The five dimensions of B cell tolerance

B cell tolerance has been generally understood to be an acquired property of the immune system that governs antibody specificity in ways that avoid auto‐toxicity. As useful as this understanding has proved, it fails to fully explain the existence of auto‐reactive specificities in healthy individuals and contribution these may have to health. Mechanisms underlying B cell tolerance are considered to select a clonal repertoire that generates a collection of antibodies that do not bind self, ie tolerance operates more or less in three dimensions that largely spare autologous cells and antigens. Yet, most B lymphocytes in humans and probably in other vertebrates are auto‐reactive and absence of these auto‐reactive B cells is associated with disease. We suggest that auto‐reactivity can be embodied by extending the concept of tolerance by two further dimensions, one of time and circumstance and one that allows healthy cells to actively resist injury. In this novel concept, macromolecular recognition by the B cell receptor leading to deletion, anergy, receptor editing or B cell activation is extended by taking account of the time of development of normal immune responses (4th dimension) and the accommodation (or tolerance) of normal cells to bound antibody, activation of complement, and interaction with inflammatory cells (fifth dimension). We discuss how these dimensions contribute to understanding B cell biology in health or disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153034/1/imr12813.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153034/2/imr12813_am.pd

Pseudomonas aeruginosa Adaptation to Lungs of Cystic Fibrosis Patients Leads to Lowered Resistance to Phage and Protist Enemies

Pathogenic life styles can lead to highly specialized interactions with host species, potentially resulting in fitness trade-offs in other ecological contexts. Here we studied how adaptation of the environmentally transmitted bacterial pathogen, Pseudomonas aeruginosa, to cystic fibrosis (CF) patients affects its survival in the presence of natural phage (14/1, ΦKZ, PNM and PT7) and protist (Tetrahymena thermophila and Acanthamoebae polyphaga) enemies. We found that most of the bacteria isolated from relatively recently intermittently colonised patients (1-25 months), were innately phage-resistant and highly toxic for protists. In contrast, bacteria isolated from long time chronically infected patients (2-23 years), were less efficient in both resisting phages and killing protists. Moreover, chronic isolates showed reduced killing of wax moth larvae (Galleria mellonella) probably due to weaker in vitro growth and protease expression. These results suggest that P. aeruginosa long-term adaptation to CF-lungs could trade off with its survival in aquatic environmental reservoirs in the presence of microbial enemies, while lowered virulence could reduce pathogen opportunities to infect insect vectors; factors that are both likely to result in poorer environmental transmission. From an applied perspective, phage therapy could be useful against chronic P. aeruginosa lung infections that are often characterized by multidrug resistance: chronic isolates were least resistant to phages and their poor growth will likely slow down the emergence of beneficial resistance mutations

What is the role of the film viewer? The effects of narrative comprehension and viewing task on gaze control in film

Film is ubiquitous, but the processes that guide viewers' attention while viewing film narratives are poorly understood. In fact, many film theorists and practitioners disagree on whether the film stimulus (bottom-up) or the viewer (top-down) is more important in determining how we watch movies. Reading research has shown a strong connection between eye movements and comprehension, and scene perception studies have shown strong effects of viewing tasks on eye movements, but such idiosyncratic top-down control of gaze in film would be anathema to the universal control mainstream filmmakers typically aim for. Thus, in two experiments we tested whether the eye movements and comprehension relationship similarly held in a classic film example, the famous opening scene of Orson Welles' Touch of Evil (Welles & Zugsmith, Touch of Evil, 1958). Comprehension differences were compared with more volitionally controlled task-based effects on eye movements. To investigate the effects of comprehension on eye movements during film viewing, we manipulated viewers' comprehension by starting participants at different points in a film, and then tracked their eyes. Overall, the manipulation created large differences in comprehension, but only produced modest differences in eye movements. To amplify top-down effects on eye movements, a task manipulation was designed to prioritize peripheral scene features: a map task. This task manipulation created large differences in eye movements when compared to participants freely viewing the clip for comprehension. Thus, to allow for strong, volitional top-down control of eye movements in film, task manipulations need to make features that are important to narrative comprehension irrelevant to the viewing task. The evidence provided by this experimental case study suggests that filmmakers' belief in their ability to create systematic gaze behavior across viewers is confirmed, but that this does not indicate universally similar comprehension of the film narrative

Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

BACKGROUND: ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis 'senses' NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs. METHODOLOGY/PRINCIPAL FINDINGS: Using biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47(phox) subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS. CONCLUSION/SIGNIFICANCE: These results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can 'sense' redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs

S100B and homocysteine in the acute alcohol withdrawal syndrome

Elevations of serum homocysteine levels are a consistent finding in alcohol addiction. Serum S100B levels are altered in different neuropsychiatric disorders but not well investigated in alcohol withdrawal syndromes. Because of the close connection of S100B to ACTH and glutamate secretion that both are involved in neurodegeneration and symptoms of alcoholism the relationship of S100B and homocysteine to acute withdrawal variables has been examined. A total of 22 male and 9 female inpatients (mean age 46.9 ± 9.7 years) with an ICD-10 diagnosis of alcohol addiction without relevant affective comorbidity were examined on admission and after 24, 48, and 120 h during withdrawal. S100B and homocysteine levels in serum were collected, and severity of withdrawal symptoms (AWS-scale), applied withdrawal medication, initial serum ethanol levels and duration of addiction were recorded. Serum S100B and homocysteine levels declined significantly (P < .05) over time. Both levels declined with withdrawal syndrome severity. Females showed a trend to a more intense decline in serum S100B levels compared to males at day 5 (P = .06). Homocysteine levels displayed a negative relationship to applied amount of clomethiazole (P < .05) and correlated with age of onset of addiction. No withdrawal seizures were recorded during the trial. As it is known for homocysteine, S100B revealed to decline rapidly over withdrawal treatment in alcoholism. This effect is more pronounced in female patients. S100B could be of relevance in the neurobiology of alcohol withdrawal syndromes. It may be indirectly related to the level of stress level or glutamatergic activity during alcohol withdrawal

Epigenetic deregulation of multiple S100 gene family members by differential hypomethylation and hypermethylation events in medulloblastoma

Deregulated expression of genes encoding members of the S100 family of calcium-binding proteins has been associated with the malignant progression of multiple tumour types. Using a pharmacological expression reactivation approach, we screened 16 S100 genes for evidence of epigenetic regulation in medulloblastoma, the most common malignant brain tumour of childhood. Four family members (S100A2, S100A4, S100A6 and S100A10) demonstrated evidence of upregulated expression in multiple medulloblastoma cell lines, following treatment with the DNA methyltransferase inhibitor, 5′-aza-2′-deoxycytidine. Subsequent analysis revealed methylation of critical CpG sites located within these four genes in an extended cell line panel. Assessment of these genes in the non-neoplastic cerebellum (from which medulloblastomas develop) revealed strong somatic methylation affecting S100A2 and S100A4, whereas S100A6 and S100A10 were unmethylated. Assessed against these normal tissue-specific methylation states, S100A6 and S100A10 demonstrated tumour-specific hypermethylation in medulloblastoma primary tumours (5 out of 40 and 4 out of 35, respectively, both 12%) and cell lines (both 7 out of 9, 78%), which was associated with their transcriptional silencing. Moreover, S100A6 hypermethylation was significantly associated with the aggressive large cell/anaplastic morphophenotype (P=0.026). In contrast, pro-metastatic S100A4 displayed evidence of hypomethylation relative to the normal cerebellum in a significant proportion primary tumours (7 out of 41, 17%) and cell lines (3 out of 9, 33%), which was associated with its elevated expression. In summary, these data characterise complex patterns of somatic methylation affecting S100 genes in the normal cerebellum and demonstrate their disruption causing epigenetic deregulation of multiple S100 family members in medulloblastoma development. Epigenetic events affecting S100 genes have potential clinical utility and merit further investigation as molecular biomarkers for this disease

Opposite Effects of HIV-1 p17 Variants on PTEN Activation and Cell Growth in B Cells

The HIV-1 matrix protein p17 is a structural protein that can act in the extracellular environment to deregulate several functions of immune cells, through the interaction of its NH2-terminal region with a cellular surface receptor (p17R). The intracellular events triggered by p17/p17R interaction have been not completely characterized yet. In this study we analyze the signal transduction pathways induced by p17/p17R interaction and show that in Raji cells, a human B cell line stably expressing p17R on its surface, p17 induces a transient activation of the transcriptional factor AP-1. Moreover, it was found to upregulate pERK1/2 and downregulate pAkt, which are the major intracellular signalling components involved in AP-1 activation. These effects are mediated by the COOH-terminal region of p17, which displays the capability of keeping PTEN, a phosphatase that regulates the PI3K/Akt pathway, in an active state through the serin/threonin (Ser/Thr) kinase ROCK. Indeed, the COOH-terminal truncated form of p17 (p17Δ36) induced activation of the PI3K/Akt pathway by maintaining PTEN in an inactive phosphorylated form. Interestingly, we show that among different p17s, a variant derived from a Ugandan HIV-1 strain, named S75X, triggers an activation of PI3K/Akt signalling pathway, and leads to an increased B cell proliferation and malignant transformation. In summary, this study shows the role of the COOH-terminal region in modulating the p17 signalling pathways so highlighting the complexity of p17 binding to and signalling through its receptor(s). Moreover, it provides the first evidence on the presence of a p17 natural variant mimicking the p17Δ36-induced signalling in B cells and displaying the capacity of promoting B cell growth and tumorigenesis

Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual epigenetic variation

The extent of variation in DNA methylation patterns in healthy individuals is not yet well documented. Identification of inter-individual epigenetic variation is important for understanding phenotypic variation and disease susceptibility. Using neutrophils from a cohort of healthy individuals, we generated base-resolution DNA methylation maps to document inter-individual epigenetic variation. We identified 12851 autosomal inter-individual variably methylated fragments (iVMFs). Gene promoters were the least variable, whereas gene body and upstream regions showed higher variation in DNA methylation. The iVMFs were relatively enriched in repetitive elements compared to non-iVMFs, and were associated with genome regulation and chromatin function elements. Further, variably methylated genes were disproportionately associated with regulation of transcription, responsive function and signal transduction pathways. Transcriptome analysis indicates that iVMF methylation at differentially expressed exons has a positive correlation and local effect on the inclusion of that exon in the mRNA transcript

Coordinated Activation of Candidate Proto-Oncogenes and Cancer Testes Antigens via Promoter Demethylation in Head and Neck Cancer and Lung Cancer

Background: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. Methodology/Principal Findings: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. Conclusions/Significance: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs i