Clonal deletion and the fate of autoreactive thymocytes that survive negative selection.

Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4(+)CD8(lo) intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4(-)CD8(-) double-negative thymocytes positive for the T cell antigen receptor αß subtype (TCRαß) that 'preferentially' migrated to the intestine, where they re-expressed CD8α and were sequestered as CD8αα(+) intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8αα(+) IELs as the developmental fate of autoreactive thymocytes that survive negative selection.

A global bifurcation organizing rhythmic activity in a coupled network.

We study a system of coupled phase oscillators near a saddle-node on invariant circle bifurcation and driven by random intrinsic frequencies. Under the variation of control parameters, the system undergoes a phase transition changing the qualitative properties of collective dynamics. Using Ott-Antonsen reduction and geometric techniques for ordinary differential equations, we identify heteroclinic bifurcation in a family of vector fields on a cylinder, which explains the change in collective dynamics. Specifically, we show that heteroclinic bifurcation separates two topologically distinct families of limit cycles: contractible limit cycles before bifurcation from noncontractibile ones after bifurcation. Both families are stable for the model at hand.

When condensed matter physics meets biology: Does superhydrophobicity benefiting the cryopreservation of human spermatozoa?

With the increasing demand in regenerative and reproductive medicine for successful conservation of living matter, the need of reliable platform in cell banking seems inevitable. Whilst the cells storage at cryogenic temperatures is a well-developed method, far less is known about the efficiency of nanotechnology in cryogenics. The primary objective of this study is to represent the first of its kind experimental results related to cryopreservation of human spermatozoa by means of superhydrophobic carbon soot coatings. The inclusion of soot-based water repellent interface during the freezing and thawing of human semen minimizes the solid-liquid interfacial area, retards the heat transfer rate and promotes the recovery of up to 80% of initial motility of post-thaw sperm cells. Our discoveries reveal a fundamentally new and exciting direction of development of cryopreservation technologies in the battle against painful biopsies and repetitive surgeries.

Superhydrophobic Soot Coated Quartz Crystal Microbalances: A Novel Platform for Human Spermatozoa Quality Assessment.

The functionality of human spermatozoa is a key factor for the success rate of natural human reproduction, but unfortunately the infertility progressively increases due to multifarious environmental factors. Such disquieting statistics requires the employment of sophisticated computer-assisted methods for semen quality analysis, whose precision, however, is unreliable in cases of patients with low sperm concentrations. In this study, we report a novel quartz crystal microbalance (QCM) based biosensor for in-situ quality assessment of male gametes, comprising a superhydrophobic soot coating as an interface sensing material. The soot deposition on the surface of a 5 MHz QCM eliminates the noise that normally arises upon immersion of the uncoated sensor in the test liquid environment, allowing the detection of human spermatozoa down to 1000⁻100,000 units/mL (1⁻100 ppb). Furthermore, the soot coated QCM delimitates in a highly repeatable way the immotile and motile sperm cells by inducing fundamentally distinct responses in respect to sensor sensitivity and signal trends. The obtained results reveal the strong potential of the superhydrophobic QCM for future inclusion in diverse laboratory analyses closely related to the in vitro fertilization procedures, with a final aim of gaining practical approaches for diagnoses and selection of male gametes.

Bifurcations in the Kuramoto model on graphs.

In his classical work, Kuramoto analytically described the onset of synchronization in all-to-all coupled networks of phase oscillators with random intrinsic frequencies. Specifically, he identified a critical value of the coupling strength, at which the incoherent state loses stability and a gradual build-up of coherence begins. Recently, Kuramoto's scenario was shown to hold for a large class of coupled systems on convergent families of deterministic and random graphs [Chiba and Medvedev, "The mean field analysis of the Kuramoto model on graphs. I. The mean field equation and the transition point formulas," Discrete and Continuous Dynamical Systems-Series A (to be published); "The mean field analysis of the Kuramoto model on graphs. II. Asymptotic stability of the incoherent state, center manifold reduction, and bifurcations," Discrete and Continuous Dynamical Systems-Series A (submitted).]. Guided by these results, in the present work, we study several model problems illustrating the link between network topology and synchronization in coupled dynamical systems. First, we identify several families of graphs, for which the transition to synchronization in the Kuramoto model starts at the same critical value of the coupling strength and proceeds in a similar manner. These examples include Erdos-Rényi random graphs, Paley graphs, complete bipartite graphs, and certain stochastic block graphs. These examples illustrate that some rather simple structural properties such as the volume of the graph may determine the onset of synchronization, while finer structural features may affect only higher order statistics of the transition to synchronization. Furthermore, we study the transition to synchronization in the Kuramoto model on power law and small-world random graphs. The former family of graphs endows the Kuramoto model with very good synchronizability: the synchronization threshold can be made arbitrarily low by varying the parameter of the power law degree distribution. For the Kuramoto model on small-world graphs, in addition to the transition to synchronization, we identify a new bifurcation leading to stable random twisted states. The examples analyzed in this work complement the results in Chiba and Medvedev, "The mean field analysis of the Kuramoto model on graphs. I. The mean field equation and the transition point formulas," Discrete and Continuous Dynamical Systems-Series A (to be published); "The mean field analysis of the Kuramoto model on graphs. II. Asymptotic stability of the incoherent state, center manifold reduction, and bifurcations," Discrete and Continuous Dynamical Systems-Series A (submitted).

A Role of Cytokine Gene Polymorphisms in Cognitive Functioning.

The changes in cognitive functions that occur with aging and in various pathological conditions are a subject of growing interest. Experimental and clinical data justify the hypothesis about the influence the immune system exerts on cognitive processes. The balance between pro-inflammatory and anti-inflammatory cytokines has been established as a necessary factor for normal cognitive functioning. Cytokine production is under strong genetic control and various single nucleotide polymorphisms (SNPs) in cytokine genes have been described. As cytokine SNPs have been demonstrated to affect the gene expression or the functional activity of the immune protein this logically led to the suggestion about the role of these polymorphisms in cognitive functioning. Studies exploring the association between different genetic variants of cytokine gene polymorphisms and cognitive abilities in healthy subjects and in demented patients show divergent results. The review of relevant literature suggests that SNPs implement their effect on cognition in large interactions with each other, as well as with many other factors, some of which still remain to be identified. This article summarizes the contemporary knowledge about the correlations between SNPs in cytokine genes and cognitive status in humans. Further research is needed to determine the precise role and the molecular mechanisms of action of the SNPs in cognitive processes.

ALK Mutation Status in EGFR-negative Non-small-cell Lung Cancer Patients in Bulgaria.

BACKGROUND: Patients with non-small-cell lung cancer (NSCLC) with anaplastic lymphoma kinase (ALK) rearrangement mutation are found to be 3-13%. AIM: To evaluate the prevalence of ALK mutations in EGFR-negative NSCLC patients in Bulgaria. MATERIALS AND METHODS: One hundred and thirty-two patients with EGFR-negative NSCLC were examined for ALK mutation analysis between January and June 2016. Data were obtained from patients' register of four major oncological hospitals in Bulgaria. RESULTS: Data were available for 124 (93.9%) patients, tumor mass was insufficient for analysis in 8 (6.1%) patients. Most of the patients were with adenocarcinoma (82 patients, 62.1%); 11 patients (8.3%) were with squamous histology and 2 patients (1.5%) were with other type of NSCLC. Histology data were missing in 37 patients (28.0%). ALK mutation was confirmed in 5 patients (3.8%), 119 (90.2%) patients had ALK wild type. ALK positive patients were with adenocarcinoma (n=3), squamous cell carcinoma (n=1) and other type (n=1) NSCLC. All ALK mutations were observed in never smokers (n=3) and former smokers (n=2). CONCLUSION: The present study is the first of this kind in Bulgaria - it investigates the prevalence of ALK mutation rate in EGFR-negative NSCLC patients, which was found to be 3.8%. The presence of EGFR, ALK or other driver mutations is a prerequisite for targeted therapy and thus needs to be accurately assessed in NSCLC.

Differences in the transduction of canonical Wnt signals demarcate effector and memory CD8 T cells with distinct recall proliferation capacity.

Protection against reinfection is mediated by Ag-specific memory CD8 T cells, which display stem cell-like function. Because canonical Wnt (Wingless/Int1) signals critically regulate renewal versus differentiation of adult stem cells, we evaluated Wnt signal transduction in CD8 T cells during an immune response to acute infection with lymphocytic choriomeningitis virus. Whereas naive CD8 T cells efficiently transduced Wnt signals, at the peak of the primary response to infection only a fraction of effector T cells retained signal transduction and the majority displayed strongly reduced Wnt activity. Reduced Wnt signaling was in part due to the downregulation of Tcf-1, one of the nuclear effectors of the pathway, and coincided with progress toward terminal differentiation. However, the correlation between low and high Wnt levels with short-lived and memory precursor effector cells, respectively, was incomplete. Adoptive transfer studies showed that low and high Wnt signaling did not influence cell survival but that Wnt high effectors yielded memory cells with enhanced proliferative potential and stronger protective capacity. Likewise, following adoptive transfer and rechallenge, memory cells with high Wnt levels displayed increased recall expansion, compared with memory cells with low Wnt signaling, which were preferentially effector-like memory cells, including tissue-resident memory cells. Thus, canonical Wnt signaling identifies CD8 T cells with enhanced proliferative potential in part independent of commonly used cell surface markers to discriminate effector and memory T cell subpopulations. Interventions that maintain Wnt signaling may thus improve the formation of functional CD8 T cell memory during vaccination.

Seasonal changes of basic erythrocyte-metric parameters in Pelophylaxridibundus (Amphibia: Ranidae) from anthropogenically polluted biotopes in Southern Bulgaria and their role as bioindicators.

The purpose of this research work is to present data that show the seasonal changes (spring-summer-autumn) of basic erythrocyte-metric parameters (ЕL: Erythrocyte length, ЕW: Erythrocyte width, ЕL/ЕW, ES: Erythrocyte size; NL: Nucleus length, NW: Nucleus width, NL/NW; NS: Nucleus size, NS/ES: Nucleus-cytoplasmic ratio) in Pelophylax ridibundus populations from three biotopes located on two rivers in Southern Bulgaria (less disrupted biotope, with domestic sewage pollution and heavy metal pollution). Differences of high statistical significance were found among the different populations. Within the population living in conditions of domestic sewage pollution, for the entire period of the investigation the erythrocytes and their nuclei had an elliptical shape (a slight elongation of ellipses in autumn) and the biggest sizes (EL, EW, ES, NL and NS were constantly higher than the less disrupted biotope), NS/ES, became significantly smaller in autumn. Throughout the period of investigation, the values of all nine cellular and nuclear parameters were statistically-significantly the lowest in the population from the biotope with heavy metal pollution. The parameters: EL, ЕW, NL, NW and ES became significantly lower, progressively and statistically, during seasonal transitions. Cells and nuclei grew ovular in shape in comparison to the populations from the other two biotopes (this process was most pronounced in autumn) and NS/ES numbers were significantly decreased in summer and autumn.

Cognitive Impairment in Multiple Sclerosis.

Multiple sclerosis (MS) is a socially significant immune-mediated disease, characterized by demyelination, axonal transection and oligodendropathy in the central nervous system. Inflammatory demyelination and neurodegeneration lead to brain atrophy and cognitive deficit in up to 75% of the patients. Cognitive dysfunctions impact significantly patients' quality of life, independently from the course and phase of the disease. The relationship between pathological brain findings and cognitive impairment is a subject of intensive research. Summarizing recent data about prevalence, clinical specificity and treatment of cognitive disorders in MS, this review aims to motivate the necessity of early diagnosis and complex therapeutic approach to these disturbances in order to reduce the social burden of the disease.

Education of murine NK cells requires both cis and trans recognition of MHC class I molecules.

Although NK cells use invariant receptors to identify diseased cells, they nevertheless adapt to their environment, including the presence of certain MHC class I (MHC-I) molecules. This NK cell education, which is mediated by inhibitory receptors specific for MHC-I molecules, changes the responsiveness of activating NK cell receptors (licensing) and modifies the repertoire of MHC-I receptors used by NK cells. The fact that certain MHC-I receptors have the unusual capacity to recognize MHC-I molecules expressed by other cells (trans) and by the NK cell itself (cis) has raised the question regarding possible contributions of the two types of interactions to NK cell education. Although the analysis of an MHC-I receptor variant suggested a role for cis interaction for NK cell licensing, adoptive NK cell transfer experiments supported a key role for trans recognition. To reconcile some of these findings, we have analyzed the impact of cell type-specific deletion of an MHC-I molecule and of a novel MHC-I receptor variant on the education of murine NK cells when these mature under steady-state conditions in vivo. We find that MHC-I expression by NK cells (cis) and by T cells (trans), and MHC-I recognition in cis and in trans, are both needed for NK cell licensing. Unexpectedly, modifications of the MHC-I receptor repertoire are chiefly dependent on cis binding, which provides additional support for an essential role for this unconventional type of interaction for NK cell education. These data suggest that two separate functions of MHC-I receptors are needed to adapt NK cells to self-MHC-I.

25 Hydroxyvitamin D and Cytokine Profile in Patients With Relapsing-Remitting Multiple Sclerosis.

In experimental allergic encephalomyelitis, the severity of the deficiency is associated with the loss of axons, and it is likely that cytotoxic T-cells 8 (CD8 T) play an important role. In relapsing-remitting multiple sclerosis, there is a correlation between the inflammatory activity in the lesion and the transection of axons. To understand the pathological mechanisms, it is important to evaluate the changes in serum concentrations of pro- and anti-inflammatory cytokines during the disease course. A total of 46 patients and 40 healthy individuals participated in an open-label, prospective, case-control study from 2012 to 2014. The serum concentrations of cytokines were measured using enzyme-linked immunosorbent assay (ELISA). An immune imbalance was observed during relapse and remission phases compared to the control group. During relapse, the levels of interferon-gamma (IFN-γ) were significantly higher compared to those in remission (p=0.017). During remission, there was an improvement in the deficiency (p<0.001), and the anti-inflammatory cytokines transforming growth factor-beta (TGF-ß) and interleukin 4 (IL4) increased compared to those in relapse (p=0.006; p=0.009). A correlation was found between the serum concentrations of tumor necrosis factor-alpha (TNF-α) and Expanded Disability Status Scale (EDSS) during relapse (correlation coefficient: 0.301; significance (Sig.) (2-tailed 0.042). During the exacerbation, there was a moderate relationship between interleukin 17 (IL17) and 25-hydroxyvitamin D (25(OH)D) (P (p-value (probability value) = 0.02)). TNF-α, IFN-γ, IL17, and TGF-ß serum levels are criteria for evaluating immune inflammatory activity during relapse and remission periods.

Vitamin D immunomodulatory potential in multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of unknown etiology whose treatment is of limited efficiency and therefore has a high social burden. As it has been suggested that myelin destruction model, the clinical manifestation and the potential of therapeutic response in MS are correlated, it is quite justifiable that we study various factors (genetic, hormonal, environmental) that take part in the autoimmune process in order to improve the control over the disrupted immune regulation. Results from epidemiological and clinical studies clearly suggest that changes in vitamin D serum concentrations are correlated with the magnitude of the risk of developing MS, the phases of relapse and remittance and with gender differences in vitamin D metabolism. Experimental and clinical studies also have established that 25-hydroxy vitamin D (25(OH)D) and 1,25-dihydroxy vitamin D (1,25(OH)2D) exert an immunomodulatory effect in the central nervous system and peripheral organs of the immune system. The standard reference range of vitamin D concentration in serum is 50-80 nmol/l--it provides normal calcium metabolism. Issues that are discussed include the vitamin D serum concentration needed to suppress the aberrant immune response in MS patients; a subgroup of MS patients suitable for vitamin D treatment, the vitamin D being applied in optimally effective and safe dosage. MS prevalence rate in Bulgaria has increased two-fold in 17 years but this is a rather short interval to be able to assume that the gene pool of the population changes. Thus further studies on possible interactions between different environmental factors and these factors' role in the disease pathogenesis are justified and necessary.

Shaping bursting by electrical coupling and noise.

Gap-junctional coupling is an important way of communication between neurons and other excitable cells. Strong electrical coupling synchronizes activity across cell ensembles. Surprisingly, in the presence of noise synchronous oscillations generated by an electrically coupled network may differ qualitatively from the oscillations produced by uncoupled individual cells forming the network. A prominent example of such behavior is the synchronized bursting in islets of Langerhans formed by pancreatic ß-cells, which in isolation are known to exhibit irregular spiking (Sherman and Rinzel, Biophys J 54:411-425, 1988; Sherman and Rinzel, Biophys J 59:547-559, 1991). At the heart of this intriguing phenomenon lies denoising, a remarkable ability of electrical coupling to diminish the effects of noise acting on individual cells. In this paper, building on an earlier analysis of denoising in networks of integrate-and-fire neurons (Medvedev, Neural Comput 21 (11):3057-3078, 2009) and our recent study of spontaneous activity in a closely related model of the Locus Coeruleus network (Medvedev and Zhuravytska, The geometry of spontaneous spiking in neuronal networks, submitted, 2012), we derive quantitative estimates characterizing denoising in electrically coupled networks of conductance-based models of square wave bursting cells. Our analysis reveals the interplay of the intrinsic properties of the individual cells and network topology and their respective contributions to this important effect. In particular, we show that networks on graphs with large algebraic connectivity (Fiedler, Czech Math J 23(98):298-305, 1973) or small total effective resistance (Bollobas, Modern graph theory, Graduate Texts in Mathematics, vol. 184, Springer, New York, 1998) are better equipped for implementing denoising. As a by-product of the analysis of denoising, we analytically estimate the rate with which trajectories converge to the synchronization subspace and the stability of the latter to random perturbations. These estimates reveal the role of the network topology in synchronization. The analysis is complemented by numerical simulations of electrically coupled conductance-based networks. Taken together, these results explain the mechanisms underlying synchronization and denoising in an important class of biological models.

Characterization of a CD44/CD122int memory CD8 T cell subset generated under sterile inflammatory conditions.

Most memory CD8 T cell subsets that have been hitherto defined are generated in response to infectious pathogens. In this study, we have characterized the CD8 T cells that survive priming conditions, devoid of pathogen-derived danger signals. In both a TCR-transgenic model and a model of contact hypersensitivity, we show that the priming of naive CD8 T cells under sterile inflammatory conditions generates memory. The corresponding memory CD8 T cells can be identified by their intermediate expression levels of CD44 and CD122. We also show that CD44/122(int) memory CD8 T cells spontaneously develop in wild type mice and that they display intermediate levels of several other memory traits including functional (IFN-gamma secretion capacity, CCL5 messenger stores), phenotypic, and molecular (T-bet and eomesodermin expression levels) features. We finally show that they correspond to an early differentiation stage and can further differentiate in CD44/122(high) memory T cells. Altogether, our results identify a new memory CD8 T cell subset that is generated under sterile inflammatory conditions and involved in the recall contact hypersensitivity reactions that are responsible for allergic contact dermatitis.

CD8beta knockout mice mount normal anti-viral CD8+ T cell responses--but why?

It has been shown previously that CD8beta in vitro increases the range and the sensitivity of antigen recognition and in vivo plays an important role in the thymic selection of CD8+ T cells. Consistent with this, we report here that CD8+ T cells from CD8beta knockout (KO) P14 TCR transgenic mice proliferate inefficiently in vitro. In contrast to these findings, we also show that CD8beta KO mice mount normal CD8 primary, secondary and memory responses to acute infection with lymphocytic choriomeningitis virus. Tetramer staining and cytotoxic experiments revealed a predominance of CD8-independent CTL in CD8beta KO mice. The TCR repertoire, especially the one of the TCRalpha chain, was different in CD8beta KO mice as compared with B6 mice. Our results indicate that in the absence of CD8beta, CD8-independent TCRs are preferentially selected, which in vivo effectively compensates for the reduced co-receptor function of CD8alphaalpha.

First return maps for the dynamics of synaptically coupled conditional bursters.

The pre-Bötzinger complex (preBötc) in the mammalian brainstem has an important role in generating respiratory rhythms. An influential differential equation model for the activity of individual neurons in the preBötc yields transitions from quiescence to bursting to tonic spiking as a parameter is varied. Further, past work has established that bursting dynamics can arise from a pair of tonic model cells coupled with synaptic excitation. In this paper, we analytically derive one- and two-dimensional maps from the differential equations for a self-coupled neuron and a two-neuron network, respectively. Using a combination of analysis and simulations of these maps, we explore the possible forms of dynamics that the model networks can produce as well as which transitions between dynamic regimes are mathematically possible.

[Aggressive immunotherapy in Susac's syndrome]. / Aggressive Immuntherapie beim Susac-Syndrom.

Susac's syndrome is a rare but important differential diagnosis of aseptic encephalitis of young women with focal neurological deficits and white matter lesions on cerebral MRI. We report on a previously healthy 36-year-old woman who presented with encephalopathy, central weakness of her right leg and multiple white matter lesions on MRI. Shortly thereafter, inner ear deafness developed and funduscopy revealed occlusions of branch retinal arteries. A diagnosis of retino-cochlear-cerebral vasculopathy or Susac's syndrome was established and steroid-based immunotherapy with high-dose corticosteroids was initiated. Steroid reduction led to repeated clinical worsening, so that immunotherapy was sequentially escalated. Finally, high-dose cyclophosphamide every 4 weeks led to sufficient control of disease activity. Recent publications have argued for an early and aggressive immunosuppression in Susac's syndrome based on clinical and histological similarities with juvenile dermatomyositis, where such a regimen has already been established. We report on these treatment guidelines with respect to the current literature and the case presented.

Treatment Optimization of Post-pneumonectomy Pleural Empyema.

INTRODUCTION: Pleural empyema after pneumonectomy still poses a serious postoperative complication. A bronchopleural fistula is often detected. Despite various therapeutic options developed during the last five decades it remains a major surgical challenge. RESULTS: There is no widely accepted treatment for post-pneumonectomy pleural empyema (PPE) and the management depends mostly on the presence or absence of broncho-pleural fistula (BPF) and the patient's general condition. In the absence of BPF, the role of surgery is still not clear because of its high morbidity and impossibility to prevent recurrences. In the earlier period, the definitive treatment consisted of open window thoracostomy followed by obliteration of the pleural cavity with antibiotic solution at the time of chest wall closure. Subsequently, the proposed different methods and modifications improved the outcome. There is an association between hospital volume and operative mortality after the lung resection. Hospital volume and the surgeon's specialty have more influence on the outcome than the individual surgeon's volume. CONCLUSIONS: Treatment management of PPE should be individualized. Definitive treatment options comprise aggressive surgery that is not possible in quite a high proportion of impaired patients. Hospital volume, surgeon's volume and surgeon's specialty may influence the prognosis.

Epidemiology, Etiology and Prevention of Postpneumonectomy Pleural Empyema.

BACKGROUND: Pleural empyema after pneumonectomy still poses a serious postoperative complication. A broncho-pleural fistula is often detected. Despite various therapeutic options developed over the last five decades it remains a major surgical challenge. MATERIALS AND METHODS: A literature search in MEDLINE database was carried out (accessed through PubMed), by using a combination of the following key-words and MeSH terms: pneumonectomy, postoperative, complications, broncho-pleural fistula, empyema, prevention. The following areas of intervention were identified: epidemiology, etiology, prevention. RESULTS: Pleural empyema in a post-pneumonectomy cavity occurs in up to 16% of patients with a mortality of more than 10%. It is associated with broncho-pleural fistula in up to 80% of them, usually in the early postoperative months. Operative mortality could reach 50% in case of broncho-pleural fistula. Unfavourable prognostic factors are: benign disease, COPD, right-sided surgery, neoadjuvant and adjuvant therapy, time of chest tube removal, long bronchial stump and mechanical ventilation. Bronchial stump protection with vascularised flaps is of utmost importance in the prevention of complications. CONCLUSION: Postpneumonectomy pleural empyema is a common complication with high mortality. The existing evidence confirms the role of bronchopleural fistula prevention in the prevention of life-threatening complications.