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At the present time, sepsis is primarily considered a disorder of the immune system caused by dominant infection, manifesting with individual response reaction of the host organism. Hence, sepsis should not be classified as a distinct pro- or anti-inflammatory syndrome, but rather as a variable continuum of overlapping immune mechanisms. The role of innate immunity in sepsis is probably the leading one, since it exerts an early and nonspecific response to any foreign agent. The nature of appropriate relationships may determine current state of the immune system. To perform a deeper study of the anti-infectious protective mechanisms, we have evaluated the relationships between the immunological parameters of peripheral blood in patients with sepsis, depending on the outcome of the disease. Total number of leukocytes, and neutrophils was determined in whole peripheral blood of the patients with sepsis, along with calculation of leukocyte counts and numbers of neutrophil extracellular traps. Serum levels of procalcitonin, peptidyl-arginine deiminase 4, and cytokines were studied by ELISA assay. To study the relationship between the indices of innate immunity, we used correlation analysis, which was carried out by the Spearman rank criterion. When conducting the statistical analysis, strong correlations between the immunological parameters of peripheral blood in patients with sepsis were not revealed, regardless of the outcome of the disease. In cases of favorable outcome, 10 relationships were identified, with an unfavorable outcome, 7 relationships were registered. All the relationships were of medium strength. Regardless of clinical outcome of sepsis, the significant relationships were established between the number of leukocytes and the level of neutrophil extracellular traps; positive relationships were also found between pro-inflammatory cytokines IL-1 IL-6, TNF IL-18. However, these relationships were significant in cases with favorable outcome, while becoming weaker and losing their significance for the group with lethal outcomes. Among the revealed relationships, the most interesting finding was an association between favorable outcome of sepsis with IL-10 contents, which is an anti-inflammatory cytokine coordinating the innate immune response. In lethal outcomes, such relationship with IL-10 was not revealed. IL-10, a cytokine with anti-inflammatory properties may limit the immune response to pathogens and, thus, potentially prevent damage to the host. Therefore, the relationship between IL-10 and other immunological parameters of peripheral blood in sepsis may affect the outcome of this condition.

Infections represent a common health problem in people at any age. Usually, a local reaction develops in response to the infection, and intensive treatment is not required. However, sometimes the response to infection is inadequate and may lead to organ dysfunction; such condition is defined as septic state. The pathophysiology of sepsis is multifactorial, and the immune system is directly involved in development of pathological processes accompanying septic conditions. Dysregulatory disorders in sepsis may be observed, in particular, those affecting immune system and, immediately, innate immunity, which presents the first line of immune defense. Currently, neutrophilic granulocytes are considered important players in development and maintenance of inflammatory processes, which is due to their ability to produce various inflammatory mediators. At the same time, at different stages of pathogenesis, immune factors and mechanisms act both as a generating factors and effectors of damage reactions, as well as the main components of the defense responses. Uncontrolled release of endogenous inflammatory mediators induced by microorganisms, and lack of damagelimiting mechanisms mutually synergize when promoting the organ dysfunction. Hence, dysfunction of the immune system may develop and deepen upon any inappropriate strategy of response to distinct infectious pathogen. The aim of our study was to assess the spectrum of microorganisms, levels of neutrophilic extracellular traps (NVL), PAD4, IL-6, IL-8 and their correlations in the patients with sepsis and septic shock, depending on the outcome of the disease. The study included 44 patients with sepsis and septic shock, and 20 apparently healthy donors. Peripheral blood samples were taken from the ulnar vein serially in dynamics, from the moment of diagnosis to clinical outcome. Microbiological and immunological studies have been carried out. The obtained data were processed with “SPSS Statistic 23.0” software. Development of septic conditions was found to be accompanied by a significant changes in the levels of studied immunological parameters (NLV, IL-6, IL-8 and PAD4). Toxicity of lysosomal enzymes and oxygen metabolites present in neutrophilic extracellular traps leads to endothelial damage, appearance of focal intravascular inflammation, which, along with damaging effects of microorganisms and their toxins, leads to development of organ failure and onset. High burden of these factors is most likely to cause lethal outcomes.

TNF mediates a variety of biological processes including cellular proliferation, inflammatory responses, and cell death and is therefore associated with numerous pathologies including autoinflammatory diseases and septic shock. The inflammatory and cell death responses to TNF have been studied extensively downstream of TNF-R1 and are believed to rely on the formation of proinflammatory complex I and prodeath complex II, respectively. We recently identified a similar multimeric complex downstream of TLR4, termed the TRIFosome, that regulates inflammation and cell death in response to LPS or Yersinia pseudotuberculosis . We present evidence of a role for the TRIFosome downstream of TNF-R1, independent of TLR3 or TLR4 engagement. Specifically, TNF-induced cell death and inflammation in murine macrophages were driven by the TLR4 adaptor TRIF and the LPS co-receptor CD14, highlighting an important role for these proteins beyond TLR-mediated immune responses. Via immunoprecipitation and visualization of TRIF-specific puncta, we demonstrated TRIF- and CD14-dependent formation of prodeath and proinflammatory complexes in response to TNF. Extending these findings, in a murine TNF–induced sepsis model, TRIF and CD14 deficiency decreased systemic inflammation, reduced organ pathology, and improved survival. The outcome of TRIF activation was cell specific, because TNF-induced lethality was mediated by neutrophils and macrophages responding to TNF in a TRIF-dependent manner. Our findings suggest that in addition to their crucial role in TNF production, myeloid cells are central to TNF toxicity and position TRIF and CD14 as universal components of receptor-mediated immune responses.

Multiple research groups have demonstrated that caspase-8 (CASP8)-mediated gasdermin D (GSDMD) cleavage drives pyroptotic cell death. Here, we discuss a novel role for the enzymatically inactive homolog of CASP8, the long isoform of cellular FLICE-like inhibitory protein (cFLIP(L)), in the regulation of this process. Specifically, cFLIP-deficiency provides a model in which to study the mechanisms regulating CASP8-mediated activation of cell death and inflammatory signaling.

Most genetic ablations of Interferon (IFN) signaling abolish both the experimentally induced IFN response and constitutive IFN, whose effects are well established in autoimmunity but understudied during infection. In host-pathogen interactions, most IFN-mediated responses are attributed to infection-driven IFN. However, IFNs confer their activity by regulating networks of Interferon Stimulated Genes (ISGs), a process that requires de novo transcription and translation of both IFN and downstream ISGs through feedback of IFN receptor signaling. Due to the temporal requirement for IFN activity, many rapid anti-microbial responses may instead result from pre-established IFN signature stemming from host-intrinsic processes. Addressing the permeating effects of constitutive IFN is therefore needed to accurately describe immunity as host-intrinsic or pathogen-induced.

Purpose Transcriptome changes can be expected in survivors after lethal irradiation. We aimed to characterize these in males and females and after different cytokine treatments 60 days after irradiation. Material and methods Male and female rhesus macaques (n = 142) received a whole-body exposure with 700 cGy, from which 60 animals survived. Peripheral whole blood was drawn pre-exposure and before sacrificing the surviving animals after 60 days. Results We evaluated gene expression in a three-phase study design. Phase I was a whole-genome screening (NGS) for mRNAs using five pre- and post-exposure RNA samples from both sexes (n = 20). Differential gene expression (DGE) was calculated between samples of survivors and pre-exposure samples (reference), separately for males and females. 1,243 up- and down-regulated genes were identified with 30–50% more deregulated genes in females. 37 candidate mRNAs were chosen for qRT-PCR validation in phase II using the remaining samples (n = 117). Altogether 17 genes showed (borderline) significant (t-test) DGE in groups of untreated or treated animals. Nine genes (CD248, EDAR, FAM19A5, GAL3ST4, GCNT4, HBG2/1, LRRN1, NOG, SYT14) remained with significant changes and were detected in at least 50% of samples per group. Panther analysis revealed an overlap between both sexes, related to the WNT signaling pathway, cell adhesion and immunological functions. For phase III, we validated the nine genes with candidate genes (n = 32) from an earlier conducted study on male baboons. Altogether 14 out of 41 genes showed a concordantly DGE across both species in a bilateral comparison. Conclusions Sixty days after radiation exposure, we identified (1) sex and cytokine treatment independent transcriptional changes, (2) females with almost twice as much deregulated genes appeared more radio-responsive than males, (3) Panther analysis revealed an association with immunological processes and WNT pathway for both sexes.

The identification and characterization of DNA-sensing pathways has been a subject of intensive investigation for the last decade. This interest, in part, is supported by the fact that the main outcome of DNA-responses is production of type I interferon (IFN-I), which, if produced in excessive amounts, leads to various pathologies. STING (Stimulator of Interferon Genes) is positioned in the center of these responses and is activated either via direct sensing of second messengers or via interaction with upstream sensors of dsDNA. STING mediates responses to pathogens as well as host-derived DNA and is, therefore, linked to various autoimmune diseases, cancer predisposition and ageing. Recent mouse models of DNA damage showed the adaptor STING to be crucial for heightened resting levels of IFN-I. In this review, we will focus on recent advances in understanding the regulation of STING-signaling and identification of its novel components.

Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.

Endogenous retroviruses and retrotransposons also termed retroelements (REs) are mobile genetic elements that were active until recently in human genome evolution. REs regulate gene expression by actively reshaping chromatin structure or by directly providing transcription factor binding sites (TFBSs). We aimed to identify molecular processes most deeply impacted by the REs in human cells at the level of TFBS regulation. By using ENCODE data, we identified ~2 million TFBS overlapping with putatively regulation-competent human REs located in 5-kb gene promoter neighborhood (~17% of all TFBS in promoter neighborhoods; ~9% of all RE-linked TFBS). Most of REs hosting TFBS were highly diverged repeats, and for the evolutionary young (0-8% diverged) elements we identified only ~7% of all RE-linked TFBS. The gene-specific distributions of RE-linked TFBS generally correlated with the distributions for all TFBS. However, several groups of molecular processes were highly enriched in the RE-linked TFBS regulation. They were strongly connected with the immunity and response to pathogens, with the negative regulation of gene transcription, ubiquitination, and protein degradation, extracellular matrix organization, regulation of STAT signaling, fatty acids metabolism, regulation of GTPase activity, protein targeting to Golgi, regulation of cell division and differentiation, development and functioning of perception organs and reproductive system. By contrast, the processes most weakly affected by the REs were linked with the conservative aspects of embryo development. We also identified differences in the regulation features by the younger and older fractions of the REs. The regulation by the older fraction of the REs was linked mainly with the immunity, cell adhesion, cAMP, IGF1R, Notch, Wnt, and integrin signaling, neuronal development, chondroitin sulfate and heparin metabolism, and endocytosis. The younger REs regulate other aspects of immunity, cell cycle progression and apoptosis, PDGF, TGF beta, EGFR, and p38 signaling, transcriptional repression, structure of nuclear lumen, catabolism of phospholipids, and heterocyclic molecules, insulin and AMPK signaling, retrograde Golgi-ER transport, and estrogen signaling. The immunity-linked pathways were highly represented in both categories, but their functional roles were different and did not overlap. Our results point to the most quickly evolving molecular pathways in the recent and ancient evolution of human genome.

Identification and studying of numerous functions of all genes of the human beings is one of the main objects of modern biological science. Due to high level of homology between mouse and human genomes the important role to reach above mentioned goal belongs to the mouse model which using in the classical genetics increase in connection with appearance of different inbred mouse lines. For instance, the differences in immune response to infectious pathogens in various mouse lines were used many times to determine immunologically competent genes. That is why the contribution of mouse model in understanding of the mechanisms of immune response to infectious pathogens is difficult to overestimate. In the current review some of the most successful and well known examples of mouse using in studies of anti-infectious response are described.

Hematopoietic syndrome of acute radiation syndrome (HSARS) is a life-threatening condition with no approved treatment. We compared recombinant human interleukin-12 (rHuIL-12; 175 ng/kg × 1) with vehicle, granulocyte-colony-stimulating factor (G-CSF; 10 µg/kg/day × 18), or rHuIL-12+G-CSF after lethal irradiation in rhesus monkeys in a Good Laboratory Practice, randomized, blinded, placebo-controlled study. Fluids, antibiotics, and blood products were not used. Survival at day 60 was significantly increased for rHuIL-12 versus G-CSF or vehicle. rHuIL-12/G-CSF combination provided no additional survival benefit over rHuIL-12. Both rHuIL-12 and rHuIL-12+G-CSF increased blood cell nadirs, induced earlier recovery of all hematopoietic lineages, and significantly decreased frequencies of severe cytopenias versus vehicle or G-CSF. In bone marrow, rHuIL-12 alone increased erythroid, myeloid, and megakaryocyte counts relative to vehicle or G-CSF. Thus, a single injection of rHuIL-12, without supportive medical intervention, significantly improved survival and promoted multilineage hematopoietic recovery in a nonhuman primate model of HSARS.

The duration and/or the magnitude of Ras-Erk activation are known to be crucial for cell-fate decisions. In T cells, sustained Erk activation correlates with differentiation/proliferation, whereas transient Erk activation parallels with unresponsiveness/apoptosis. The mechanism by which Son of sevenless (Sos) proteins and Ras guanyl-releasing protein 1 (RasGRP1) contribute to dynamics of Erk activation in mature T cells is not yet known. Here, we have assessed this issue using stimuli inducing either transient or sustained TCR signaling and RNA interference mediated suppression of Sos1, Sos2, and RasGRP1 expression in primary human T cells. We found that transient Erk activation depends on RasGRP1 but not on Sos. Conversely, sustained Erk signaling and T-cell activation depend on both Sos1 and RasGRP1. In summary, our data show for the first time that the two guanine nucleotide exchange factors expressed in T cells are differentially involved in the regulation of the duration of Erk phosphorylation and T-cell activation.

Cell death and inflammation are intimately linked during homeostasis, host defense, and tumorigenesis. Pathogenic Yersinia inhibits the MAP kinase TGFβ-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8–mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. The ensuing cell death is rapid and morphologically is similar to pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. The IL1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations provide signal 1 for transcriptional upregulation and signal 2 for the inflammasome assembly. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1β production. Our results uncover a form of caspase-8–mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir. Altogether, this knowledge will be fundamental for our understanding of the mechanism of regulation of cell death and the discovery of new drug targets that can span areas from infections to tissue injury both in and ex vivo. This work was supported by the NIH grant AI056234 and Russian Science Foundation Grant 15-15-00100 (to A.P.)

Recognition of microbial components is critical for activation of TLRs, subsequent innate immune signaling, and directing adaptive immune responses. The DNA sensor TLR9 traffics from the endoplasmic reticulum to endolysosomal compartments where it is cleaved by resident proteases to generate a competent receptor. Activation of TLR9 by CpG-motif containing oligodeoxynucleotides (CpG ODNs) is preceded by agonist endocytosis and delivery into the endolysosomes. The events that dictate this process remain largely unknown; furthermore, it is unclear whether the receptors involved in mediating uptake of exogenous DNA are conserved for both naturally derived pathogenic DNA and synthetic ODNs. In this study, we report that peritoneal macrophages from a wild-derived inbred mouse strain, MOLF/Ei, are hyporesponsive to CpG ODN but are fully responsive to bacterial DNA, thus implying that microbial recognition is not fully recapitulated by a synthetic analog. To identify the gene responsible for the CpG ODN defect, we have performed genome-wide linkage analysis. Using N2 backcross mice, we mapped the trait with high resolution to a single locus containing Mrc1 as the gene conferring the trait. We show that mannose receptor 1 (MRC1; CD206) is involved in CpG ODN uptake and trafficking in wild-derived MOLF/Ei peritoneal macrophages. Furthermore, we show that other strains of wild-derived mice also require MRC1 for CpG-induced cytokine responses. These findings reveal novel functions for MRC1 and demonstrate that wild-derived mice are important and indispensable model for understanding naturally occurring regulators of inflammatory responses in innate immune pathways.

Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses. Here, we have analyzed signaling dynamics induced upon TCR ligation in primary human T cells. We used CD3 antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs), two widely employed methods to stimulate T cells in vitro. We show that classical sAbs stimulation induces a transient and abortive response, whereas iAbs induce sustained TCR-mediated signaling, resulting in productive T-cell responses previously observed only in antigen-specific murine systems. In summary, our analysis documents TCR signaling kinetics and suggests that iAbs are better suited for studying TCR-mediated signaling as they mimic antigen specific systems.

Thanks to the numerous studies that have been carried out recently in the field of cytosolic DNA sensing, STING (Stimulator of Interferon Genes) is now recognized as a key mediator of innate immune signaling. A substantial body of evidence derived from in vivo mouse models demonstrates that STING-regulated pathways underlie the pathogenesis of many diseases including infectious diseases and cancers. It has also become evident from these studies that STING is a promising therapeutic target for the treatment of cancer. However, mouse strains commonly used for modelling innate immune response against infections or tumors do not allow investigators to accurately reproduce certain specific characteristics of immune response observed in human cells. In this review, we will discuss recent data demonstrating that the use of wild-derived genetically distinct inbred mice as a model for investigation into the innate immunity signaling networks may provide valuable insight into the STING-regulated pathways specific for human cells. The maximum complexity of STING-mediated mechanisms can probably be seen in case of DNA virus-induced carcinogenesis in which STING may perform unexpected biological activities. Therefore, in another part of this review we will summarize emerging data on the role of STING in human DNA virus-related oncopathologies, with particular attention to HPV-associated cervical cancer, aiming to demonstrate that STING indeed "starts a new chapter" in research on this issue and that wild-derived mouse models of STING-mediated response to infections will probably be helpful in finding out molecular basis for clinical observations.

cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP-deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3' UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.

Meeting abstracts IL-12 is a heterodimeric, pro-inflammatory cytokine that enhances the cytotoxic activity of natural killer (NK) cells and cytotoxic CD8+ T-lymphocytes, and induces an IFN-α-dominated Th1 CD4+ T-lymphocyte response. IL-12 as an immunotherapeutic agent administered subcutaneously

Infection with the trematode parasite Schistosoma mansoni results in a distinct heterogeneity of disease severity, both in humans and in an experimental mouse model. Severe disease is characterized by pronounced hepatic egg-induced granulomatous inflammation in a proinflammatory cytokine environment, whereas mild disease corresponds with reduced hepatic inflammation in a Th2 skewed cytokine environment. This marked heterogeneity indicates that genetic differences play a significant role in disease development, yet little is known about the genetic basis of dissimilar immunopathology. To investigate the role of genetic susceptibility in murine schistosomiasis, quantitative trait loci analysis was performed on F2 progeny derived from SJL/J and C57BL/6 mice, which develop severe and mild pathology, respectively. In this study, we show that severe liver pathology in F2 mice 7 wk after infection significantly correlated with an increase in the production of the proinflammatory cytokines IL-17, IFN-γ, and TNF-α by schistosome egg Ag-stimulated mesenteric lymph node cells. Quantitative trait loci analysis identified several genetic intervals controlling immunopathology as well as IL-17 and IFN-γ production. Egg granuloma size exhibited significant linkage to two loci, D4Mit203 and D17Mit82, both of which were inherited in a BL/6 dominant manner. Furthermore, a significant reduction of hepatic granulomatous inflammation and IL-17 production in interval-specific congenic mice demonstrated that the two identified genetic loci have a decisive effect on the development of immunopathology in murine schistosomiasis.

In a phenotypic screen of the wild-derived mouse strain MOLF/Ei, we describe an earlier and more potent toll-like receptor (TLR)–mediated induction of IL-6 transcription compared with the classical inbred strain C57BL/6J. The phenotype correlated with increased activity of the IκB kinase axis as well as p38, but not extracellular signal-regulated kinase or c-Jun N-terminal kinase, mitogen-activated protein kinase (MAPK) phosphorylation. The trait was mapped to the Why1 locus, which contains Irak2, a gene previously implicated as sustaining the late phase of TLR responses. In the MOLF/Ei TLR signaling network, IRAK-2 promotes early nuclear factor κB (NF-κB) activity and is essential for the activation of p38 MAPK. We identify a deletion in the MOLF/Ei promoter of the inhibitory Irak2c gene, leading to an increased ratio of pro- to antiinflammatory IRAK-2 isoforms. These findings demonstrate that IRAK-2 is an essential component of the early TLR response in MOLF/Ei mice and show a distinct pathway of p38 and NF-κB activation in this model organism. In addition, they demonstrate that studies in evolutionarily divergent model organisms are essential to complete dissection of signal transduction pathways.

Mouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophila intracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophila growth, whereas macrophages derived from A/J mice allow >10 3 -fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5 allele. The B6 restrictive Naip5 allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophila growth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophila growth restriction in MOLF/Ei macrophages was mapped to the Naip5 region as well, indicating that the originally predicted change in the A/J Naip5 allele may not be critical for restriction. In the product of the A/J Naip5 permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophila intracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.

Although inflammatory cytokines produced by activation of Toll-like receptors (TLRs) are essential for early host defense against infection, they also mediate a vast array of pathologies, including autoimmune disease, hypersensitivity reactions, and sepsis. Thus, numerous regulatory mechanisms exist in parallel with proinflammatory pathways to prevent excessive release of these potent effector molecules. We report elucidation of a novel regulatory function for interleukin receptor–associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1, also known as SIMPL) through quantitative trait locus mapping of the TLR response in wild-derived mouse strains. This gene emerged as a negative regulator of TLR2-mediated interleukin (IL)-6 production in MOLF/Ei mice, which expressed IRAK1BP1 mRNA in an allele-specific manner when crossed with the C57BL/6J strain. Human peripheral blood mononuclear cells and primary macrophages from two other wild-derived mouse strains also induced IRAK1BP1 mRNA by 4 hours after stimulation with agonists of various TLRs. Examination of its effects on IL-6 and other cytokines demonstrated that IRAK1BP1 regulates transcription of a specific subset of TLR-responsive genes, producing an overall antiinflammatory profile. Our results reveal that IRAK1BP1 is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. Furthermore, these results show that the genetic diversity of wild-derived mouse strains makes them a valuable model of important human gene functions that have been lost in some laboratory-inbred strains.

The vertebrate immune system has evolved to recognize nucleic acids of bacterial and viral origin. Microbial DNA, as well as synthetic oligonucleotides based on these motifs, activates innate immune pathways mediated by the family of Toll-like receptors (TLR) initiating a cascade of signals in immune cells necessary for responses to pathogens. However, not all of the proteins that participate in TLR-mediated responses have been identified. In studies described herein, we observed significant variation in innate immune responses among selected wild-derived strains of mice. Specifically, we show that mice of MOLF/Ei, Czech/Ei, and MSM/Ms strains are hypo-responsive to polyinosinic-polycytidylic acid (poly(I:C)) because of a mutation in Tlr3. In addition, we discovered a hypo-response to cytosine guanine dinucleotide in MOLF/Ei mice and established that it is not linked to Tlr9, but to another locus. Further inquiry revealed that this hypo-response is transmitted as a monogenic dominant trait that can be mapped and cloned through positional cloning methods. These results suggest the existence of a novel molecule that can alter pro-inflammatory signals or activate additional signal transduction pathways. In addition, they support the wild-derived mouse strain as a forward genetic tool for the identification of novel immunological phenotypes.

With the stimulator of IFN genes (STING) C terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. In this article, we identify novel mutations in NTD of Sting of the MOLF strain in response to HSV and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFN-β caused by failure of MOLF STING to translocate from the endoplasmic reticulum. These data provide evidence that the NTD of STING affects DNA responses via control of trafficking. They also show that the genetic diversity of wild-derived mice resembles the diversity observed in humans. Several human alleles of STING confer attenuated IFN-I production similar to what we observe with the MOLF Sting allele, a crucial functional difference not apparent in classical inbred mice. Thus, understanding the functional significance of polymorphisms in MOLF STING can provide basic mechanistic insights relevant to humans.

Dendritic cells (DCs) are a heterogeneous group of mononuclear phagocytes with versatile roles in immunity. They are classified predominantly based on phenotypic and functional properties, namely their stellate morphology, expression of the integrin CD11c, and major histocompatibility class II molecules, as well as their superior capacity to migrate to secondary lymphoid organs and stimulate naïve T cells. However, these attributes are not exclusive to DCs and often change within inflammatory or infectious environments. This led to debates over cell identification and questioned even the mere existence of DCs as distinct leukocyte lineage. Here, we review experimental approaches taken to fate map DCs and discuss how these have shaped our understanding of DC ontogeny and lineage affiliation. Considering the ontogenetic properties of DCs will help to overcome the inherent shortcomings of purely phenotypic- and function-based approaches to cell definition and will yield a more robust way of DC classification.

A mathematical model that describes the effects of acute radiation exposure on thrombopoiesis in primates and humans is presented. Thrombopoiesis is a complex multistage dynamic process with potential differences between species. Due to known differences in cellular radiosensitivities, nadir times, and cytopenia durations, direct extrapolation from rhesus to human platelet dynamics is unrealistic. Developing mathematical models of thrombopoiesis for both humans and primates allows for the comparison of the system's response across species. Thus, data obtained in primate experiments can be extrapolated to predictions in humans. Parameter values for rhesus macaques and humans were obtained either from direct experimental measurements or through optimization procedures using dynamic data on platelet counts following radiation exposure. Model simulations accurately predict trends observed in platelet dynamics: at low radiation doses platelet counts decline after a time lag, and nadir depth is dose dependent. The models were validated using data that was not used during the parameterization process. In particular, additional experimental data was used for rhesus, and accident and platelet donor data was used for humans. The model aims to simulate the average response in rhesus and humans following irradiation. Variation in platelet dynamics due to individual variability can be modeled using Monte Carlo simulations in which parameter values are sampled from distributions. This model provides insight into the time course of the physiological effects of radiation exposure, information which could be valuable for disaster planning and survivability analysis and help in drug development of radiation medical countermeasures.

Despite significant progress in understanding the origin of soluble CD14 (sCD14), its physiological function remains largely unknown. Recent research has produced contradictory observations suggesting that sCD14 may have either beneficial or detrimental properties in protection against LPS-induced endotoxin shock. To resolve this controversy and to establish a mouse model suitable for elucidation of the functions of human CD14 (hCD14) in vivo, we generated several lines of transgenic mice bearing different copy numbers of the hCd14 transgene on a murine Cd14-/- background. The hCD14 was entirely capable of complementing loss of mouse CD14 to mediate cellular responses to LPS. Serum levels of sCD14 in a founder with multiple copies of the transgene were several times higher than in transgenic animals with a single copy of Cd14. Furthermore, mice with high levels of hCD14 were hypo-responsive to LPS and survived a lethal dose of LPS. Further inquiry into the mechanism of the hypo-response to LPS revealed that protection is associated with the higher amounts of circulating LPS. Most of this circulating LPS can be immunoprecipitated with anti-CD14 antibodies. These results suggest that sCD14 blocks circulating LPS by limiting the amount of monocyte-bound LPS and thus reduces inflammatory responses.

Toll-like receptors (TLR) and IL-12 represent key elements of innate immunity. Using C57BL/10 ScCr mice it was shown that TLR4 is important for control of infection with respiratory syncytial virus (RSV). Since these mice have an additional defect in the IL-12R, we reinvestigated immunity to RSV in several C57BL/10 and BALB/c mouse strains lacking a functional TLR4, a functional IL-12-IL-12R interaction or both. In the absence of a functional IL-12 axis, early virus control was impaired in C57BL/10 mice, but not in BALB/c mice. By contrast, TLR4 had no impact on RSV elimination. Pulmonary NK cell recruitment was impaired in IL-12 deficient BALB/c mice and NK cytotoxicity was reduced in IL-12/IL-12R-deficient mice of both genetic backgrounds. Absence of TLR4 had no impact on NK cell recruitment or NK activity nor on recruitment of other pulmonary inflammatory cells. Activation of RSV-specific T cell immunity, including T cell mediated immunopathology, was normal in all mutant strains. These findings clearly argue against a significant role for TLR4 and define a limited role for IL-12 in primary murine RSV infection.

C57BL/10ScCr (Cr) mice carry a deletion of the Toll-like receptor 4 (tlr4) gene (i.e. they are tlr4(0/0)) and are thus refractory to LPS effects. Insertion of wild-type tlr4 transgene into the tlr4(0/0) Cr germ line endowed LPS susceptibility in the two transgenic lines created, indicating that TLR4 is the only limiting factor for LPS responsiveness in Cr mice. The absolute levels of tlr4 mRNA expressed by the heterozygous transgenic (tlr4(Tr/0)), wild-type C57BL/10ScSn (Sn) (tlr4(+/+)) and heterozygous F1 (Sn x Cr) (tlr4(+/0)) mice varied markedly. However, the pattern of distribution of expression in the different organs was the same in all strains. In different biological assays (B cell mitogenicity, cytokine induction and lethal toxicity) the degree of LPS response obtained in the different strains of mice correlated with the levels of tlr4 mRNA expression. In macrophages, investigation of the LPS-induced cytokine (IL-6) response revealed a linear relationship between the response and the logarithm of TLR4-MD-2 levels.

The cytoplasmic protein Stimulator of Interferon Genes (STING) plays an essential role in sensing cytoplasmic DNA of intracellular pathogens and initiating IFN type I responses in myeloid cells. It can also directly bind cyclic dinucleotides produced by intracellular bacteria or by the DNA-binding cyclic GMP-AMP (cGAMP) synthase. To date, the signaling mechanisms involving STING activation has been studied in macrophages and dendritic cells. Taking into consideration the fact that STING is highly expressed in the thymus and spleen, we decided to investigate its functional role in T cells. We revealed that STING can be activated in T cells by DMXAA (5,6-dimethylxanthenone-4-acetic acid), which binds to the same site in murine STING as cGAMP and triggers phosphorylation of TBK1 and IRF3 resulting in IFNβ production and increased expression of interferon stimulated genes. In contrast to T lymphocytes obtained from C57B6 WT mice, STING−/− T cells displayed no phosphorylation of TBK1/IRF3, and produced no detectable IFNβ upon stimulation. DMXAA treatment also resulted in IFNgamma secretion by WT cells, while DMXAA-treated knockouts produced only a small amount of IFNgamma comparable to unstimulated WT T cells. Both CD4+ and CD8+ subsets of WT T cells responded to DMXAA with phosphorylation of TBK1/IRF3 and IFNβ production, but only CD4+ cells produced IFNgamma. By performing RNA sequencing, numerous differences in gene expression (including interferon stimulated genes) between WT and STING−/− T cells before and after DMXAA stimulation were identified. The work was supported by the Russian Science Foundation (grant no. 115-15-00100 ).

Pathogen recognition by the innate immune system initiates the production of proinflammatory cytokines but can also lead to programmed host cell death. Necroptosis, a caspase-independent cell death pathway, can contribute to the host defense against pathogens or cause damage to host tissues. Receptor-interacting protein (RIP1) is a serine/threonine kinase that integrates inflammatory and necroptotic responses. To investigate the mechanisms of RIP1-mediated activation of immune cells, we established a genetic screen on the basis of RIP1-mediated necroptosis in wild-derived MOLF/EiJ mice, which diverged from classical laboratory mice over a million years ago. When compared with C57BL/6, MOLF/EiJ macrophages were resistant to RIP1-mediated necroptosis induced by Toll-like receptors. Using a forward genetic approach in a backcross panel of mice, we identified cylindromatosis (CYLD), a deubiquitinase known to act directly on RIP1 and promote necroptosis in TNF receptor signaling, as the gene conferring the trait. We demonstrate that CYLD is required for Toll-like receptor-induced necroptosis and describe a novel mechanism by which CYLD is down-regulated at the transcriptional level in MOLF/EiJ macrophages to confer protection from necroptosis.

Background Recombinant human interleukin 12 (rHuIL-12) regulates hematopoiesis and cell-mediated immunity. Based on these hematopoietic and immunomodulatory activities, a recombinant human IL-12 (rHuIL-12) is now under development to address the unmet need for a medical countermeasure against the hematopoietic syndrome of the acute radiation syndrome (HSARS) that occurs in individuals exposed to lethal radiation, and also to serve as adjuvant therapy that could provide dual hematopoietic and immunotherapeutic benefits in patients with cancer receiving chemotherapy. We sought to demonstrate in healthy subjects the safety of rHuIL-12 at single, low doses that are appropriate for use as a medical countermeasure for humans exposed to lethal radiation and as an immunomodulatory anti-cancer agent. Methods Two placebo-controlled, double-blinded studies assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of rHuIL-12. The first-in-human (FIH) dose-escalation study randomized subjects to single subcutaneous injections of placebo or rHuIL-12 at 2, 5, 10, and 20 μg doses. Due to toxicity, dose was reduced to 15 μg and then to 12 μg. The phase 1b expansion study randomized subjects to the highest safe and well tolerated dose of 12 μg. Results Thirty-two subjects were enrolled in the FIH study: 4 active and 2 placebo at rHuIL-12 doses of 2, 5, 10, 12, and 15 μg; 1 active and 1 placebo at 20 μg. Sixty subjects were enrolled in the expansion study: 48 active and 12 placebo at 12 μg dose of rHuIL-12. In both studies, the most common adverse events (AEs) related to rHuIL-12 were headache, dizziness, and chills. No immunogenicity was observed. Elimination of rHuIL-12 was biphasic, suggesting significant distribution into extravascular spaces. rHuIL-12 triggered transient changes in neutrophils, platelets, reticulocytes, lymphocytes, natural killer cells, and CD34+ hematopoietic progenitor cells, and induced increases in interferon-γ and C-X-C motif chemokine 10. Conclusion A single low dose of rHuIl-12 administered subcutaneously can elicit hematological and immune-mediated effects without undue toxicity. The safety and the potent multilineage hematopoietic/immunologic effects triggered by low-dose rHuIL-12 support the development of rHuIL-12 both as a radiation medical countermeasure and as adjuvant immunotherapy for cancer. Trial registration ClinicalTrials.gov: NCT01742221

For the sake of coherence, this account is written chiefly in the person of the first author, though it represents the joint recollection of both authors, who were party to the cloning of Lps. Numerous dates and events were reconstructed based upon meticulous records kept by Betsy Layton, Administrative Assistant to the first author, who also participated in the cloning effort. Scientific citations are indicated in superscript and are to be found under References; comments are indicated as { n} and are to be found under Notes (section before References).

Langerhans cells (LC) are skin-specific members of the dendritic cell (DC) family. DC are unique among APC for their capacity to activate immunologically naive T cells, but little is known about their chemotactic recruitment of T cells. We now report that LC produce macrophage inflammatory protein-1 gamma (MIP-1 gamma), a newly identified CC chemokine. MIP-1 gamma mRNA was detected in epidermal cells freshly procured from BALB/c mice, and depletion of I-A+ epidermal cells (i.e., LC) abrogated that expression. MIP-1 gamma mRNA was detected in the XS52 LC-like DC line as well as by 4F7+ splenic DC and granulocyte-macrophage CSF-propagated bone marrow DC. XS52 DC culture supernatants contained 9 and 10.5 kDa immunoreactivities with anti-MIP-1 gamma Abs. We observed in Boyden chamber assays that 1) XS52 DC supernatant (added to the lower chambers) induced significant migration by splenic T cells; 2) this migration was blocked by the addition of anti-MIP-1 gamma in the lower chambers or by rMIP-1 gamma in the upper chambers; and 3) comparable migration occurred in both CD4+ and CD8+ T cells and in both activated and nonactivated T cells. We conclude that mouse DC (including LC) have the capacity to elaborate the novel CC chemokine MIP-1 gamma, suggesting the active participation of DC in recruiting T cells before activation.

304 Tonicity signals drive and anti-inflammatory Th17 cell properties J Matthias, R Noster, M Poltorak, D Soll and C Zielinski 1 Technical University of Munich, Munich, Germany, 2 Technical University of Munich, Munich, Germany and 3 Charite, Berlin, Germany Sodium chloride has recently been demonstrated to strongly enhance the generation of Th17 cells in both mouse and man, which provided mechanistic evidence for the epidemiological correlation of enhanced dietary salt intake and the increasing incidence of autoimmune diseases. Our data support the Th17 cell promoting effect of sodium chloride on human Th17 cell priming. However, Th17 cells acquired anti-inflammatory properties upon stimulation in sodium chloride enriched micromilieus. In addition, sodium chloride also strongly suppressed cytotoxic functions of human T cells. Together, our data suggest that despite the reported association of salt with induction of pathogenic Th17 cells, sodium chloride instead is a potent inducer of the anti-inflammatory Th17 cell subset and also paralyzes cytotoxic T cell properties. Therefore, our data suggest that reduction of sodium chloride levels by either dietary restriction or specific targeting of downstream signaling events could counteract T cell immunoparalysis and exhaustion in settings of chronic inflammatory skin diseases or malignancies. 305 Heat shock protein 60 & 70 expression in psoriasis vulgaris before and after NB-UVB therapy A Kotb Dermatology Research, National Research Center, Cairo, Egypt Psoriasis is a common chronic, relapsing, non-infectious inflammatory skin disease. Heat shock proteins (Hsps) play an important role in essential functions in cells under physiological conditions and play a major role in protecting cells against damage in stressful conditions & they appeared in some studies to play an important role in pathogenesis of many inflammatory skin conditions including psoriasis, atopic dermatitis and systemic lupus erythrematoses. The aim was to detect the expression of HSP60 & HSP70 in psoriasis and its correlation to the disease severity and to review potential role of HSP60 & HSP70 in pathogenesis and therapy of psoriasis. Patients with Psoriasis Vulgaris were treated with NB-UVB & the skin biopsies were taken before & after therapy. The immunoreactivity of HSP60 & HSP70 was detected using IRIDI score. It was confirmed that HSP60 & HSP70 was expressed in psoriatic lesions and their expression is increased with the disease severity but not related to either age or sex & it decreased markedly after exposure to NB-UVB twice weekly for 12 weeks. In conclusion, HSP60 & HSP70 play a role in the pathogenesis of psoriasis & both can be used as a target for therapy.

Although the pathogenesis of schizophrenia is unknown, there are data which indicate that the disease may be due to neurodevelopmental disturbances. Cell recognition molecules such as N-CAM and L1 antigen are involved in cell-cell interactions during development and in plasticity of the nervous system and could therefore be altered in relation to ongoing or established pathological processes. Using the Western blot technique, we found significant increases in N-CAM immunoreactive proteins and decreases in L1 antigen in the CSF of schizophrenic patients as compared to normal controls. The decrease in L1 antigen was observed in the 140-kDa band, and N-CAM was increased only in the 120-kDa band. The 120-kDa band of N-CAM and the 140-kDa band of L1 antigen were prominent components of CSF, but in serum these bands were minor or not detectable. Neuroleptic treatment did not significantly change either N-CAM or L1 antigen concentrations in CSF. It is possible that these CSF proteins are derived from CNS cells as secreted soluble N-CAM isoforms and L1 peptides. Our results suggest the possibility of a specific pattern of abnormal cellular function in the CNS in schizophrenia.

Imipramine was administered chronically to LEW/N, outbred and F344/N rats which were then exposed to the aseptic irritant carrageenin in order to determine whether the decreased hypothalamic expression of CRH m-RNA previously shown to be associated with imipramine affects peripheral immune processes. Both LEW/N and outbred but not F344/N rats had vigorous inflammatory responses to carrageenin, and imipramine was associated with significant decreases in the local cellular inflammatory response to carrageenin. Imipramine was also associated with changes in the expression of peripheral blood cell MHC class II expression in LEW/N and outbred rats. These results suggest that at doses comparable to those used clinically imipramine has significant effects on response to an inflammatory stimulus.

The L -tryptophan eosinophilia myalgia syndrome ( L -Trp-EMS), related to ingestion of impure L -Trp, occurred in epidemic proportions in the Unite

Immunological reactions after intracerebroventricular syn-, allo- and xenogenic transplantation of adrenal medulla were investigated histologically. In xenografts only, T cell infiltration and graft rejection were observed. Syngrafts and allografts were not rejected and were not infiltrated by T cells, although expression of MHC class II antigen was observed at all survival times. Major histocompatibility complex (MHC) class I immunoreactivity was strongly expressed in adrenal cortex syngrafts, which could play a role in the rejection of grafts containing mixed cell populations. The survival of chromaffin cells in allografts was decreased as compared to syngrafts, and there were fewer allograft animals with large numbers of surviving chromaffin cells. There was some increased cellularity (microglia and macrophages) in allografts even though no T cell infiltration was found. Therefore, it appears that this limited survival of intracerebral adrenal medulla allografts is not due to T cell-mediated graft rejection.

We previously reported that a chimeric protein consisting of the human p55 TNF receptor covalently linked to a murine IgG1 Fc heavy chain acts as an efficient TNF inhibitor, as a result of its high binding affinity for native TNF trimers of both murine and human origin. A transgenic mouse line constitutively expressing the inhibitor from a cytomegalovirus promoter was established. All organs examined expressed the transgene. TNF inhibitory activity was easily detected in plasma of transgenic animals but not in plasma of nontransgenic littermates. This founder line was not found to have any obvious phenotype. Specifically, transgenic mice born to wild-type or transgenic females were indistinguishable from nontransgenic littermates with respect to their size at birth, at three months and at six months of age, with respect to the size and morphology of their lymphoid organs and with respect to their hematocrit, white blood cell count, and differential. Although TNF is known to be constitutively expressed by cells of the thymus and trophoblast, the lack of a clear phenotype in association with the constitutive expression of a TNF inhibitor suggests that TNF may be dispensable in early development.

The A7 cell line is an astrocyte-like cell immortalized by SV40 large T antigen, using retroviral-mediated gene transfer. These cells were transplanted into rat brains, and the graft-host interaction was investigated immunohistochemically. The A7 cells survived focally 2, 6 and 8 weeks after transplantation and retained the immunocytochemical properties observed in vitro. No immunological response was observed. GAP-43 and N-cadherin immunoreactivities were not expressed by A7 cells, but were seen in the matrix within the area of the graft and in the surrounding brain tissue. This indicates that A7 cells may stimulate expression of GAP-43 and N-cadherin immunoreactivity by host tissue. Expression of Thy 1.1 was not observed within the graft site after 2 weeks of survival, but 6 and 8 weeks after transplantation Thy 1.1 was observed within the graft area, indicating the possible co-existence of grafted cells and host tissue. Although indirect, these observations suggest that the A7 cells induce changes in host brain, including possible growth or regeneration of host tissue into the graft area.

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentiation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.