Therefore, the molecular mechanisms described above may have been

selected because they achieve Treg cell lineage stability and prevent off-target, innocuous antigen-specific responses during inflammation.[46] In contrast, Th17 cells represent a potent inflammatory Th cell subset endowed with the ability to augment adaptive responses, tissue inflammation, and neutrophil recruitment, and are therefore often juxtaposed with Treg cells as frequent culprits of autoimmune disease.[25] Indeed, studies from both Rudensky and colleagues and Littman and colleagues validated the functional importance selleck products of Treg or Th17 cell regulatory elements through comparison with genome-wide association study data. For example, both sites of Treg-specific chromatin accessibility, and binding sites for the core Th17 cell transcription factors overlapped with different mutations linked to ulcerative colitis and rheumatoid arthritis, diseases in which Th17 cells and Treg cells have opposing roles and where dysregulation of either cell type can result in disease.[12, 14] Intuitively then, when not dysregulated by genetic lesions or environmental toxins, Th17 cell environmental

responsiveness and lineage plasticity can allow for the harnessing of their potent INCB024360 inflammatory potential to fight infection and resolve tissue damage while assuring their appropriate restraint and reprogramming under homeostatic conditions. Similarly, Th1 and Th2 cells have encoded appropriate environmental responsiveness and stability into their transcriptional programmes, enabling the maintenance of type-specific memory responses with some capacity for adaptation. Both TBET and GATA3 reinforce their own expression directly, next through transcriptional positive feedback loops, and indirectly, through enhancement of cytokine

receptor expression and autocrine signals upstream of MRF activation.[47] The TBET target HLX, and perhaps TBET itself can activate TBET gene expression.[23, 48] For both TBET and GATA3, retroviral expression can induce transcription of the endogenous genes.[23, 49] As with FOXP3 autoregulation, these cell intrinsic positive feedback loops confer a degree of environmental buffering and thereby bolster lineage fidelity. Indeed, Th1 or Th2 cells that have undergone several rounds of division, demethylated CpG motifs at key lineage genes, and established transcriptional autoregulatory loops, become highly committed.[50, 51] In contrast, newly differentiated Th1 and Th2 cells are highly responsive to reprogramming following exposure to alternative lineage-instructing cytokines.

CD4+ T cells were identified as CD3+CD8− by surface staining. Intracellular IL-17 (FITC labelled IL-17A,

eBioscience, San Dieago, CA, USA) and IFN-γ (PE-labelled, BD Pharmingen) cytokines were measured using a fixation and permeabilisation kit 15 in a standard ICS assay. Absolute IL-17 numbers were determined as the percentage of cells staining positive for IL-17 secretion multiplied by the absolute CD4+ T-cell count of the patient/control at the time of sampling. FoxP3 expression was determined using anti-human FoxP3 staining set (Clone PCH101, eBioscience). Briefly, Selleck AZD9668 cells were surface stained with FITC-labelled CD4+ (clone SK3 BD Pharmingen) and PE-labelled CD25 (clone MEM-181,

AbD Serotec, Oxford, UK). Cells were then washed and fix/permeabilised and stained using Fix/Permeabilisation Foxp3 staining kit for FoxP3 or the appropriate isotype control antibody 15. Absolute numbers of Treg cells were determined as the percentage of cells staining for defined Treg cell markers multiplied by the absolute CD4+ T-cell count of the patient/control at the time of sampling. Statistical analysis was performed using Graphpad PRISM software (Graphpad Prism, version 4, CA, USA). Unpaired multiple comparison tests were performed using non-parametric Kruskal–Wallis test. Paired analysis was performed using Student’s t test. p-Values of 0.05 and below were considered statistically significant. G.T. was supported by an educational grant from Gilead Pharmaceuticals. selleck inhibitor The authors 3-mercaptopyruvate sulfurtransferase acknowledge financial support from the Department of Health via the National Institute for Health Research

(NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. We thank our patients for their active participation in the study. The author contributions were as follows: Experiments were conceived and designed by G.T., B.P. and A.V. and performed by G.T. Data were analysed by G.T. and A.V. The manuscript was prepared by G.T., A.V. and B.P. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“T regulatory (Treg) cells are critical for maintaining immune homeostasis and establishing tolerance to foreign, non-pathogenic antigens including those found in commensal bacteria and food. Because of their multiple suppressive mechanisms, Tregs represent a promising strategy for engineering tolerance to self and non-self antigens in chronic inflammatory diseases.

Definitive evidence that IFN was escaping the uterus was provided by Oliveira et al.75 who demonstrated a 500–1000-fold increase in antiviral activity in the uterine vein compared to the uterine artery or jugular blood of early pregnant ewes. These results provided strong support for the early evidence showing low, but detectable levels of IFN-τ7 and antiviral activity8 in the blood. Work from this same group later demonstrated that the antiviral activity was indeed caused by release of IFN-τ.76

These important studies were the first to definitively click here demonstrate that IFN-τ had a direct systemic effect, and that this effect could increase CL lifespan. Interestingly, Tuo et al.78 had previously shown

that exogenous IFN-τ had dramatic effects on immune cell recirculation and redistribution in lambs by reducing CD4+, CD5+ and gamma delta + T cells in the peripheral circulation without changing numbers of CD8+ T cells. This effect occurred within 6–12 hr of treatment and peripheral immune cell populations returned to pre-treatment control values by 48 hr. Furthermore, IFN-τ was shown to cause a dose-dependent reduction in lymphocyte proliferation79 and to suppress lymphocyte blastogenesis80in vitro. In contrast, IFN-τ stimulated NK Vemurafenib manufacturer cell activity in sheep PBMC.81 Taken together, these experiments provide evidence that, while ruminants and humans possess different mechanisms for supporting CL function during early pregnancy, there exists the distinct possibility that they may share functions as a result of the fact that they are both present in the peripheral circulation during the very earliest stages of pregnancy recognition signaling and both can apparently bind and alter function of circulating immune cells. Support for this hypothesis is currently limited owing to few of studies examining the effects of either hCG or IFN-τ on circulating immune cell function. However, work carried out in MRIP later pregnancy

in cattle clearly supports similarities between humans and cattle in alterations in peripheral and endometrial immune cell populations.12 For example, in cattle there was an increase in peripheral cells exhibiting the T regulatory phenotype (CD4+ CD25+) as well as recruitment of these cells to the endometrium. T regulatory cells secrete IL-4 and can induce tolerance to paternal alloantigens and inhibition of T regulatory function is associated with compromised pregnancy.12 We recently conducted a transcriptional profiling experiment to identify genes regulated in PBL by pregnancy and progesterone in cattle82 (Ott and Gifford unpublished). Results from these studies clearly indicated that a large number of known interferon-stimulated genes increase in PBL of early pregnant cows. In addition, some genes not previously thought to be IFN responsive were also increased.

“
“Dengue is a mosquito-borne viral disease selleck of humans,

and animal models that recapitulate human immune responses or dengue pathogenesis are needed to understand the pathogenesis of the disease. We recently described an animal model for dengue virus (DENV) infection using humanized NOD-scid IL2rγnull mice (NSG) engrafted with cord blood haematopoietic stem cells. We sought to further improve this model by co-transplantation of human fetal thymus and liver tissues into NSG (BLT-NSG) mice. Enhanced DENV-specific antibody titres were found in the sera of BLT-NSG mice compared with human cord blood haematopoietic stem cell-engrafted NSG mice. Furthermore, B cells generated during the acute phase and in memory from splenocytes of immunized BLT-NSG mice secreted DENV-specific IgM antibodies with neutralizing activity. Human T cells in engrafted BLT-NSG mice secreted

interferon-γ in response to overlapping DENV peptide pools and HLA-A2 restricted peptides. The BLT-NSG mice will allow assessment of human immune responses to DENV vaccines and the effects of previous immunity on subsequent DENV infections. Dengue virus (DENV) is a mosquito-borne member of Deforolimus mouse the Flavivirus genus and includes four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). The virus infects approximately 50 million individuals each year, leading to over 500 000 hospitalizations. Infection results in a range of symptoms from mild fever to acute febrile illness (dengue fever). In a small percentage of cases, however, individuals develop a severe capillary leakage syndrome, dengue haemorrhagic fever and dengue shock syndrome, which can be life-threatening.1,2 Studies in humans suggest that dengue haemorrhagic fever and dengue shock syndrome are more likely to occur in individuals experiencing BCKDHB their second DENV infections and in infants born to DENV-immune mothers. Experimental manipulation of in vivo immune responses to DENV is a critical step in exploration of the role of previous immunity in subsequent DENV infection

and testing of candidate vaccines and therapeutics. Progress in understanding the pathogenesis of dengue haemorrhagic fever has come largely from controlled well-designed clinical studies of patients with mild and severe forms of dengue disease in endemic areas.3–10 Most patients who present to hospital live in endemic areas and are experiencing a secondary infection; however, the serotype of the previous DENV infection is difficult to determine. Furthermore, controlled virus challenge studies are not feasible in humans, and it is difficult to assess the contribution of antibodies or T cells to DENV pathogenesis. Immunodeficient mice bearing components of a human immune system (humanized mice) present a novel approach for studying human immune responses to DENV.

Our patient was successfully treated with 6 months of ganciclovir therapy. Our studies supported the early and prolonged ganciclovir therapy in WAS patients with CMV infection Staurosporine purchase for a better outcome. This patient

also developed urticaria and angioedema caused by ingestion of cow milk. His IgE levels were 3750 IU/ml. This feature was rarely described in WAS. Whether cow’s milk allergy (CMA) is associated with WAS remains unclear. As most of the patients with WAS have markedly elevated serum IgE levels, and CMA can be IgE-mediated, IgE could be an important contributory factor in the pathogenesis of CMA in WAS. Further studies of CMA in WAS patients could lead to new insights into the immune pathomechanism of CMA. In summary, we reported the clinical manifestations and long-term follow-up of seven unrelated Thai patients with molecular confirmation of WAS. Six different mutations including one nonsense mutation were identified, expanding the mutational spectrum of WASP. The patient with this novel mutation had CMV infection, which was successfully treated with long-term ganciclovir. He also developed angioedema and urticaria as a result of cow’s milk allergy. This study was supported by the Royal Golden Jubilee

Ph.D. Program to PA (Grant No. PHD/0202/2552), the National Science and Technology Development Agency, the Thailand Research Fund, and the learn more National Research University Project, office of the Higher Education Commission (HR1163A). “
“Human NK cells can be subdivided into CD56dim and CD56bright NK cells, which exhibit different phenotypical and functional characteristics. As murine NK cells lack CD56 or a distinct correlate, direct comparative studies of NK cells in mice and humans are limited. Although CD27 is currently proposed as a feasible subset marker in mice, we assume that the usage of this marker alone is insufficient. We rather investigated the expression of the chemokine receptor CXCR3 for its suitability for distinguishing murine NK-cell subsets with simultaneous consideration

of CD27. Compared with CXCR3− NK cells, exerting stronger cytotoxic capability, CXCR3+ NK cells displayed an activated phenotype with a lower expression of Ly49 receptors, corresponding to human CD56bright NK cells. Also in common with human CD56bright NK cells, murine CXCR3+ NK cells Lck exhibit prolific expansion as well as robust IFN-γ, TNF-α and MIP-1α production. We additionally demonstrated changes in both CXCR3 and CD27 expression upon NK-cell activation. In summary, CXCR3 serves as an additional applicable marker for improved discrimination of functionally distinct murine NK-cell subsets that comply with those in humans. NK cells are BM-derived granular lymphocytes that are distinct from T and B cells. As a part of the innate immune system, NK cells play a role in early defence of infection and tumor rejection without prior sensitization.

In selected experiments, rapamycin 1 or 10 ng/mL or CsA 0.1 or 1.0 mcg/mL was added into cultures containing 100 IU/mL human recombinant IL-2. Multiscreen-IP 96-well microtiter plates (Millipore, Bedford, MA) were coated with a mouse anti-human CD3 mAbs (2 μg/mL) Venetoclax manufacturer and mouse anti-human IFN-γ capture mAbs (4 μg/mL). Freshly isolated T cells (1×105 cells/well in 200 μL) were cultured for 36 h, isolated,

washed and incubated with a biotinylated mouse anti-human IFN-γ mAbs (2 μg/mL). After washing, HRP-labeled streptavidin (DAKO, Carpinteria, CA) was added for 1 h and subsequently the spots were developed with AEC substrate (Sigma-Aldrich, St. Louis, MO) and analyzed in an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH). Cytokine secretion is expressed

as spots/well. CD4+ T cells were stained with up to four directly conjugated fluorescent antibodies or control antibodies for 30 min at 4°C. After extensive washing the cells were fixed and permeabilized using the Fixation & Permeabilization kit (eBioscience), and intracellular staining of FOXP3 and CTLA-4 was performed according to the manufacturer’s recommendations. Data were acquired on a FACsCalibur (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR). For cell sorting experiments, CD4+ cells stained for desired cell surface markers were isolated using a FACSAria or FACSVantage (BD Selleckchem Dabrafenib Biosciences) apparatus. PCR was performed using the TaqMan Gene Expression Assay Kit (TaqMan, MGC probes, Applied Biosystems,

Foster City, CA) and the 7300 real-time PCR system. Gene-specific primers for the analysis of human Tbet and GAPDH by real-time PCR were obtained from Applied Biosystems. Migration of lymphocyte subpopulations in response to IP-10 (CXCL10) was quantified at single-cell resolution using microfluidic devices and time-lapse microscopy, as described previously 46. Briefly, photoresist (SU8, Microchem, Newton, MA), click here was patterned within silicon wafers, which were used as a mold to produce a PDMS (Fisher Scientific, Fair Lawn, NJ) device, which was then bonded onto standard 1×3 in. glass slides (Fisher Scientific). The microfluidic network inside each device consisted of an array of up to 450 parallel channels (6×6 μm cross-section and 800 μm long) connected to one main channel, (50 μm tall, 400 μm wide and 10 mm long) with inlets and outlets. The devices were first primed with a solution of IP-10 (100 nM) and fibronectin (250 nM) for 15 min. After priming, sorted populations of either CXCR3+ or CXCR3− CD4+CD25+CD127dim/− Tregs (∼1×105/condition) suspended in 15 μL of media were introduced into the main channel through tubing connected to the main inlet. The cells were flushed through the main channel until media was seen to emerge from the main outlet.

Furthermore, macrophages may shift from one phenotype to another [17]. In considering the role of macrophages in brain injury, it may be important to distinguish between macrophage subsets. Thus, in vitro studies have demonstrated that M1

macrophages are neurotoxic, while M2 macrophages promote regenerative neuronal growth [24]. CCL2, which is expressed post-TBI in the brain and cerebrospinal fluid, has been thought FK506 purchase to elicit primarily M1 macrophages, and the presence of macrophages/microglia early after TBI by histology is often associated with the expression of TNF, IL-6, and IL-1 [1, 13, 25-27]. These findings previously suggested that there is a prominent M1 phenotype in early macrophage recruitment following TBI. Characterization of macrophages in TBI by histology has been complicated by difficulty in distinguishing them from microglia; there is no known marker that is expressed by macrophages but not microglia or vice versa. By flow cytometry, however, the two cell populations can be distinguished by the

level of CD45 expression. Using this approach, we have examined the nature of macrophages responding to TBI in mice. To facilitate macrophage subset identification, we examined TBI in YARG mice, in which yellow fluorescent protein (YFP) is expressed under the promoter for the M2 marker, Arg1 Angiogenesis inhibitor [28, 29], and Yet40 mice, in which YFP is expressed under the promoter for the M1 marker, IL-12p40. We here demonstrate that a subset of brain wound macrophages upregulate

Arg1 and home to the site of injury. At day 1 after injury, 21 ± 1.5% of the ipsilateral hemisphere macrophages express high levels of Arg1, but the number of Arg1+ cells falls thereafter and cannot be detected after 1 week. Whole genome expression analysis of Arg1+ and Arg1− macrophages following TBI Epothilone B (EPO906, Patupilone) revealed that these macrophage subsets differ in their expression of over 1300 genes, with notable differences in genes encoding chemokines. The pattern of gene expression in neither population is characteristic of in vitro derived M2 or M1 cells. Our results indicate that the macrophage response to TBI is heterogeneous, and the early response includes at least two distinct subsets. As assessed by expression of Arg1, the ratio of these subsets changes with time. To assess the immune response following TBI, we used an adult murine controlled cortical impact model. Histological analysis of brain sections following TBI confirmed cortical injury, which extended into the hippocampus (Fig. 1A). Hematoxylin and eosin (H&E) staining revealed increased cellular recruitment to cortical tissues adjacent to the lesion (Fig. 1A). Immunohistochemical staining for F4/80 showed that macrophages/microglia are widely present at the pericontusional site (e.g. in areas of the cortex adjacent to the lesion) (Fig. 1B).

Efficient responses to the fungus require a complex network of immunological mechanisms. Together with alveolar macrophages and neutrophils, which constitute a primary line of innate cellular defence against A. fumigatus,1,2 the crucial role of the adaptive immunity has been extensively demonstrated.3 Indeed, besides the well-characterized protective role of T helper type 1 (Th1) lymphocytes,4–7 the newly described regulatory T cells and interleukin-17 (IL-17) -producing cells (Th17) represent important mediators of the inflammatory and anti-inflammatory

Gefitinib datasheet host responses against A. fumigatus.8 However, dendritic cells (DCs) also play a fundamental function in initiating and modulating the specific immune responses upon recognition of A. fumigatus.5,9,10 After internalization of A. fumigatus conidia, DCs mature and acquire the capacity to polarize

naive T cells and, in turn, to promote a protective response.9 In keeping with these findings, in vivo results on the migration of lung DCs into lymphoid organs, where they drive an appropriate T-cell response to fungal antigens,11 have brought DCs centre stage as promising targets for intervention in immunotherapy and fungal vaccine development.12 In addition, it is important SB203580 solubility dmso to consider several studies that have recently pointed to DCs and type I interferons (IFNs) as special players in the immune response tailored to combat tumours and infections.13–15 Indeed, although the anti-microbial properties of these cytokines have not been fully characterized yet, type I IFNs represent important immunomodulators of the innate, as well as the adaptive, arm of the immune system. Type I IFN can promote

the differentiation of human blood monocytes into DCs and contribute to their maturation.16,17 This leads to the generation of DCs able to stimulate a primary human antibody response, a Th1 proliferation,18 and a cross-priming of CD8 T cells against viral antigens.19 In addition, one crucial outcome of type I IFN-induced effects is the ability to directly stimulate IFN-γ production in natural killer and T cells,20–22 which in turn promotes the development of a cell-mediated immune response. Based on these immunoregulatory properties, in this work the expression and the Phosphatidylinositol diacylglycerol-lyase capacity of type I IFN, namely IFN-β, to modulate the T-cell polarizing capacity of A. fumigatus-infected DCs was investigated in an attempt to evaluate the effects induced by this cytokine on anti-fungal immunity. Although the phagocytosis of the fungus was not affected by IFN-β treatment, the maturation induced by A. fumigatus infection was enhanced in IFN-β-primed DCs, as evaluated by analysing the immunophenotype and the release of pro-inflammatory and regulatory cytokines. Accordingly, IFN-β endowed DCs with potent Th1 polarizing capacity because an enhanced IFN-γ production in T cells co-cultured with A. fumigatus-infected DCs was observed in the presence of IFN-β.

Long-term success can be secured only by adaptability. It is increasingly clear that to cope with our expanding knowledge of T cell biology, immunologists must be as flexible as the cells they love to study. S. M. A. and R. A. O. are supported by grants from the UK Medical Research Council, the Wellcome Trust and the UK Multiple

Sclerosis Society. S. M. A. holds a Research Councils UK fellowship in translational medicine. L. S. T. is supported learn more by MRC- and BBSRC-funded PhD studentships and by financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College

London and King’s College Hospital NHS Foundation Trust. The authors declare no conflict of interest. “
“Interleukin (IL)-17A is increased both in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. Administration of pristane to genetically intact mice results in the production of autoantibodies and proliferative glomerulonephritis, resembling human lupus nephritis. These studies sought to define the role of IL-17A in experimental lupus induced by pristane administration. Pristane was administered to wild-type (WT) and IL-17A−/− mice. Local and systemic immune responses were assessed after 6 days and 8 weeks, and autoimmunity, glomerular inflammation and renal Thymidylate synthase injury were measured at 7 months. IL-17A production increased significantly 6 days after pristane BYL719 injection, with innate immune cells, neutrophils (Ly6G+) and macrophages (F4/80+) being the predominant source of IL-17A. After 8 weeks, while systemic IL-17A was still readily detected

in WT mice, the levels of proinflammatory cytokines, interferon (IFN)-γ and tumour necrosis factor (TNF) were diminished in the absence of endogenous IL-17A. Seven months after pristane treatment humoral autoimmunity was diminished in the absence of IL-17A, with decreased levels of immunoglobulin (Ig)G and anti-dsDNA antibodies. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4+ T cells and intrarenal expression of T helper type 1 (Th1)-associated proinflammatory mediators were decreased in IL-17A−/− mice. WT mice developed progressive proteinuria, but functional and histological renal injury was attenuated in the absence of IL-17A. Therefore, IL-17A is required for the full development of autoimmunity and lupus nephritis in experimental SLE, and early in the development of autoimmunity, innate immune cells produce IL-17A. “
“A bacteriophage lambda DNA vaccine expressing the small surface antigen (HBsAg) of hepatitis B was compared with Engerix B, a commercially available vaccine based on the homologous recombinant protein (r-HBsAg).

The mock-immunized group that received an AJ challenge were reduced to two mice in the group because of a technical error during challenge. The resulting blood-stage infections were followed by microscopic examination of Giemsa’s solution-stained thin blood smears taken daily using venous blood from the tail. In order to determine the day at which parasites first became detectable in the blood, at least 10 000 red blood cells were examined per smear. For the generation of sporozoites, Anopheles stephensi mosquitoes were allowed to feed on anaesthetized mice that had been inoculated with 1 × 106 iRBCs IP 6 days

previously. Prior to feeding, mouse blood was checked for R788 datasheet the presence of gametocytes, and their viability assessed by the observation of exflagellation of microgametocytes in fresh blood Atezolizumab cost preparations. Seven to 10 days post-feed, mosquito mid-guts

were dissected and the presence of oocysts confirmed. Sixteen days post-feed, mosquito salivary glands were dissected into a 50 : 50 solution of FCS and Ringer’s solution, crushed in a glass and Teflon tissue homogeniser, and the numbers of sporozoites in the homogenate assessed by counting with a haemocytometer. In order to assess sporozoite viability, only those sporozoites displaying circular gliding motility were considered viable. There were no discernable differences in the viability of CB and AJ sporozoites, and sporozoites of both strains were handled in exactly

the same manner prior to immunization and challenge inoculation. All mice were kept on 0·05% para-aminobenzoic acid (PABA)-supplemented water ad libitum and were housed at 21°C on a 12 h-light–dark cycle. Anopheles stephensi mosquitoes were fed with 0·05% PABA-supplemented 10% glucose solution and were housed at 27°C and 70% humidity on a 12-h light–dark cycle. We used R version 2·7·0; The R Foundation for Statistical Computing; http://www.R-project.org) for data analysis. To analyse patterns of parasitaemia during infections, we used mixed effects models because, by treating each infection as a ‘random’ effect, we can account for repeated measures from each infection and overcome pseudoreplication problems associated with such data. These Adenylyl cyclase models were fitted with Poisson error distributions and minimized following stepwise deletion of the least significant term, using log-likelihood ratio tests to evaluate the change in model deviance, until only significant terms remained. We present F-ratios for fixed effects remaining in minimal models. Mann–Whitney tests were used to compare patency data. Cumulative, summary data were analysed with linear models, using anova (F ratios) to evaluate significance of terms. The days on which parasites became detectable by microscopy (patent) in the blood of mice subjected to various immunization and challenge regimens are shown in Table 1.