However, the picture emerging now is one check details of multiple IL-23-responsive cell types, pro-inflammatory cytokine induction, and pathogenic “licensing” following an IL-23-dominated interaction between the T cell and the antigen-presenting cell (APC). This review will focus on our changing view of IL-23-dependent autoimmune pathologies with a particular emphasis on the responder cells and their IL-23-induced factors that ultimately mediate tissue destruction. Regardless of the underlying mechanism by which autoimmunity is initiated, the inevitable outcome is a chronic immune response against self-antigen

accompanied by the accumulation of inflammatory mediators. Extensive pathology in the affected organs is characteristic of late-stage autoimmunity and this devastating process is often well underway when a disease is diagnosed. It is this stage of autoimmunity that is most relevant when considering therapeutic intervention, as patients are rarely aware, prior to health complaints, that

an autoimmune manifestation will ultimately take place. We are now in possession of substantial evidence selleckchem that implicates pro-inflammatory cytokines in a wide range of auto-immune pathologies. The early success of anti-TNF-α therapy in rheumatoid arthritis galvanized the notion that a number of other autoimmune diseases, in which similar mechanisms may operate, could also be treated by blockade of the cytokines thought to be responsible for pathogenesis [1]. MycoClean Mycoplasma Removal Kit These pro-inflammatory cytokines are produced by CD4+ T helper cells, which orchestrate immune responses by sending out secreted signals to other immune cells and stromal cells. Not only the cytokines expressed, but the mechanisms controlling the generation of the cytokine-secreting cells themselves have been heavily scrutinized, with the long-term goal being to treat autoimmune disease by neutralizing the effector cytokines

secreted by autoaggressive T cells. We now know that the differentiation of effector T cells is in itself dependent on cytokines present at the time of their activation. The subsequent polarization, which takes place when T-cell receptor, costimulatory, and cytokine signals combine (reviewed in [2]), can result in a broad range of biological functions within the activated T cells. When we consider the sheer number of cytokine combinations theoretically available to a T cell, it is perhaps surprising that so few cellular “phenotypes” have been characterized. Immunologists appear to be keen on categorizing different subsets of T cells, with a rather rigid attribution of biological function being applied to each subset. One could argue that this trend began some 40 years ago, when T cells were subdivided into CD4+ helpers and CD8+ cytotoxic killers.

In order to select for TCRL Abs, we generated biotinylated versio

In order to select for TCRL Abs, we generated biotinylated versions of HLA-DR2-derived RTLs, RTL1000 (DR2–MOG-35-55) and RTL340 (DR2–MBP-85-99). These constructs were produced by in vitro refolding of purified inclusion bodies and were found to be very pure, homogenous and monomeric by SDS-PAGE and size exclusion

chromatography analyses (Fig. 1A). HLA-DR2 (DRA1*0101 and DRB1*1501) contains a disulfide bond between conserved cysteines in the β1 domain (residues 15 and 79 of the DR-B chain) 32. The formation of this native conserved disulfide bond within the RTL molecule was verified by gel-shift assay (Fig. 1B). SDS-PAGE analyses of reduced and non-reduced RTL1000 samples revealed that the non-reduced sample had a smaller apparent

molecular weight, buy LEE011 indicating the presence of an internal disulfide bond leading to a more compact structure. High biotinylation levels are essential for a successful screening of the desired Abs using our phage display screening strategy. The RTL constructs were found to have high biotinylation levels, identical to the compared 100% biotinylated MBP standard (Fig. 1C). In previous reports, RTLs were found to deliver peptide-specific rudimentary signals through the TCR of human Th1 cells 19 and a murine T-cell hybridoma 20. We verified the interaction of biotinylated RTL1000 with the cognate TCR of the H2-1 T-cell hybridoma specific for the DR2–MOG-35-55 complex. As shown in Fig. 1D, MOG-35-55-specific activation of

the H2-1 hybridoma was inhibited by pre-incubation of H2-1 with RTL1000. Control RTL340 (DR2–MBP-85-99) did not inhibit this antigen-specific response, indicating selective RTL1000 ligation of the TCR leading to inhibitory signaling. We conclude that the RTL1000 construct mimics the minimal MHC-II domains necessary for specific interaction with the TCR and therefore it was used as a soluble recombinant protein for the selection of Abs directed to the α1β1 DR2–MOG-35-55 T-cell epitope in a TCRL fashion. For selection of TCRL Abs directed to MHC-II, we used a strategy of screening a large Ab phage library consisting of a repertoire of 3.7×1010 human recombinant Fab fragments 33. see more RTL1000 was used as a minimal DR2–MOG-35-55 complex recognized by autoreactive T cells. We applied the library to panning on soluble RTL1000. Seven hundred-fold enrichment in phage titer was observed following four rounds of panning. The specificity of the selected phage Abs was determined by ELISA comparison of streptavidin-coated wells incubated with biotinylated RTL1000 (DR2–MOG-35-55) or RTL340 (DR2–MBP-85-99) (Fig. 2A). Fab clones with peptide-dependent, MHC-restricted binding were picked for further characterization.

Interleukin-21 is secreted by activated T cells, including the Th

Interleukin-21 is secreted by activated T cells, including the Th1, Th2 and Th17 cell subsets.24 However, relative to the Th1 and Th2 subset, Th17 cells secrete significantly higher amounts of IL-21.24 IL-21 AZD2281 plays an important role as an autocrine signal for the differentiation of Th17 cells and the absence of the IL-21 receptor leads to a reduction in activated Th17 cells.25 IL-21 plays pleiotropic effects within the immune system where it mediates autoantibody production on B cells, generates mature cytotoxic natural killer cells, and enhances

CD8+ T cell activity.43 Interleukin-22 is secreted by Th17 cells in response to IL-23.27 The receptor for IL-22 is expressed on epithelial and endothelial cells but not on immune cells.44 It is believed that Th17 cells use IL-22 to mediate local R788 clinical trial tissue inflammation as seen in mouse models of psoriasis45 or possibly facilitate the influx of Th17 cells as IL-22 helps disrupt the blood-brain barrier and promotes Th17 cell infiltration into the central nervous system.46 IL-22 also plays protective roles in acute liver inflammation47 and can induce lipopolysaccharide-binding protein from hepatocytes.48 Secretion of IL-9 has been reported by Th2,49 Treg cells50 and more recently by Th17 cells.28 IL-9 plays protective effects against nematode infections when secreted by Th2 cells,49 suppresses EAE when secreted by Treg cells,50 and mediates EAE when

secreted by Th17 cells.28 Differentiated Th17 cells express the IL-9 receptor

and IL-9 may act as an autocrine signal amplifying Th17-mediated disease, as the transfer of IL-9R-deficient T cells into wild-type mice delayed the onset of EAE.28 IL-9, however, also enhances the suppressive effects of Treg cells and IL-9R deficient mice develop more severe EAE.51 The polarization of naive CD4+ Th cells into Treg cells and Th17 requires TGF-β. Unopposed TGF-β stimulation in the context of antigen presentation induces Foxp3 expression and Treg commitment and immunoregulation. However, in the context of inflammation signalled by the presence of IL-6, TGF-β drives Th17 differentiation and inflammation.52 Furthermore, IL-6 inhibits the generation of Foxp3 and the differentiation of Tregs. It also facilitates Th17 effector cells by reducing the functional capacity of Tregs.53 These observations suggest a reciprocal relationship Cell press between Tregs and Th17 differentiation depending on the presence of inflammatory danger signal IL-6.54 This reciprocity of activation/deactivation of inflammation may explain the prominence of Th17 pathway in the development of autoimmunity. The reciprocity between Th17 and Treg cell development is seen at multiple levels of CD4+ activation. At the level of T subset pathway regulators, RORγt (the critical Th17 pathway inducing transcription factor) and Foxp3 (critical transcription factor for Treg cells) have been shown to interact physically and inhibit one another.


it is not clear how the loss of TDP-43 results i


it is not clear how the loss of TDP-43 results in cell dysfunction or cell loss. TDP-43 was first identified as a protein that binds to DNA, and it is now considered to regulate RNA metabolism.[17] Using a method that identifies the mRNA binding to a specific protein, Hydroxychloroquine in vitro many RNAs that might be regulated by TDP-43 have been identified.[18, 19] These studies have shown that TDP-43 binds to long mRNA molecules with large introns and regulates the splicing and amounts of mRNA in several ways.[18, 19] Consequently, the depletion of TDP-43 might alter pre-mRNA metabolism. Indeed, the alteration of RNA profiles has been reported from cultured cells and model animals with depleted TDP-43. In ALS, alterations of mRNA expression profiles have been reported,[20-22] although the association between TDP-43 and these alterations of mRNA observed in ALS remain to be clarified. To our knowledge, POLDIP3 is the only gene in which the splicing is directly regulated by TDP-43 and is altered in spinal motor

neurons with ALS but not in brain with frontotemporal lobar degeneration.[23, 24] In addition, immunohisotochemical analysis indicated that several genes selleck inhibitor processed by TDP-43 express key molecules for function or survival of spinal motor neurons and show decreasing amounts of products.[25] However, it is unclear how the function of TDP-43 correlates with the depletion of these products. Thus, the specific functions of TDP-43 have not been fully evaluated in vitro or in ALS patients. These disturbances of RNA metabolism might not be explained simply by the

loss of TDP-43 function on pre-mRNA. Therefore, some researchers have speculated that TDP-43 serves another function associated with RNA metabolism.[26] TDP-43 forms foci in the nucleus and associates with several nuclear bodies, suggesting that TDP-43 plays a role in Cediranib (AZD2171) the functioning of nuclear bodies. Nuclear bodies are classified and identified by their unique protein components.[27] In addition, most of these bodies are tightly associated with a unique RNA and regulate that particular RNA metabolism.[28, 29] In contrast to cytoplasmic organelles, nuclear bodies do not have a membranous structure that separates their contents from nucleoplasm. Thus, the components of nuclear bodies are frequently exchanged between the bodies and the nucleoplasm. The dynamism of the components is a unique characteristic of nuclear bodies. The protein components decrease their mobility in nuclear bodies as compared to that in nucleoplasm. Thus, the bodies are recognized based on the increased concentration of the component protein. The nucleolus and Cajal bodies are the most well-known nuclear bodies. The nucleolus is the center for maturation of rRNA, whereas Cajal bodies are sites for the maturation of U snRNAs and consist of coilin.

The development of various techniques and microRNA reagents has e

The development of various techniques and microRNA reagents has enabled work to progress very rapidly in this area. In the present article the authors describe the methods they have used that have enabled them to contribute to our current understanding of the role of microRNAs in diabetic nephropathy. ”
“This is an update of a previous CARI Guideline on management of anaemia in CKD patients. ”
“Idiopathic membranous nephropathy (IMN) is the most common cause of nephrotic syndrome in adults. The term idiopathic or primary as opposed to secondary, is used when no cause can be deduced from the medical history, physical examination, or laboratory tests commonly performed to assess a

patient with proteinuria. The M-type phospholipase A2 receptor (PLA2R) was identified as an important selleck chemicals llc antigenic target

in the pathogenesis of IMN and the presence of circulating PLA2R antibodies was closely association with disease activity in patients with IMN.[1] It is becoming increasingly clear and more widely accepted that IMN is an organ-specific autoimmune disease involving the kidneys. Prognosis in patients with IMN and nephrotic syndrome is more variable. Around 30% of patients develop spontaneous GSK3235025 in vitro remission 1–2 years after diagnosis.[2] However, 30–40% of patients progress toward end-stage renal disease (ESRD) within 5–15 years.[3] Immunosuppressant therapy has been reported to induce disease remission and reduce the risk of progression to ESRD or death.[4] Alkylating agents and corticosteroids have been shown to be effective in nephrotic IMN patients in many trials, and these agents should be considered the gold standard of therapy. Despite the favourable results with alkylating agents, there is a reluctance to prescribe them due to the short-term and potential long-term adverse effects. Short-term effects include myelosuppression and the risk of infertility, which is a concern for patients of childbearing age. The

risk of cancer remains a long-term Liothyronine Sodium concern. Leflunomide (LEF) is an immunomodulatory drug that inhibits mitochondrial enzyme dihydroorotate dehydrogenase (an enzyme involved in de novo pyrimidine synthesis). In addition, it plays a key role in the de novo synthesis of pyrimidine ribonucleotide uridine monophosphate, and it has been reported to have antiproliferative and anti-inflammatory actions. This double action is thought to slow the progression of autoimmune diseases and approved for use in rheumatoid arthritis. The introduction of new immunosuppressive agents and biologicals has provided hope for effective and safer treatment of patients with IMN. However, the efficacy and safety of LEF for patients with IMN with nephrotic syndrome is still controversial. The natural history of IMN is quite variable, and many studies have reported a relatively good outcome in untreated patients.

Strains were evaluated in vitro and in mice. Twenty-two healthy v

Strains were evaluated in vitro and in mice. Twenty-two healthy volunteers received single oral doses of either strain in a physiological study of safety, shedding, and immunogenicity. Volunteers were observed in the hospital for seven days and had daily blood cultures, routine safety blood tests (complete blood count with differential; hepatic and renal function), and fecal cultures; none had fever, positive blood cultures, prolonged shedding, or serious or unexpected events. Four of 12 volunteers who received the actA/plcB-deleted strain had minor, transient, asymptomatic serum transaminase elevations (maximum increase 1.4×

upper normal). Six of six volunteers who received ≥4 × 109 colony forming units had detectable mucosal immune responses to listerial antigens, PF-02341066 in vivo but not to the vectored influenza antigen. Approximately half the volunteers had modest interferon-γ ELISpot responses to a complex listerial antigen, but none had increases over their baseline responses to the influenza antigen. Comparison with prior work suggests that foreign antigen expression, and perhaps also freezing, may adversely affect the organisms’ immunogenicity. Live attenuated bacteria expressing foreign antigens present a promising approach in the development of human immunotherapies and vaccines. Favorable attributes of

Listeria monocytogenes include its innate immunostimulatory EX-527 properties, efficient cytosolic entry of antigen presenting cells, and its ability to stimulate vigorous CD4 and CD8 T cell responses. L. monocytogenes antigen processing and presentation via

the major histocompatability complex class I pathway makes it an attractive vector for delivery of viral and cancer antigens. Animal studies show that L. monocytogenes vectors can generate T cell-mediated immune responses to lymphocytic choriomeningitis virus, influenza virus, HIV-1 Gag, and human papilloma virus-16 antigens (1–5). Despite these promising results, there are safety concerns related to using recombinant wild type new L. monocytogenes as a vaccine vector due to the high degree of morbidity and mortality that results from naturally acquired infection. Several groups have developed strategies for attenuating L. monocytogenes by deletion of specific virulence genes (1, 6–8) and two strains have been evaluated in published human studies: ΔactA/plcB (9) and ΔprfA (10) via oral and parenteral routes, respectively. In our prior oral clinical study, 20 healthy adult volunteers received escalating single doses of live attenuated L. monocytogenesΔactA/plcB safely. No individual experienced a serious adverse event. Three of 20 individuals had mild elevations in serum transaminases (maximum 2.5× upper limit of normal) that were temporally associated with vaccination and could not be otherwise explained. Subsequently, another biotechnology group developed an L.

Another study showed that prestimulation of ITAM-coupled receptor

Another study showed that prestimulation of ITAM-coupled receptors and integrins can inhibit TLR responses indirectly through induction of inhibitors such as IL-10, STAT3, SOCS3, ABIN-3, and A20 69. The inhibitory capacity of receptors previously believed to only activate cells, emphasizes the complex signaling networks and cross-talk

in signal transduction pathways, and will contribute to a tightly balanced immune response. Coevolution of interacting species drives molecular evolution through continual natural selection for adaptation and counter adaptation. Hence, pathogens coevolving with humans have developed multiple mechanisms to evade immune recognition. Selleckchem Sirolimus A pathogen that encodes a functional Bioactive Compound Library datasheet ligand for a phagocyte inhibitory receptor could enhance survival

by suppressing effector functions such as phagocytosis, ROS, and cytokine production. It has been shown that Staphylococcus aureus binds specifically to PIR-B, a suppressor of TLR-mediated inflammatory responses, and PIR-B-deficient macrophages display enhanced inflammatory responses to S. aureus90. The specific bacterial protein that binds to PIR-B remains to be determined. Bacterially encoded ligands have also been found for Siglec-5 and Siglec-9 30, 91. The group B Streptococcus cell wall-anchored β protein specifically binds Siglec-5, and it was shown that Siglec-5 activation through mafosfamide β protein results in less phagocytosis, less oxidative burst, fewer neutrophil extracellular traps (NETs 92) and reduced IL-8 production in neutrophils 91. Other examples of bacterially encoded proteins that act as a functional ligand for inhibitory receptors include interaction of surface protein A1 on Moraxella catarrhalis or opacity-associated proteins on Neisseria meningitidis with

CEACAM1 93. Evolutionary selection of pathogens that produce ligands for inhibitory receptors indicates that it can lead to an evolutionary advantage, which in turn underlines the importance of inhibitory receptors as regulators of phagocyte cell function. Considering the number of inhibitory receptors on phagocytes, it is likely that many more bacterially encoded ligands for inhibitory receptors will be discovered. Interestingly, activating family members have been described for many inhibitory receptors and often a cell will express both inhibitory and activating members of the same receptor 94. These so-called paired receptors include Siglecs 95, CD200R 96, PIR 97, SIRP 97, KIR, and Ly49 94. In the light of the discussion above, it is fascinating to speculate that the evolution of these activating counterparts is driven by the continuous battle between pathogens and host. An important study by Abi-Rached and Parham demonstrate that activating KIR members are derived from inhibitory KIRs 98.

We discuss here important pro-inflammatory molecules and leucocyt

We discuss here important pro-inflammatory molecules and leucocyte populations that were identified as key players in the murine model of DENV-2 infection using the mouse-adapted strain P23085. The inflammatory response triggered by this model of DENV infection frequently leads to tissue damage and death. However, it is possible in this model to assess and distinguish mechanisms necessary for the host response

to deal with infection from those that cause unwanted, misplaced and uncontrolled inflammation and drive disease. By understanding where/how host–pathogen interactions lead to disease, we may be able to suggest novel strategies to restrain severe systemic and local inflammatory responses. Chemokines are members of a structurally related family of cytokines involved in leucocyte buy Veliparib traffic during infection and inflammation. They are classified according to the relative position of conserved N-terminal cysteine residues, in which CC

chemokines represent the most abundant family and have the first two cysteines placed adjacently.[72] Chemokine receptors are expressed on the surface of leucocytes and are G protein-coupled receptors containing seven transmembrane domains.[73] Experimental and epidemiological evidence suggests an important role for chemokines, especially those from the CC family, and their receptors in infectious diseases such as HIV and herpes simplex virus 1.[74, 75] The expression of CC chemokines dominates over the expression of CXC chemokines during

viral infections, although this observation does not represent a general rule.[75] Among the CC chemokines, CCL3/MIP-1α and CCL5/regulated upon activation, normal T cell-expressed and secreted (RANTES) are widely associated with viral infections [74, 76] During intranasal influenza virus infection in mice, CCL2/monocyte chemotactic protein-1 (MCP-1) is detected in the lungs at various time-points post-infection, whereas other chemokines, including CCL3 and CCL5, are not expressed.[77] On the other hand, respiratory syncytial virus-infected mice display high levels of expression of numerous Orotic acid chemokines in the lungs, including CCL3 and CCL5.[78] Among flaviviruses, CC chemokine receptors play an important role in leucocyte recruitment to the central nervous system.[79] Besides a deleterious pro-inflammatory role that CC chemokines could play in central nervous system, a well-studied example involves acute infection by West Nile virus in mice, in which the lack of CCR2 and CCR5 leads to decreased leucocyte recruitment, increased viral load in the central nervous system and enhanced mortality. West Nile virus infection induces high and continuous levels of CCL2 and CCL5, which are required for the local accumulation of NK cells, macrophages and T lymphocytes to control infection.

Study subjects were prospectively recruited from visitors to Seou

Study subjects were prospectively recruited from visitors to Seoul National University Bundang Hospital between 2009 and 2012. One hundred and twelve FD patients and 269 controls were enrolled. In SLC6A4 5-HTTLPR polymorphism, the frequency of S/S genotype was significantly

lower than that of L/L + L/S genotype in FD compared to controls (P < 0.05). After stratification according to Helicobacter pylori infection, the S/S genotype was significantly associated with H. pylori-positive epigastric pain syndrome (EPS) patients (adjusted odds ratio (OR) 0.46; 95% confidence interval (CI) 0.22–0.99; P = 0.048). In TRPV1 945G>C polymorphism, the frequency of C/C genotype was lower in FD compared to controls (P = 0.057). The C carrier and C/C genotype was significantly associated with postprandial find more distress

syndrome (PDS) and EPS, respectively (adjusted OR 0.47 and 0.43; 95% CI 0.25–0.90 and 0.20–0.93; P = 0.021 and 0.033). After stratification, the significant associations remained in H. pylori-positive PDS and EPS patients (adjusted OR 0.37 and 0.28; 95% CI 0.16–0.88 and 0.09–0.85; P = 0.024 and 0.025). The genetic polymorphism of SLC6A4 5-HTTLPR and TRPV1 945G>C could be one of the pathophysiological factors of FD, especially in the case of H. pylori-positive patients in Korea. ”
“Polo-like kinase (PLK) proteins play critical roles in the control of cell cycle progression, either favoring or inhibiting cell proliferation, and in DNA damage response. Although either overexpression or down-regulation of PLK proteins occurs frequently in various cancer types, no comprehensive analysis on their function in human hepatocellular carcinoma (HCC) has been performed to date. In the present study, we define roles for PLK1, PLK2, PLK3, and PLK4 during hepatocarcinogenesis. Levels of PLK1, as assessed by means of real-time reverse-transcription PCR and western blot analysis, were progressively increased from nonneoplastic surrounding liver tissues to HCC, reaching the highest

expression in tumors with poorer outcome (as defined by the length of patients’ survival) compared with normal livers. In sharp contrast, PLK2, O-methylated flavonoid PLK3, and PLK4 messenger RNA and protein expression gradually declined from nontumorous liver to HCC, with the lowest levels being detected in HCC with shorter survival. In liver tumors, PLK2-4 down-regulation was paralleled by promoter hypermethylation and/or loss of heterozygosity at the PLK2-4 loci. Subsequent functional studies revealed that PLK1 inhibition led to suppression of cell growth in vitro, whereas opposite effects followed PLK2-4 silencing in HCC cell lines. In particular, suppression of PLK1 resulted in a block in the G2/M phase of the cell cycle and in massive apoptosis of HCC cells in vitro regardless of p53 status.

The potential of further studies of brown algae in these importan

The potential of further studies of brown algae in these important areas has been increasingly hindered by the absence of tools for manipulation of gene expression that would facilitate further mechanistic analysis and gene function studies at a molecular level.

The aim of this study was to establish a method that would allow the analysis of gene function through RNAi-mediated gene knockdown. We show that injection of double-stranded RNA (dsRNA) FK506 corresponding to an α-tubulin gene into Fucus serratus Linnaeus zygotes induces the loss of a large proportion of the microtubule cytoskeleton, leading to growth arrest and disruption of cell division. Injection of dsRNA targeting β-actin led to reduced rhizoid growth, enlarged cells and the failure to develop apical hair cells. The silencing effect on actin expression was maintained for 3 months. These results

indicate that the Fucus embryo possesses a functional RNA interference system that can be exploited to investigate gene function during embryogenesis. ”
“Understanding responses of marine algae to changing ocean temperatures requires knowledge of the impacts of elevated temperatures and the likelihood of adaptation to thermal stress. The potential for rapid evolution of thermal tolerance is dependent see more on the levels of heritable genetic variation in response to thermal stress within a population. Here, we use a quantitative genetic breeding design to establish whether there is a heritable variation in thermal sensitivity in two populations of a habitat-forming intertidal macroalga, Hormosira banksii (Turner) Descaisne. Gametes from multiple Oxalosuccinic acid parents were mixed and growth and photosynthetic performance were measured in the resulting embryos, which were incubated under control and elevated temperature (20°C and 28°C). Embryo growth was reduced at 28°C, but significant interactions between male genotype and temperature in one population indicated the presence of genetic variation

in thermal sensitivity. Selection for more tolerant genotypes thus has the ability to result in the evolution of increased thermal tolerance. Furthermore, genetic correlations between embryos grown in the two temperatures were positive, indicating that those genotypes that performed well in elevated temperature also performed well in control temperature. Chlorophyll a fluorescence measurements showed a marked decrease in maximum quantum yield of photosystem II (PSII) under elevated temperature. There was an increase in the proportion of energy directed to photoinhibition (nonregulated nonphotochemical quenching) and a concomitant decrease in energy used to drive photochemistry and xanthophyll cycling (regulated nonphotochemical quenching). However, PSII performance between genotypes was similar, suggesting that thermal sensitivity is related to processes other than photosynthesis.