser scanning confocal microcopy (LSCM), left eye lenses were decapsulated, fixed and processed for scanning electron microscopy (SEM). Scaled 3D-computer assisted drawings (CADs) and animations were constructed from the data to depict the changes in suture patterns and fiber end architecture.Lenses from 48 RCS dystrophic rats (RCS/Lav) and 24 genetically matched control animals  of control lenses showed the expected peripheral pattern of labeling at all ages. Dystrophic RCS lenses at 2 weeks were comparable to controls, however by 3–4 weeks they displayed scattered foci of F-actin within the BMC. At all time points thereafter, F-actin was rearranged into a ‘rosette’ pattern of prominent foci at cell vertices.The data are consistent with the hypothesis that migration of basal fiber ends is altered in a two stage process wherein initially, migration patterns undergo a rapid shift resulting in abnormal suture sub-branch formation. Subsequent cytological alterations are consistent with an eventual cessation of migration, precluding proper targeting of basal ends to their sutural destinations and leading to cataract plaque formation. Congenital retinal dystrophy in the Royal College of Surgeons (RCS) rat is a recessively inherited anomaly that results in the progressive degeneration of photoreceptor cells in the retina during the later stages of retinal development  -3. One oIn the retina, the first damage occurs approximately 12 days postnatal in the rod outer segments and by 18 days postnatal, the outer segment layer is twice as thick when compared to normal. There is also the presence of outer segment debris within this thickened layer. At about 4 weeks postnatal, this layer reaches its maximum thickness and both the inner segments as well as the photoreceptor nuclei begin to degenerate. By about 40 days postnatal, both inner and outer rod segments have completely disintegrated ,9-11. Th+/Lav=genetically matched controls) were acquired courtesy of Dr. M. LaVail, Retinal Degeneration Rat Model Resource (RDRMR), University of California, San Francisco, CA. Animals were housed in the Comparative Research Center of Rush University Medical Center (RUMC), Chicago, IL, and bred as per the schemata laid out by the RDRMR for breeding RCS strains. The animals were cared for in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and all experiments were performed under aseptic and sterile conditions as approved by the Institutional Care and Use Committee (IACUC) of RUMC, Chicago, IL. The RCS experimental animals were randomized into six groups  and genetically matched controls were randomized into three groups . Each group had eight animals with n=72. At the specific age points, animals were euthanized using an intra-peritoneal dose of sodium pentobarbital (1 ml @398 mg/ml).RCS rat breeding pairs . The equatorial diameter of all lenses was measured and lenses were photographed using a digital camera  operated by the Q Capture Pro Software  on a Pentium PC platform. All right eye (OD) lenses were processed for cytochemistry and all left eye (OS) lenses were processed for visualization via scanning electron microscopy (SEM). Statistical analysis was performed to determine significant changes in lens equatorial diameter.All OD lenses were fixed in 3% paraformaldehyde in 0.07 M phosphate buffered saline (PBS) for 2 h. Fixed lenses were secured on specimen mounting blocks posterior side-up using cyanoacrylate adhesive, embedded in 2.5% agarose gel and sectioned at 100 μm thickness in a vibrating knife microtome . Sections were fixed for an additional 30 min in 3% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 30 min and labeled for F-actin using Phalloidin-TRITC at 1:50 dilution . Labeled sections were mounted on glass coverslips  with Vectashield mounting medium  to prevent photobleaching. The coverslips were sealed to glass slides using a commercially available lacquer.All OS lenses were decapsulated from the anterior aspect to expose the posterior fiber ends. Decapsulated lenses were fixed in 2.5% glutaraldehyde fixative in 0.07 M sodium cacodylate buffer for 3 days with fresh fixative changes daily. Following fixation, lenses were washed in 0.1 M sodium cacodylate buffer, post-fixed in 1% aqueous osmium tetroxide at 4 °C, washed in buffer and dehydrated through a graded ethanol series. Following dehydration with 100% ethanol, the lenses were passed through a graded ethanol/Freon 113 series, critical-point dried in 100% Freon 23  in a Balzers Critical Point Drier 020 , secured on aluminum stubs with colloidal silver adhesive, sputter coated with gold  and examined in a scanning electron microscope . Digital electron micrographic magnification series were acquired from 50×-4000× for each specimen.Whole lens measurements, in conjunction with data from gross lens photographs and Confocal/SEM micrographs were used to create scaled 3 Dimensional-Computer Assisted Design (3D-CAD) drawings and animated models using Corel DRAW Graphics Suite  on a Pentium PC platform. These explain and depict the temporal sequence of changes to suture patterns, transparency and basal end architecture in the RCS rat lens during the development of PSCs.+/Lav lenses showed an average increase from 3.30 mm at 2–3 weeks, 4 mm at 4–6 weeks, and 4.51 mm at 7–8 weeks of age  with age from an average of 2.96 mm at 2–3 weeks of age to 3.43 mm at 4–6 weeks of age and 3.76 mm by 7–8 weeks post natal, while the control RCS-rdys of age . In gene+/Lav) showed the expected inverted Y suture pattern at all age groups . By 7–8 weeks postnatal, F-actin distribution reflected the structural damage seen by SEM and revealed areas of fiber end loss and abnormal stellate shape of the ends  formation. The changes in suture pattern included the formation of one or two suture sub-branches as early as 3 weeks postnatal and the addition of more sub-branches by 4 weeks. These changes in suture pattern were extremely rapid and were noticeable well before the PSC could be observed. In a previous study, we documented the rapid formation of such abnormal suture sub-branches in Wistar rats  and haveThe initiation of PSC formation begins with a small sutural or central opacity being observed as early as 5 weeks postnatal. The opacity continued to grow in dimensions until it circumscribed the extent of the posterior suture branches by about 6 weeks postnatal and completely obscured the posterior suture by 7–8 weeks postnatal . InteresAs expected, the evaluation of posterior fiber ends revealed distinct morphological changes during cataractogenesis, specifically, the shape, end to end packing, organized arrangement, and the filopodial configuration and direction showed a marked variation from normal. The first noticeable change at 3 weeks postnatal was the altered shape of the fiber ends. The irregularly spheroidal outlines were occasionally interspersed with areas characterized by ‘domed’ ends. Although these ends exhibited an appreciable difference in their morphology, they continued to maintain contact with the surrounding ends, presumably via adherens junctions. In fact, prior investigations have shown that the basal membrane complex (BMC) has ample cadherin which mediates interactions within and between cohorts of migrating fiber ends in normal lenses ,29. In 4It is known that the leading edge of a migrating cell is a site of rapid actin polymerization forming membrane extensions in the form of lamellipodia or filopodia that play a crucial role in directing the cell during migration -32. LikeCellular morphology is often dictated by the cytoskeleton, specifically actin configurations. To better understand the morphological changes that were observed in the RCS rat, cytochemical techniques were used to elucidate F-actin distribution in the BMC of the posterior fiber ends. As expected, F-actin arrangement in lenses up to 3 weeks postnatal was comparable to normal distributions seen in other rodent strains, and the outlines very succinctly revealed the normal shape, size and arrangement of the fiber ends, concomitant to that seen in the SEM data. By 4–6 weeks postnatal, F-actin underwent a very unique redistribution and was no longer present as an unbroken outline around the fiber ends; rather bright foci of actin fluorescence were visible at fiber end vertices in a distinctive ‘rosette’ arrangement . The relElongating lens fibers and migrating fiber ends typically show an increased association of F-actin at the membranes ,35, withAnother possible interpretation is that the rearrangement of F-actin occurs as a compensatory mechanism that prevents fiber end degeneration during PSC formation. As observed within the cataract plaque, the fiber ends revealed a dilated appearance  with disIn conclusion, this study elucidated the structural changes that affect migrating fiber ends in the RCS rat during the initiation and development of a posterior subcapsular cataract. The rapid alteration of lens fiber end migration leads to abnormal suture sub-branch formation with a concomitant change in fiber end morphology and F-actin configuration, ultimately resulting in PSC formation. Continued studies in our laboratory are presently focused on elucidating other BMC molecules responsible for these structural changes and agents that could potentially be responsible for these distinct alterations.


Inflammatory stimuli are causative for insulin resistance in obesity as well as in acute inflammatory reactions. Ongoing research has identified a variety of secreted proteins that are released from immune cells and adipocytes as mediators of insulin resistance; however, knowledge about their relevance for acute inflammatory insulin resistance remains limited. In this study we aimed for a clarification of the relevance of different insulin resistance mediating factors in an acute inflammatory situation.Insulin resistance was measured in a cohort of 37 non-diabetic patients undergoing cardiac surgery by assessment of insulin requirement to maintain euglycaemia and repeated measurements of an insulin glycaemic index. The kinetics of cortisol, interleukin 6 (IL6), tumour necrosis factor α (TNFα), resistin, leptin and adiponectin were assessed by repeated measurements in a period of 48 h.Insulin resistance increased during the observation period and peaked 22 h after the beginning of the operation. IL6 and TNFα displayed an early increase with peak concentrations at the 4-h time point. Serum levels of cortisol, resistin and leptin increased more slowly and peaked at the 22-h time point, while adiponectin declined, reaching a base at the 22-h time point. Model assessment identified cortisol as the best predictor of insulin resistance, followed by IL6, leptin and adiponectin. No additional information was gained by modelling for TNFα, resistin, catecholamine infusion rate, sex, age, body mass index (BMI), operation time or medication.Serum cortisol levels are the best predictor for inflammatory insulin resistance followed by IL6, leptin and adiponectin. TNFα, and resistin have minor relevance as predictors of stress dependent insulin resistance. The Western lifestyle has created a pandemic of obesity, which has dramatically increased the prevalence of insulin resistance and diabetes mellitus. Efforts to understand the linkage between the accumulation of body fat and the occurrence of insulin resistance have identified a variety of adipose tissue derived secreted proteins as mediators for insulin resistance. Some of these so-called adipokines such as leptin or adiponectin are adipocyte specific, while other mainly inflammatory cytokines are secreted by immune cells that infiltrate the adipose tissue in an obesity dependent manner. Diabetes has therefore also been considered a chronic inflammatory disease . The relWhile these factors were characterised in the chronic metabolic environment of obesity, our knowledge about their relevance as mediators of insulin resistance in acute inflammatory situations remains limited. Stress induced insulin resistance has classically been ascribed to increased serum levels of cortisol which promotes gluconeogenesis and inhibits peripheral glucose disposal in a stress dependent manner. However, TNFα, IL6, resistin or leptin are induced and adiponectin levels are reduced by inflammatory stimuli, which makes it likely that similar mechanisms are relevant in chronic and acute inflammation ,5. The cWe prospectively enrolled 37 non-diabetic patients scheduled for cardiac surgery with cardiopulmonary bypass and requirement of extracorporeal circulation. Patients were excluded from the study if they met the following criteria: pregnancy, diabetes mellitus, fasting glucose > 126 mg/dl, use of antidiabetic medication or glucocorticoids.Patients were fasting since the evening of the preoperative day. Insulin resistance was recorded by the individual insulin requirements to maintain euglycaemia. Blood glucose was assessed on an hourly bases and insulin infusion rate consequently adjusted to maintain glucose levels between 80 and 126 mg/dl. In addition, repeated measurements of C peptide as an indicator of endogenous insulin production were recorded as well as insulin serum levels, representing the circulating sum of endogenously produced and exogenous applied insulin. An insulin glycaemic index was calculated at each time point (insulin × glucose/22.5). Blood samples were drawn directly before surgery (baseline), at arrival in the intensive care unit (ICU) (4 to 6 h time point), 6 h post arrival in the ICU (10 to 12 h time point) and the morning of the first and second postoperative days. As some patients were discharged to a normal ward at the first postoperative day, blood was only collected from 26 patients on the second postoperative day. Blood samples were stored on ice and directly centrifuged for serum collection. No glucose containing solutions were given during the day of the procedure, while all patients were started on a continuous infusion of glucose 10% with a rate of 10 ml/h at the morning of the first postoperative day. Low rate exogenous applied glucose did therefore only affect the last blood sampling at the second postoperative day. No additional parenteral or enteral nutrition was supplied during the observation period. The applied catecholamine doses were recorded at the blood collection time point in mg/h. The study protocol was approved by the Ethics Committee of the Ludwig-Maximilians-University Munich, Germany. All patients gave informed written consent.Blood samples were stored at -70°C until analysis. Serum levels of TNFα, IL6, leptin, adiponectin and resistin were determined with a commercial enzyme-linked immunosorbent assay . Serum concentrations of cortisol, insulin, and C peptide were quantified using a multichannel immunoanalyzer based on electrochemiluminescence as the principle of signal generation  by the Department of Clinical Chemistry .Spearman correlation was performed for associations between baseline characteristics. A linear mixed effects model was fit to model the influence of the metabolic factors on insulin resistance (log insulin glycaemic index). The factors were included forward by a likelihood ratio test. A random effect per patient accounts for the subjective level of each patient (which includes the baseline levels) and for the dependence of the measurements within each patient. All measurements of a patient were used together with the time after surgery when they occurred (rounded to the nearest hour) Curves were fitted by the non-parametric locally-weighted scatterplot smoothing (LOWESS) smoother, which uses locally-weighted polynomial regression. Statistical analysis was performed using R 2.6.1.Baseline characteristics of the study participants are shown in Table Blood glucose was monitored on an hourly basis throughout the observation period. All patients required insulin treatment to maintain euglycaemia. Consequently, blood glucose was kept stable throughout the observation period Figure , while iAt baseline (preoperative blood sample) we found correlations between IL6, resistin and TNFα   . In addition, baseline leptin and adiponectin were found to correlate positively or negatively with body mass index (BMI)  .During the observation period inflammatory cytokines rapidly increased with peak concentrations of TNFα and IL6 found at the 4 to 6 h time point Figure . Serum lWe next asked which parameter would best predict insulin resistance. Using a linear mixed effects model we included the parameters by a forward selection using a likelihood ratio test to predict the individual insulin resistance as measured by the insulin glycaemic index. The model thereby includes the baseline and all following values of each parameter in a time dependent manner for each patient. The kinetic of each parameter was than analyzed for its relevance to predict insulin resistance in the same patient.Serum cortisol was found to be the strongest predictor for the insulin glycaemic index , followed by IL6 , leptin  and adiponectin  (Table To better understand the relevance of different mediators of insulin resistance we have performed kinetic studies in an acute inflammatory setting in humans. Inflammation caused by cardiac surgery increased insulin resistance in a time dependent manner which was paralleled by an induction of cortisol, TNFα, IL6, resistin and leptin while adiponectin serum levels were decreased. Testing the relevance of each parameter to predict insulin resistance we found best performance for serum cortisol followed by serum IL6, leptin and adiponectin. No additional information was gained by modelling for TNFα, resistin, catecholamine infusion rate, gender, BMI, operation time, heart/lung bypass time or medication, suggesting a minor relevance of these parameters for inflammatory insulin resistance in our model.Cortisol is the major adaptive signalling regulator of stress. Cortisol increases glucose availability by augmentation of hepatic glucose production via transcriptional and post-transcriptional activation of gluconeogenic enzymes including glucose-6-phosphatase and phosphoenolpyruvate . In addiAn ongoing debate considers the relevance of IL6 as a mediator of insulin resistance in humans ,13. InitLeptin is a predominant regulator of energy metabolism with additional immune regulatory functions . Leptin Adiponectin has been identified as an insulin sensitising adipocyte derived protein, which is decreased in obesity . AdiponeResistin has been established as an adipocyte derived mediator of insulin resistance in mice . Some buAlthough TNFα has been found to be a mediator of insulin resistance in acute and chronic models of inflammation ,37, TNFαBetter understanding of the relevant mediators of inflammatory insulin resistance will provide potentially clinical relevant information. Following the concept of relative adrenal insufficiency substitution of hydrocortisone has widely been used in critically ill patients. A recent study has now re-evaluated this approach and reported minor survival rates in hydrocortisone treated patients with septic shock due to infectious causes . Taking This study has several limitations. We used an insulin glycaemic index to quantify insulin resistance, which is less precise than an insulinic clamp, the gold standard for the assessment of insulin resistance. However, the extended observation period limited the use of a clamp setting in our study. Future experiments are needed to confirm our results under clamp conditions.The proposed model only offers associations but cannot provide causal relationship between the different parameters and insulin resistance. In addition, we cannot role out that other mediators of insulin resistance such as catecholamines, glucagon or growth hormone might also have contributive effects. The study is further limited by a relatively small sample size. Further studies in larger cohorts are needed to confirm the obtained results and further differentiate the relevance of specific factors preferentially under insulinic clamp conditions.Serum cortisol levels are the best predictor for inflammatory insulin resistance followed by IL6, leptin and adiponectin. TNFα, and resistin have minor relevance as predictors of stress dependent insulin resistance.• Cortisol is the best predictor of stress induced insulin resistance during the course of cardiac surgery.• Insulin resistance is further predicted by IL6, leptin and adiponectin.• TNFα, and resistin do not predict stress induced insulin resistance in our model.ACE-I: angiotensin converting enzyme inhibitor; ARB: angiotensin II receptor blocker; AMPK: adenosine monophosphate-activated protein kinase; GLUT4: glucose transporter 4; IL6: interleukin 6; IRS-1: insulin receptor substrate 1; JNK: C-Jun N-terminal kinase; NF-κB: nuclear factor κB; SOCS3: suppressor of cytokine signalling 3; TNFα: tumour necrosis factor α.The authors declare that they have no competing interests.ML contributed to study design, execution, and manuscript preparation. UCB contributed to study design, manuscript editing, IMBF sample collection and ELISA performance. MV contributed to insulin, C peptide and cortisol assays. VH contributed to statistical analysis. KN contributed to patient recruitment. BG contributed to study design and manuscript editing. EK contributed to patient recruitment. KP contributed to study design and manuscript editing.


In addition, several foreign antigens including bacteria such as Porphyromonas gingivalis and Chlamydia pneumoniae and viruses such as enterovirus and cytomegalovirus have been associated with atherosclerosis as potentially causative or bystander participants, adding another level of complexity to the analysis of the pathophysiology of atherosclerosis. The present review summarises the most important scientific findings published within the last two decades on the importance of antigens, antigen stimulation, and adaptive immune responses in the development of atherosclerotic plaques.Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of monocytes/macrophages, smooth muscle cells, and lymphocytes within the arterial wall in response to the release of proinflammatory molecules. Such accumulation results in the formation of the atherosclerotic plaque, which would eventually evolve to complications such as total artery occlusion, rupture, calcification, or aneurysm. Although the molecular mechanism responsible for the development of atherosclerosis is not completely understood, it is clear that the immune system plays a key role in the development of the atherosclerotic plaque and in its complications. There are multiple antigenic stimuli that have been associated with the pathogenesis of atherosclerosis. Most of these stimuli come from modified self-molecules such as oxidised low-density lipoproteins (oxLDLs), beta2glycoprotein1 ( It is difficult toidentify the factors responsible for the initiation of the atheroma lesion and/orthe order in which these factors contribute to plaque formation. Nevertheless, it is known that endothelial dysfunction and high levels of circulatingcholesterol, as oxLDL, play a key role in the proinflammatory process thattriggers the first steps in the development of atherosclerotic plaques , 2. WhatAs theinflammatory process becomes chronic, smooth muscle cells also start to migratefrom the media into the intima layer of the vessel, in response to chemokinesand aided by the release of membrane metalloproteinases (MMPs) that enable themto break through the elastic lamina into the subendothelial space  9, 10]., 10.9, 1Neointimaformation is a complex phenomenon that occurs in response to vessel wall damagein which repair and injury mechanisms give birth to areas rich inproinflammatory cells and collagen deposition , 12. It Atherosclerosisbelongs to the group of chronic inflammatory diseases in which the cellularcomponents of the immune system play a key role in its development andcomplications. The chronic accumulation of monocytes/macrophages, smooth musclecells, and T-lymphocytes in response to the accumulation and release ofproinflammatory molecules within the arterial wall constitutes the hallmark ofa developing atherosclerotic plaque . AlthougAs stated above, bothendogenous and exogenous antigens are involved in atherogenesis. neoantigen, a term used to describe modifiedself-antigens for which normally the immune system is tolerant but which uponmodification have the potential to excerpt autoreactive immune responses. Modifiedlipoprotein particles have the capacity to interact with the endothelium,triggering their accumulation and retention in the subendothelial space. Theretained oxLDL particles induce macrophage foam cell formation, smooth musclecell migration and proliferation [OxLDL remains oneof the most studied antigens in atherogenesis . LDL doeferation  and, in feration .In atherosclerosis,increased levels of oxLDL have been found in the serum of patients admitted with acute myocardial infarction, and immunohistochemical studies revealed thepresence of oxLDL within atherosclerotic plaques –85. ProtThe initialinteraction of proteoglycans with the LDL within the arterial wall leads lipidmodifications that expose GAG binding sites. Cholesterol, cholesteryl estersand phospholipids are the main targets for lipid modifications and their rolein the development of the disease has been extensively evaluated. Thesemodifications play an important role, as stated above, in LDL retention withinthe arterial wall which is also facilitated by interactions with surfacemolecules such as apoE and lipoprotein lipase expressed on macrophages .However, moststudies have focused on the role of oxidised phospholipids (oxPLs) . Peroxidβ (IL-1β), tumor necrosis factor alpha (TNF-α)and monocyte colony stimulating factor (M-CSF) can induce the expression of theoxLDL receptor (oxLDL-R) on the surface of endothelial cells, thus furthercontributing to oxLDL accumulation [It has also beenestablished that oxLDL acts as a chemoattractant for monocytes allowing them toinfiltrate the lesion site. OxLDL has been shown to induce monocyte adhesion tothe endothelium by upregulating the expression of adhesion molecules on theirsurface, by inducing macrophage major histocompatibility class II (MHC-II)& LeuM3 cell surface expression and by accelerating monocytedifferentiation in to macrophages . Endothemulation , 98.κB synthesis [Scavengerreceptors expressed by macrophages play a pivotal role in LDL accumulation; suchreceptors include CD36 (a membrane glycoprotein), CD68, CXCL16, the scavengerreceptors A & B1 (SR-A & SR-B1), and the lectin-type oxidised low-densitylipoprotein receptor 1 (LOX1) , 99. It ynthesis , 103.6 kDa) favours the exposure of many epitopesrecognised by the antibodies generated, mainly IgM. OxLDL is known to be a verypotent immunogen and the antibodies generated in response to its modificationsare able to bind to many other similarly modified endogenous proteins [Streptococcus pneumoniae [The large size ofthe LDL molecule . Immunised micedeveloped high titres of anti-MDA-LDL antibodies and the treatmentsignificantly reduced lesion size at the aortic sinus by more than half when comparedwith their control littermates immunised with PBS. However, they did not find differencesbetween the groups with respect to cellular composition of the atheroscleroticplaques [−/−) immunised withhomologous malondialdehyde-modified LDL (MDA-LDL) induced the synthesis of antibodiesof different classes against distinctive epitopes on oxLDL and that thisantibody response is significantlycorrelated with a reduction by approximately 40% of lesion size.However, they also showed that immunisation with MDA-LDL raised equivalentamounts of both T helper 1 (Th1)-related IgG2a and Th2-dependent IgG1antibodies [−/− mice with homologous plaque homogenates or homologous MDA-LDL.They found that both antigen preparations reduced lesion development. The protectiveeffect was associated with a specific raise of T-cell-dependent IgG antibodiesagainst MDA-LDL and oxidised phospholipids which are correlated with the reduction in plaque sizeand circulating cholesterol levels [The effect of LDLimmunisation on atheroprotection has also been assessed using mouse models ofthe disease. George et al. was the first to report the effect of MDA modifiedLDL immunisation in apoE-deficient mice (apoEplaques . Later otibodies . On the l levels . Despiteβ2-glycoprotein I (β2GpI), a positively chargedplasma protein circulating in the blood and also present in platelets and endothelialcells in atherosclerotic plaques [β2GpI which occurs when the proteinbinds to negatively charged phospholipids present in the atheroscleroticplaques. When transgenic animals are immunised with β2GpI, the atherosclerosis processis accelerated [β2GpIcan function as a scavenger receptor to mediate lipid engulfment bymacrophages. Furthermore, histological studies showed that β2GpI is located inthe subendothelial space in areas rich in CD4+ T cells [β2GpI reactive T cells can promote the generation offatty streaks in LDL−/− mice, indicating that cellular autoimmunityis involved in the pathogenesis of atherosclerosis [Rheumatic patientssuffering from the antiphospholipid syndrome produce large amounts of antiphospholipidantibodies. The standard phospholipid used to detect antiphospholipidantibodies is cardiolipin, which is prone to peroxidation and is also animportant component of the oxLDL molecule . A cofac plaques . Bindingelerated . It has  T cells . A recenclerosis .Lp(a) is an antigenof relevance to atherosclerosis development . Lp(a) iA further self-antigeninvolved in lupus-related atherosclerosis is LPL . It is aA recent studysuggests a possible role of AGE as facilitators of antigenic stimulation inatherosclerosis by promoting the maturation of dendritic cells (DCs) . AGE proαsecretion and promotion of atherosclerosis. HSPs are highly conserved amongdifferent species. Antibodies involved in the atherosclerotic developmentrecognise both human and microbial HSPs [HSPs are releasedfrom stressed endothelial cells and can act as chaperones in the process ofdenaturation of other proteins. They can induce the production of specificantibodies which usually accelerate atherosclerotic plaque development whenused to immunise experimental animals . Human aial HSPs .The potentialrelationship between bacterial infections and the induction of atherosclerosishas been studied in different groups of cardiovascular patients including thosewho develop the disease but that lack the conventional risk factors associatedwith it such as hypercholesterolemia, high blood pressure, smoking and diabetes. It has Porphyromonas gingivalis which has beendetected within atherosclerotic plaques [P. gingivalis fimbriae was shown to be mediated by ligation of TLR2, TLR4, CD14,and beta2-integrins and also by the upregulation of nuclear factor kappa-B(NF-κB). It has also been observed that P.gingivalis fimbriae may promote atheroscleroticplaque rupture by inducing the secretion of MMPs [Bacteroidesforsynthus, where the protein A secreted by the bacterium acts through CD14and TLR2 ligations to induce atherosclerosis, whereas in the case of Streptococcus mutans it is the proteinAgI/II that acts through CD14 and TLR4 [An example is  plaques . The inf of MMPs –122. Othand TLR4 .Chlamydophila pneumoniae (C. pneumoniae) infection whereLPS and bacterial HSP act as ligands to TLRs. C. pneumoniae is an intracellular prokaryotic pathogen thatinfects humans provoking distinct forms of pneumonia, and it has been also proposedthat it may cause chronic inflammatory diseases such as atherosclerosis [C. pneumoniae within atheromalesions has been detected by PCR and immunohistochemistry. However, detectionis sometimes difficult due to phases of activity and latency of the pathogen [C. pneumoniae than control patients [C. pneumoniae has been also extractedand cultured from atherosclerotic plaques [C. pneumoniae [C. pneumoniae can persistently infect epithelial cells andmacrophages within human atherosclerotic plaques causing a chronic and nonlyticinfection [+ T-helper (Th)1 cells andantibodies, although other components such as CD8+ T cells also playa key role [C.pneumoniae unless the therapy was started early during the acute infection [Similar mechanismshave been described for clerosis . Chlamydpathogen , 124. Thpathogen  but the patients . Moreove plaques , 128. Exeumoniae . C. pneukey role –132. Thekey role . Howeverkey role , 134. InHelicobacter pylori—a cause ofgastrointestinal infections—has been alsofound to be present in atherosclerotic lesions but completely absent fromhealthy arteries [The microorganism arteries , 53. HowE. coli-derived GroEL, an HSP [Cross-reactivityhas also been observed with antibodies against human HSP60 and , an HSP . The gen, an HSP –137.Viruses have alsobeen postulated as promoters of atherosclerosis. One of the most closely linkedto this disease is cytomegalovirus. This virus infects the majority of thehuman population by targeting SMCs and endothelial cells producing a latenttype of infection –142. US2Another pathogenassociated with atherosclerosis is enterovirus, especially the enterovirusgroup coxsackie B virus . High le+ and a small fraction of thepopulation consists of CD8+ T cells. CD4+ cells isolatedfrom human plaques have been found to express the αβ T cell receptor (αβ TCR) [+ T cells in the process of affinity maturation and isotype class switching ofB-cell clones specific to atherogenic antigens [T lymphocytes arepresent in atherosclerotic plaques at all stages of its development . Most T αβ TCR)  that recαβ TCR) . They arantigens .+ T cells generates one of the two major types of functionally different effectorT cells, the T-herper1 (Th1) or the Th2. The response of the former cells isconsidered to be proinflammatory in the context of atherosclerosis and is characterisedby the secretion of IFN-γ, IL-12 and TNF-α which are all involved in macrophageactivation. IFN-γ also drives Th1 cell differentiation that can be inhibited byIL-10 [γ. Th1 is the predominant T cell subset found in atherosclerotic lesions[The unbalancebetween pro- and anti-inflammatory immune responses appears to be responsiblefor the development of atherosclerosis. The activation of naïve CD4byIL-10 . Th2 res lesions, 153.The type ofantibody produced, driven by Th1 or Th2 immune responses, also plays a key rolein atherogenesis . The synOn the other hand,Th2 activation and proliferation appears to be triggered by epitope-specificstimulation , 156–160γ also accelerates atherosclerotic plaque development [Noticeably, thetherapeutic correction of the balance between these two types of responses hasbeen pivotal for the development of novel interventions, such as vaccinesagainst the development of the disease. Experiments carried out using inbredstains of mice show that C57BL/6 mice are more prone to develop Th1 responses andmore atherosclerosis than BALB/c mice which are prone to develop Th2 responses andconsequently atheroresistant . It has elopment , an effeelopment .γ [Switching theimmune response to a Th2 type can be achieved by the expression of theanti-inflammatory cytokine IL-10 which suppresses the effect of proinflammatorycytokines such as IL-12 and IFN-γ , 169. Thγ . T-bet dγ . These fα, and IL-22 expressions. Their discovery hascontributed to explain crucial regulatory mechanisms which until now theclassic control by Th1 and Th2 or Treg cell-mediated mechanisms could notexplain. Th17 cells have been suggested to play a key role in inflammation andautoimmunity. They have also been involved in the pathogenesis ofhypersensitivity reactions. The study of their role in host defence mechanismshas just recently started and promises to be another area of high interest incardiovascular biology research (see the following articles for a comprehensivereview of the recently published literature on Th17 [β1), and Foxp3 levels in thesepatients as compared to stable angina and controls suggesting a potential rolefor Th17/Treg imbalance in plaque destabilization and the onset of ACS [Just recently thefirst report appeared on the possible role of Th17 cells in cardiovasculardisease . Th17 ce on Th17 –177). Cht of ACS .β2-GpI and the presence of circulating antibodies againstthem, that in most cases correlates with the clinical outcome, have been usedto justify the classification of atherosclerosis as an inflammatory diseasewith an important autoimmune component. No less important is the role offoreign antigens derived, among others, from bacteria and viruses which might playa causal and/or at least a bystander effect contributing to the chronicinflammatory process and its complications. Finally, the role of T lymphocytesand the pro- and anti-inflammatory balances controlled by their differentsubsets has been shownto be crucial in the development of the disease. These responses are ultimatelydriven by the nature of the initial stimuli (the antigen) and supported by acomplex cascade of events involving cytokines, components of the extracellularmatrix, and even gene expression regulators such as transcription factors. Ourcurrent understanding of the immunopathogenic mechanisms involved inatherosclerotic plaque development has witnessed an enormous advance in the lastdecade, and some of this knowledge constitutes the foundation for the design ofthe next generation of drugs to combat cardiovascular disease and reduce its devastatingconsequences for the benefit of mankind.The multifactorialnature of atherosclerosis also applies to the number and quality of antigenscapable of inducing proinflammatory/proatherogenic immune responses. Theevidence accumulated so far supports the view that oxLDL is one of the mostimportant atherogenic antigens, by virtue of being the main trigger ofmonocytes/macrophage and SMC infiltration, proliferation, and conversion in tofoam cells in the neointima layer. The key role of other self-derived antigenssuch as HSPs and