Yoram Louzoun

Functional connectivity of the prefrontal cortex and the amygdala in posttraumatic stress disorder

BACKGROUND Persistent, intrusive re-experiencing in posttraumatic stress disorder (PTSD) is commonly construed as a failure of cingulate inhibition (i.e., extinction) over a hyperresponsive amygdala, based primarily on animal research of fear conditioning and the finding of cingulate hypoperfusion in PTSD. METHODS We examined functional connectivity in patients with PTSD and healthy trauma survivors during repeated symptom provocation using H(2)O(15) positron emission tomography. RESULTS Memory retrieval networks (right prefrontal cortex, hippocampus, and visual cortex) were common to both groups. Networks supporting autonomic and emotional control and preparatory motor action (amygdala, anterior cingulate, subcallosal gyrus, and premotor cortex) differed between the two groups and became progressively disparate with successive presentations of the traumatic script. Patterns of effective connectivity demonstrated the predominance of direct influences of the amygdala on visual cortex, subcallosal gyrus, and anterior cingulate in PTSD but not in control subjects. There was little evidence for failure of inhibition of cingulate or subcallosal cortex over the amygdala. CONCLUSIONS These patterns might represent excessive influences of the amygdala over regions involved in autonomic, and higher-order visual memory processing in PTSD. The present data suggest that inferences of direct correspondence between animal studies and pathophysiology of PTSD should be made with caution.

Rep-Seq: uncovering the immunological repertoire through next-generation sequencing

Recent scientific discoveries fuelled by the application of next‐generation DNA and RNA sequencing technologies highlight the striking impact of these platforms in characterizing multiple aspects in genomics research. This technology has been used in the study of the B‐cell and T‐cell receptor repertoire. The novelty of immunosequencing comes from the recent rapid development of techniques and the exponential reduction in cost of sequencing. Here, we describe some of the technologies, which we collectively refer to as Rep‐Seq (repertoire sequencing), to portray achievements in the field and to present the essential and inseparable role of next‐generation sequencing to the understanding of entities in immune response. The large Rep‐Seq data sets that should be available in the near future call for new computational algorithms to segue the transition from ‘classic’ molecular‐based analysis to system‐wide analysis. The combination of new algorithms with high‐throughput data will form the basis for possible new clinical implications in personalized medicine and deeper understanding of immune behaviour and immune response.

Mathematical modelling and evaluation of the different routes of transmission of lumpy skin disease virus

Lumpy skin disease (LSD) is a severe viral disease of cattle. Circumstantial evidence suggests that the virus is transmitted mechanically by blood-feeding arthropods. We compared the importance of transmission via direct and indirect contact in field conditions by using mathematical tools. We analyzed a dataset collected during the LSD outbreak in 2006 in a large dairy herd, which included ten separated cattle groups. Outbreak dynamics and risk factors for LSD were assessed by a transmission model. Transmission by three contact modes was modelled; indirect contact between the groups within a herd, direct contact or contact via common drinking water within the groups and transmission by contact during milking procedure. Indirect transmission was the only parameter that could solely explain the entire outbreak dynamics and was estimated to have an overall effect that was over 5 times larger than all other possible routes of transmission, combined. The R0 value induced by indirect transmission per the presence of an infectious cow for 1 day in the herd was 15.7, while the R0 induced by direct transmission was 0.36. Sensitivity analysis showed that this result is robust to a wide range of assumptions regarding mean and standard deviation of incubation period and regarding the existence of sub-clinically infected cattle. These results indicate that LSD virus spread within the affected herd could hardly be attributed to direct contact between cattle or contact through the milking procedure. It is therefore concluded that transmission mostly occurs by indirect contact, probably by flying, blood-sucking insects. This has important implications for control of LSD.

Resting regional cerebral perfusion in recent posttraumatic stress disorder

BACKGROUND Brain imaging research in posttraumatic stress disorder has been largely performed on patients with chronic disease, often heavily medicated, with current or past alcohol and substance abuse. Additionally, virtually only activation brain imaging paradigms have been done in posttraumatic stress disorder, whereas in other mental disorders both resting and activation studies have been performed. METHODS Twenty-eight (11 posttraumatic stress disorder) trauma survivors underwent resting state hexamethylpropyleneamineoxime single photon emission computed tomography and magnetic resonance imaging 6 months after trauma. Eleven nontraumatized subjects served as healthy controls. RESULTS Regional cerebral blood flow in the cerebellum was higher in posttraumatic stress disorder than in both control groups. Regional cerebral blood flow in right precentral, superior temporal, and fusiform gyri in posttraumatic stress disorder was higher than in healthy controls. Cerebellar and extrastriate regional cerebral blood flow were positively correlated with continuous measures of depression and posttraumatic stress disorder. Cortisol level in posttraumatic stress disorder was negatively correlated with medial temporal lobe perfusion. Anterior cingulate perfusion and cortisol level were positively correlated in posttraumatic stress disorder and negatively correlated in trauma survivors without posttraumatic stress disorder. CONCLUSIONS Recent posttraumatic stress disorder is accompanied by elevated regional cerebral blood flow, particularly in the cerebellum. This warrants attention because the cerebellum is often used as a reference region in regional cerebral blood flow studies. The inverse correlation between plasma cortisol and medial temporal lobe perfusion may herald hippocampal damage.

The Importance of Thermodynamic Equilibrium for High Throughput Gene Expression Arrays

We present an analysis of physical chemical constraints on the accuracy of DNA micro-arrays under equilibrium and nonequilibrium conditions. At the beginning of the article we describe an algorithm for choosing a probe set with high specificity for targeted genes under equilibrium conditions. The algorithm as well as existing methods is used to select probes from the full Saccharomyces cerevisiae genome, and these probe sets, along with a randomly selected set, are used to simulate array experiments and identify sources of error. Inasmuch as specificity and sensitivity are maximum at thermodynamic equilibrium, we are particularly interested in the factors that affect the approach to equilibrium. These are analyzed later in the article, where we develop and apply a rapidly executable method to simulate the kinetics of hybridization on a solid phase support. Although the difference between solution phase and solid phase hybridization is of little consequence for specificity and sensitivity when equilibrium is achieved, the kinetics of hybridization has a pronounced effect on both. We first use the model to estimate the effects of diffusion, crosshybridization, relaxation time, and target concentration on the hybridization kinetics, and then investigate the effects of the most important kinetic parameters on specificity. We find even when using probe sets that have high specificity at equilibrium that substantial crosshybridization is present under nonequilibrium conditions. Although those complexes that differ from perfect complementarity by more than a single base do not contribute to sources of error at equilibrium, they slow the approach to equilibrium dramatically and confound interpretation of the data when they dissociate on a time scale comparable to the time of the experiment. For the best probe set, our simulation shows that steady-state behavior is obtained in a relaxation time of approximately 12-15 h for experimental target concentrations approximately (10(-13) - 10(-14))M, but the time is greater for lower target concentrations in the range (10(-15)-10(-16))M. The result points to an asymmetry in the accuracy with which up- and downregulated genes are identified.

Estimating hypermutation rates from clonal tree data

To understand the mechanisms underlying the varying patterns of mutations that occur during immune and autoimmune responses, estimates of the somatic hypermutation rate are critical. However, despite its significance, precise estimates of the mutation rate do not currently exist. Microdissection studies of mutating B cell clones provide an opportunity to measure this rate more accurately than previously possible. Each microdissection provides a number of clonally related sequences that, through the analysis of shared mutations, can be genealogically related to each other. The shape of these clonal trees is influenced by many processes, including the hypermutation rate. We have developed two different methods to estimate the mutation rate based on these data. These methods are applied to two sets of experimental data, one from an autoimmune response and one from the antihapten response to (4-hydroxy-3-nitrophenyl)acetyl (NP). Comparable mutation rates are estimated for both responses, 0.7–0.9 × 10−3 and 0.9–1.1 × 10−3 bp−1 division−1 for the autoimmune and NP responses, respectively. In addition to comparing the results of the two procedures, we investigate the effect on our estimate of assumptions, such as the fraction of lethal mutations.

Taking Advantage: High-Affinity B Cells in the Germinal Center Have Lower Death Rates, but Similar Rates of Division, Compared to Low-Affinity Cells

B lymphocytes producing high-affinity Abs are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high-affinity B cells are selected during the immune response has never been elucidated. Although it has been shown that high-affinity cells directly outcompete low-affinity cells in the germinal center (GC), whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity cells proliferate more rapidly or are more likely to enter cell cycle, thereby outgrowing lower affinity cells. Alternatively, higher affinity cells could be relatively more resistant to cell death in the GC. By comparing high- and low-affinity B cells for the same Ag, we show here that low-affinity cells have an intrinsically higher death rate than do cells of higher affinity, even in the absence of competition. This suggests that selection in the GC reaction is due at least in part to the control of survival of higher affinity B cells and not by a proliferative advantage conferred upon these cells compared with lower affinity B cells. Control over survival rather than proliferation of low- and high-affinity B cells in the GC allows greater diversity not only in the primary response but also in the memory response.

Sleep-Wake Transitions in Premature Neonates Predict Early Development

OBJECTIVE: To identify patterns of sleep-wake transitions in the neonatal period that might differentiate premature infants who would show better or worse outcomes in multiple developmental domains across the first 5 years of life. METHODS: Participants were 143 low birth weight premature infants (mean birth weight: 1482 g; mean gestational age [GA]: 31.82 weeks). Sleep states were observed at a GA of 37 weeks in 10-second epochs over 4 consecutive evening hours and were analyzed through mathematical clustering. Neurobehavioral maturation was evaluated with the Neonatal Behavior Assessment Scale at discharge, emotional regulation was assessed during infant-mother and infant-father interactions at 3 and 6 months, cognitive development was measured at 6, 12, and 24 months, and verbal IQ, executive functions, and symbolic competence were tested at 5 years. RESULTS: Three types of state-transition patterns were identified, and no differences in birth weight, GA, or medical risk between the 3 groups were found. Infants whose sleep-state transitions were mainly characterized by shifts between quiet sleep and wakefulness exhibited the best development, including greater neonatal neuromaturation, less negative emotionality, better cognitive development, and better verbal, symbolic, and executive competences at 5 years. In comparison, infants who cycled mainly between states of high arousal, such as active sleep and cry, or between short episodes of active and quiet sleep showed poorer outcomes. CONCLUSIONS: Defining sleep organization on the basis of transitions between states proved useful for identifying risk and resilience indicators in neonatal behavior to predict trajectories of neurobehavioral, emotional, and cognitive growth.

The evolution of mathematical immunology

Summary:  The types of mathematical models used in immunology and their scope have changed drastically in the past 10 years. Classical models were based on ordinary differential equations (ODEs), difference equations, and cellular automata. These models focused on the ‘simple’ dynamics obtained between a small number of reagent types (e.g. one type of receptor and one type of antigen or two T‐cell populations). With the advent of high‐throughput methods, genomic data, and unlimited computing power, immunological modeling shifted toward the informatics side. Many current applications of mathematical models in immunology are now focused around the concepts of high‐throughput measurements and system immunology (immunomics), as well as the bioinformatics analysis of molecular immunology. The types of models have shifted from mainly ODEs of simple systems to the extensive use of Monte Carlo simulations. The transition to a more molecular and more computer‐based attitude is similar to the one occurring over all the fields of complex systems analysis. An interesting additional aspect in theoretical immunology is the transition from an extreme focus on the adaptive immune system (that was considered more interesting from a theoretical point of view) to a more balanced focus taking into account the innate immune system also. We here review the origin and evolution of mathematical modeling in immunology and the contribution of such models to many important immunological concepts.

Editing Anti-DNA B Cells by Vλx

Receptor editing is performed by replacement of Vκ genes that contribute to autoreactivity. In addition, the Cκ locus can be deleted by Vκ rearrangement to intronic or 3′ of Cκ RS sequences (also referred to as κ deletion elements). B cells that delete the Cκ can then express λ light chains. However, the λ locus, either of man or mouse, does not allow V gene replacement. Nor does it appear to be deleted. Therefore, editing of autoreactive λ B cells may require alternative pathways. We have found that in anti-DNA heavy chain transgenic mice (tgs) VH3H9/56R, B cells that express anti-DNA receptors comprised of λ1 in association with an anti-DNA heavy chain often coexpress a κ chain that prevents DNA binding. We speculate that such isotypically included cells may have low anti-DNA receptor densities, a feature that may lead to self-tolerance. Here we describe a mechanism of preventing DNA binding by expression of a rarely used member of the Vλ family, Vλx. The λx B cells of the tgs also express CD25 and may represent B cells that have exhausted light chain editing possibilities.