Dynamic simulation of regulatory networks using SQUAD.

BACKGROUND: The ambition of most molecular biologists is the understanding of the intricate network of molecular interactions that control biological systems. As scientists uncover the components and the connectivity of these networks, it becomes possible to study their dynamical behavior as a whole and discover what is the specific role of each of their components. Since the behavior of a network is by no means intuitive, it becomes necessary to use computational models to understand its behavior and to be able to make predictions about it. Unfortunately, most current computational models describe small networks due to the scarcity of kinetic data available. To overcome this problem, we previously published a methodology to convert a signaling network into a dynamical system, even in the total absence of kinetic information. In this paper we present a software implementation of such methodology. RESULTS: We developed SQUAD, a software for the dynamic simulation of signaling networks using the standardized qualitative dynamical systems approach. SQUAD converts the network into a discrete dynamical system, and it uses a binary decision diagram algorithm to identify all the steady states of the system. Then, the software creates a continuous dynamical system and localizes its steady states which are located near the steady states of the discrete system. The software permits to make simulations on the continuous system, allowing for the modification of several parameters. Importantly, SQUAD includes a framework for perturbing networks in a manner similar to what is performed in experimental laboratory protocols, for example by activating receptors or knocking out molecular components. Using this software we have been able to successfully reproduce the behavior of the regulatory network implicated in T-helper cell differentiation. CONCLUSION: The simulation of regulatory networks aims at predicting the behavior of a whole system when subject to stimuli, such as drugs, or determine the role of specific components within the network. The predictions can then be used to interpret and/or drive laboratory experiments. SQUAD provides a user-friendly graphical interface, accessible to both computational and experimental biologists for the fast qualitative simulation of large regulatory networks for which kinetic data is not necessarily available.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

tagO is involved in the synthesis of all anionic cell-wall polymers in Bacillus subtilis 168.

Sequence homologies suggest that the Bacillus subtilis 168 tagO gene encodes UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme responsible for catalysing the first step in the synthesis of the teichoic acid linkage unit, i.e. the formation of undecaprenyl-PP-N-acetylglucosamine. Inhibition of tagO expression mediated by an IPTG-inducible P(spac) promoter led to the development of a coccoid cell morphology, a feature characteristic of mutants blocked in teichoic acid synthesis. Indeed, analyses of the cell-wall phosphate content, as well as the incorporation of radioactively labelled precursors, revealed that the synthesis of poly(glycerol phosphate) and poly(glucosyl N-acetylgalactosamine 1-phosphate), the two strain 168 teichoic acids known to share the same linkage unit, was affected. Surprisingly, under phosphate limitation, deficiency of TagO precludes the synthesis of teichuronic acid, which is normally induced under these conditions. The regulatory region of tagO, containing two partly overlapping sigma(A)-controlled promoters, is similar to that of sigA, the gene encoding the major sigma factor responsible for growth. Here, the authors discuss the possibility that TagO may represent a pivotal element in the multi-enzyme complexes responsible for the synthesis of anionic cell-wall polymers, and that it may play one of the key roles in balanced cell growth.

A dendritic cell vaccine pulsed with autologous hypochlorous Acid-oxidized ovarian cancer lysate primes effective broad antitumor immunity: from bench to bedside.

PURPOSE: Whole tumor lysates are promising antigen sources for dendritic cell (DC) therapy as they contain many relevant immunogenic epitopes to help prevent tumor escape. Two common methods of tumor lysate preparations are freeze-thaw processing and UVB irradiation to induce necrosis and apoptosis, respectively. Hypochlorous acid (HOCl) oxidation is a new method for inducing primary necrosis and enhancing the immunogenicity of tumor cells. EXPERIMENTAL DESIGN: We compared the ability of DCs to engulf three different tumor lysate preparations, produce T-helper 1 (TH1)-priming cytokines and chemokines, stimulate mixed leukocyte reactions (MLR), and finally elicit T-cell responses capable of controlling tumor growth in vivo. RESULTS: We showed that DCs engulfed HOCl-oxidized lysate most efficiently stimulated robust MLRs, and elicited strong tumor-specific IFN-γ secretions in autologous T cells. These DCs produced the highest levels of TH1-priming cytokines and chemokines, including interleukin (IL)-12. Mice vaccinated with HOCl-oxidized ID8-ova lysate-pulsed DCs developed T-cell responses that effectively controlled tumor growth. Safety, immunogenicity of autologous DCs pulsed with HOCl-oxidized autologous tumor lysate (OCDC vaccine), clinical efficacy, and progression-free survival (PFS) were evaluated in a pilot study of five subjects with recurrent ovarian cancer. OCDC vaccination produced few grade 1 toxicities and elicited potent T-cell responses against known ovarian tumor antigens. Circulating regulatory T cells and serum IL-10 were also reduced. Two subjects experienced durable PFS of 24 months or more after OCDC. CONCLUSIONS: This is the first study showing the potential efficacy of a DC vaccine pulsed with HOCl-oxidized tumor lysate, a novel approach in preparing DC vaccine that is potentially applicable to many cancers. Clin Cancer Res; 19(17); 4801-15. ©2013 AACR.

Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

Differential requirement for CD4 help in the development of an antigen-specific CD8+ T cell response depending on the route of immunization.

Previous studies in our laboratory have shown that DBA/2 mice injected i.p. with syngeneic P815 tumor cells transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8+CD62L- T cells expressing exclusively the Vbeta10 segment. We have used this model to study the regulatory mechanisms involved in the development of the CW3-specific CD8+ response, with respect to different routes of immunization. Whereas both intradermal (i.d.) and i.p. immunization of DBA/2 mice with P815-CW3 cells led to a strong expansion of CD8+CD62L-Vbeta10+ cells, only the i.d. route allowed this expansion after immunization with P815 cells transfected with a minigene coding for the antigenic epitope CW3 170-179 (P815 miniCW3). Furthermore, depletion of CD4+ T cells in vivo completely abolished the specific response of CD8+CD62L-Vbeta10+ cells and prevented the rejection of P815-CW3 tumor cells injected i.p., whereas it did not affect CD8S+CD62L-Vbeta10+ cell expansion after i.d. immunization with either P815-CW3 or P815 miniCW3. Finally, the CW3-specific CD8+ memory response was identical whether or not CD4+ T cells were depleted during the primary response. Collectively, these results suggest that the CD8+ T cell response to P815-CW3 tumor cells injected i.p. is strictly dependent upon recognition of a helper epitope by CD4+ T cells, whereas no such requirement is observed for i.d. injection.

Cloning and expression of rat pancreatic beta-cell malonyl-CoA decarboxylase.

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

24 Hours in the Life of HIV-1 in a T Cell Line.

HIV-1 infects CD4+ T cells and completes its replication cycle in approximately 24 hours. We employed repeated measurements in a standardized cell system and rigorous mathematical modeling to characterize the emergence of the viral replication intermediates and their impact on the cellular transcriptional response with high temporal resolution. We observed 7,991 (73%) of the 10,958 expressed genes to be modulated in concordance with key steps of viral replication. Fifty-two percent of the overall variability in the host transcriptome was explained by linear regression on the viral life cycle. This profound perturbation of cellular physiology was investigated in the light of several regulatory mechanisms, including transcription factors, miRNAs, host-pathogen interaction, and proviral integration. Key features were validated in primary CD4+ T cells, and with viral constructs using alternative entry strategies. We propose a model of early massive cellular shutdown and progressive upregulation of the cellular machinery to complete the viral life cycle.

Protein kinase C-activating tumor promoters enhance the differentiation of astrocytes in aggregating fetal brain cell cultures.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a marked, rapid, and sustained increase in the activity of the astrocyte-specific enzyme glutamine synthetase (GS). This effect was accompanied by a small increase in RNA synthesis and a progressive reduction in DNA synthesis. Only mitotically active cultures were responsive to PMA treatments. Since in aggregate cultures astrocytes are the preponderant cell type, both in number and mitotic activity, it can be concluded that PMA induces and/or enhances the terminal differentiation of astrocytes. The developmental expression of GS was also greatly stimulated by mezerein, a potent nonphorbol tumor promoter, but not by 4 alpha-phorbol 12,13-didecanoate, a nonpromoting phorbol ester. Since both tumor promoters, PMA and mezerein, are potent and specific activators of C-kinase, it is suggested that C-kinase plays a regulatory role in the growth and differentiation of normal astrocytes.

Strength and limitations of regulatory T Cells for immunotherapy in transplantation.

In many experimental models, CD4+CD25+Foxp3+ regulatory T cells (nTreg) have been identifi ed as key players in promoting peripheral transplantation (Tx) tolerance. We have been focusing on therapies based on antigen-specifi c nTreg that can control effector T cells (Teff) and prevent allograft rejection. The use of nTreg in immunotherapeutic protocols for solid organ Tx is however limited by their overall low numbers as well as the low precursor frequency of alloantigen cross-reactive nTreg expected to be found in a normal individual. Moreover, although we previously described robust protocols to generate and expand antigen-specifi c nTreg in vitro, the process requires careful selection of highly pure nTreg and cumbersome ex-vivo manipulations, rendering this strategy not easily applicable in clinical solid organ Tx. In this study, we aimed to expand Treg directly in vivo and determine their suppressive function, effi cacy and stability in promoting donor-specifi c tolerance in a stringent murine Tx model. Our data suggest that IL-2-based therapies lead to a signifi cant increase of Treg in vivo. The expanded Treg suppressed Teff proliferation (albeit slightly less effi ciently than nTreg isolated from control mice) and allowed prolonged graft survival of major MHC-mismatched skin grafts in wild-type non-lymphopenic recipients. The expanded Treg alone were however not suffi cient to induce tolerance in stringent experimental conditions. Rapamycin reduced the frequency of Teff but did not impede expansion of Treg. Pro-infl ammatory stimuli hindered the expansion of Treg and resulted in an increase in the frequency of CD4+IFN-γ+ and CD4+IL17+ T cells. We propose that IL-2-based treatments would be an effi cient method for expanding functional Treg in vivo without affecting other immune cell populations, thereby favorably shifting the pool of alloreactive T cells towards regulation in response to an allograft. However, we also highlight some potential limitations of Treg expansion such as concomitant infl ammatory events.

Overlapping, additive and counterregulatory effects of type II and I interferons on myeloid dendritic cell functions.

Dendritic cells (DCs) are central player in immunity by bridging the innate and adaptive arms of the immune system (IS). Interferons (IFNs) are one of the most important factors that regulate both innate and adaptive immunity too. Thus, the understanding of how type II and I IFNs modulate the immune-regulatory properties of DCs is a central issue in immunology. In this paper, we will address this point in the light of the most recent literature, also highlighting the controversial data reported in the field. According to the wide literature available, type II as well as type I IFNs appear, at the same time, to collaborate, to induce additive effects or overlapping functions, as well as to counterregulate each one's effects on DC biology and, in general, the immune response. The knowledge of these effects has important therapeutic implications in the treatment of infectious/autoimmune diseases and cancer and indicates strategies for using IFNs as vaccine adjuvants and in DC-based immune therapeutic approaches.

T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-gamma), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome.

Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.

Modeling stochasticity and robustness in gene regulatory networks.

MOTIVATION: Understanding gene regulation in biological processes and modeling the robustness of underlying regulatory networks is an important problem that is currently being addressed by computational systems biologists. Lately, there has been a renewed interest in Boolean modeling techniques for gene regulatory networks (GRNs). However, due to their deterministic nature, it is often difficult to identify whether these modeling approaches are robust to the addition of stochastic noise that is widespread in gene regulatory processes. Stochasticity in Boolean models of GRNs has been addressed relatively sparingly in the past, mainly by flipping the expression of genes between different expression levels with a predefined probability. This stochasticity in nodes (SIN) model leads to over representation of noise in GRNs and hence non-correspondence with biological observations. RESULTS: In this article, we introduce the stochasticity in functions (SIF) model for simulating stochasticity in Boolean models of GRNs. By providing biological motivation behind the use of the SIF model and applying it to the T-helper and T-cell activation networks, we show that the SIF model provides more biologically robust results than the existing SIN model of stochasticity in GRNs. AVAILABILITY: Algorithms are made available under our Boolean modeling toolbox, GenYsis. The software binaries can be downloaded from http://si2.epfl.ch/ approximately garg/genysis.html.