S2). In contrast,

levels of IP-10 correlated well with ear swelling, because PD-0332991 mw only CTLA-4-Ig treatment in the sensitization phase – but not the challenge phase – could reduce the levels of IP-10. These data suggest that the release of IL-4, IL-1β, MIP-2 and IP-10 locally in the inflamed ear is regulated differently by CTLA-4-Ig; whereas IL-4, MIP-2 and IP-10 are suppressed when CTLA-4-Ig is present only in the sensitization phase, our data show that MIP-2 and IL-4 can also be suppressed when CTLA-4-Ig is present during the challenge phase alone. In this study we show that CTLA-4-Ig treatment suppresses hapten-induced inflammation in two skin inflammation models. The effect of CTLA-4-Ig has been shown previously in the DNFB-induced selleck products CHS model but not in the oxazolone-induced CHS

model. The short-term effect on ear swelling was detected in both the DNFB-induced model, where 25 mg/kg was sufficient to suppress the response completely, while in the oxazolone model 125 mg/kg was necessary to obtain the same degree of suppression. DNFB has been described previously to induce a T helper type 1 (Th1)-mediated response [16, 19], whereas oxazolone is shown to mediate a mixed phenotype characterized by both Th1 and Th2 cells [20]. The different efficacy of CTLA-4-Ig in the two models may be attributed to the notion that CTLA-4-Ig may suppress Th1 responses more efficiently than Th2 responses [16]. Furthermore, in our hands oxazolone-induced inflammation is dominated more by neutrophils than T cells (compared to the DNFB-model); thus, it is formally possible that the effect of CTLA-4-Ig is less efficient in the oxazolone model because of the considerable involvement of neutrophils. Alternatively, the need for a higher dose to suppress oxazolone-induced inflammation could also reflect the stronger overall response by oxazolone Interleukin-2 receptor compared to DNFB (see Fig. 1). In addition to the short-term effect, we found that CTLA-4-Ig induces a long-lasting suppression

of inflammation in both models even in the absence of any detectable, circulating CTLA-4-Ig during the secondary response. The lack of circulating CTLA-4-Ig 3 weeks after administration is expected, as the half-life of human CTLA-4-Ig has been estimated to be 30 h in mice [13, 21]. Interestingly, sustained immune modulation by CTLA-4-Ig has also been shown in other settings, including transplantation, where short-term CTLA-4-Ig therapy led to long-term tissue- and organ-graft survival and induction of tolerance [14, 22-24]. Furthermore, it has been shown in vitro that CTLA-4-Ig induces a long-lasting hypo-responsiveness in human mixed leucocyte cultures (MLC) [12]. The precise mechanisms by which CTLA-4-Ig mediates the sustained suppression are not entirely clear.

3 While Foxp3 gene expression is limited to Tregs in mice, it can also be expressed by activated human effector T cells (Teffs).4–6 In this regard, recent evidence suggests that human CD4+ FoxP3+ T cells are composed of at least three phenotypically and functionally distinct subpopulations: FoxP3Low resting Tregs (rTregs), FoxP3HI activated Tregs (aTregs) (both of which are suppressive in vitro),

and cytokine-secreting (i.e. IL-2 and IFN-γ) FoxP3Low non-suppressive T cells.4,6 Although the relevance of human FoxP3 cell subsets remains to be established in health and disease, it is generally considered Midostaurin datasheet that a decrease in the number and/or function of Tregs plays a role in autoimmune disease pathogenesis by allowing uncontrolled immune effector activities.6–8

In contrast, an abnormal increase in Treg number and/or function may result in abnormal suppression of immune effector functions and defective clearance of pathogens or tumours.9,10 Maintaining a tight control of Treg activities appears critical to (i) ensuring an adequate immune response against pathogens, (ii) avoiding excessive immune activation which may be deleterious to the host, and (iii) maintaining immune tolerance against self-antigens. Recent evidence suggests selleck that, upon stimulation of the immune system, there is an initial phase of Teff expansion (first 1–2 weeks) followed by a second phase (weeks 3–4) of expansion of Tregs which then control the Teff response.11 Expansion of Teffs and expansion of Tregs both require the same Edoxaban conditions of antigen stimulation, but express distinct kinetics. Thus, effectors predominate early to achieve pathogen clearance, without the interference of regulatory cells.12 Once the pathogen has been cleared from the host, increased numbers of regulatory cells (resulting from the second phase of expansion) can suppress the effectors, and the immune system can return to its

steady state. Pro-inflammatory cytokines, such as IL-1, IL-6 and tumour necrosis factor alpha (TNF-α), have been found to promote Treg proliferation/expansion, and in parallel to support proliferation of Teffs.13,14 In addition, all three cytokines have been shown to make Teffs relatively resistant to suppression by Tregs.15–17 Not previously described, however, is a cytokine that can preferentially promote activation of Teffs while inhibiting Treg expansion. Type 1 interferons (IFN-I) are innate cytokines that are transiently induced during viral infection and have unique roles in defence against viruses, but their persistent stimulation may contribute to autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory myositis and Sjögren’s syndrome.

[6] Similar observations have been made in some experimental models of nephron deficiency.[75, 76] Furthermore, the prevalence of chronic kidney disease is also significantly greater in obese than non-obese individuals.[77] Recently, Gurusinghe et al. demonstrated that an increase in body weight as a result of fat feeding EX 527 price in nephron deficient mice resulted in greater

increase in night-time arterial pressure and renal fibrosis than nephron-replete obese controls.[78] This highlights the potential for detrimental effects of excessive weight gain in individuals with a nephron deficiency. This is particularly concerning as in a multi-centre study conducted in the United States by Reese et al. found that a third of the donor population were either clinically hypertensive, obese, or had a GFR of <60 mL/min per 1.73 m2.[79] The authors suggested that due to the increasing demand for live organ donation, the stringent PD0325901 purchase criteria for selection of organ donors are being relaxed resulting in acceptance of growing numbers of medically complex donors.[79] Such practice will undoubtedly result in a greater number of donors developing advanced renal and cardiovascular disease, thus increasing the economic burden associated with treatment of

these conditions. The mechanisms via which a low nephron number causes hypertension remain unclear. An increase in reabsorption of sodium is central to the development of hypertension following nephron deficiency. However, a decrease in filtered load as suggested by Brenner[2] cannot be the sole explanation for the hypothesized retention of sodium. Recently, Vallon and colleagues put forward a hypothesis Interleukin-3 receptor to explain the onset of hyperfiltration in the setting of Type I diabetes,[80] which may be important in discerning some of the mechanisms contributing to glomerular hyperfiltration and to hypertension in models of nephron deficiency. They proposed that an increase in sodium-glucose transport was the primary stimulus for hypertrophy of

the proximal tubules.[80] The increase in reabsorption of sodium-glucose in the proximal tubules would then decrease distal delivery which would be interpreted as an inadequate GFR at the level of the macula densa and would cause a TGF-dependent increase in SNGFR.[80] Compensatory growth of the proximal tubules also occurs in the setting of a reduced renal mass and we propose that the compensatory increase in reabsorption of sodium contributes to retention of sodium and drives the initial increase in blood pressure. Indirect support for this hypothesis is provided in our model of nephron deficiency induced by fetal uninephrectomy in the sheep. We found that, following uninephrectomy in the sheep fetus at 100 days of gestation (term 150 days), the weight of the remnant kidney increased markedly such that it was not different to the total kidney weight of the sham animals at the age of 6 months age.

Nukuzuma, unpublished data). Proliferation characteristics of COS-tat cells may provide important background information for studies using these cell lines. Thus, we first compared the cell proliferation of three COS-tat cell lines

with those of parental check details COS-7 cells. COS-7 cells (ATCC CRL 1651) and COS-tat cell clones (8) were cultivated in EMEM containing 10% FBS (hereafter called culture medium). Cell cultures were maintained at 37°C in a humidified incubator containing 5% CO2 in air. The relative number of live cells was determined by measuring mitochondrial succinate dehydrogenase activity using MTT assay. COS-7 cells and COS-tat cell clones were each plated in five wells of 96-well culture plates at a concentration of 2 × 103 cells/well in 100 μL culture medium and incubated at 37°C in a CO2 incubator. MTT assay was performed using a Cell Proliferation Kit I (MTT) (Roche, Penzberg, Germany) according to

the manufacturer’s instructions. After an incubation period of 5 days, 10 μL MTT solution was added to each well to a final concentration of 0.5 mg/mL, and the plates incubated for 4 hr. Then, find more 100 μL solubilization solution was added to each well, and the plates placed in an incubator overnight. The formazan products were solubilized, and spectrophotometric data were measured using an enzyme-linked immunosorbent assay reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 550 nm with a reference wavelength of 650 nm. The significance of inter-group differences was statistically determined by Student’s t-test. As shown in Table 1, the enzyme activity of COS-tat7 and COS-tat15, and COS-tat22 cells was lower than that of parental COS-7 cells and this difference Molecular motor was statistically significant (P < 0.01). Of note, the enzyme activity of COS-tat22 cells was lower than that of COS-tat7 and COS-tat15 cells (P < 0.01). To measure the doubling time, COS-7 cells and COS-tat cell clones were plated in 6-well culture plates at a concentration of 4 × 104 cells/well in 2 mL culture medium. After an incubation period of 72 hr, cell numbers were counted. The

doubling time of COS-7, COS-tat7, COS-tat15, and COS-tat22 were 21.6, 24.6, 22.8, and 30.8 hr, respectively. The doubling time of COS-7 COS-tat cells were in agreement with the proliferation characteristics of the cells as judged by MTT assay. Taken together, these results indicate that stable expression of Tat leads to down-regulation of cell proliferation. We next compared the production of PML-type JCV in COS-tat cell clones with that in parental COS-7 cells. Since JCV capsids have the property of agglutinating human type O erythrocytes, HA assay has been traditionally employed to determine the virus titer (12). COS-7 and COS-tat cell clones were cultured in 35-mm dishes containing 2 mL culture medium until the cells were 50–80% confluent.

01) and 6-to-12-hour-dwell (p < 0.02) of PD patients with oral pyridoxamine compared with PD patients with Selleckchem GDC-973 no oral pyridoxamine. Conclusion: Pyridoxamine is a potent candidate of a protective agent against progressive deterioration of peritoneal function in PD patients. CHANG JER-MING1, CHEN SZU-CHIA1,2, CHEN HUNG-CHUN1 1Division of Nephrology, Department of Internal Medicine, Kaohsiung Medical University Hospital; 2Department of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University Introduction: Neurological complications are prevalent, and contribute

largely to the morbidity and mortality in patients with renal failure. Quantitative

sensory testing (QST) is a reliable test of large and small fiber sensory modalities, documented well by the American Diabetes Association endorsement of QST in 1992 as a valid test in epidemiologic studies and drug trials of diabetic neuropathy.QST can assess and quantify sensory nerve function noninvasively. QST has potential as a neurophysiologic tool. Using QST, sensory deficits may be quantified and the data can be used in parametric PS-341 mouse statistical analysis. Previous study showed thermal sensation was abnormal in 30% of 64 non-diabetic hemodialysis patients, which was a much higher prevalence than that which has previously been reported. However, the role of small fiber neuropathy remained

uncertain in peritoneal dialysis (PD) population. We evaluated the prevalence and associated risk factors of abnormal thermal sensation in PD. Methods: This study enrolled selleckchem 19 persistent PD patients. Thermal sensitivity was assessed by QST. Demographic and medical data including age, gender and comorbid conditions were obtained from medical records or interviews with patients. Statistical analysis was performed using SPSS 17.0 for windows (SPSS Inc. Chicago, USA). Results: The mean age of the 19 patients was 51.3 ± 10.7 years. The median of PD duration was 68.4 months. Thermal sensation was abnormal in 68.4% (13/19) of the patients. Compared with patients with normal thermal sensation, patients with abnormal thermal sensation were found to have older age (54.9 ± 7.6 vs. 43.3 ± 12.7 years; P = 0.023). Conclusion: Our study showed a high prevalence of abnormal thermal sensation in PD. Old age was associated with abnormal thermal sensation. The limited patient number restricted our study power. Future prospective studies were needed to survey the long-term outcomes in large PD population.

First, significant blood volumes are needed to measure rare lymphocyte populations that are at the centre of this disease. There is as yet no consensus on the precise autoreactive T cell peptide–major histocompatibility complex (MHC) recognition specificities in humans or, indeed, on the likelihood that they are shared between different subjects. The low affinities of autoreactive T cells pose unique challenges for detection, especially with regard to teasing out signal from noise, and it remains incompletely determined whether fresh or frozen samples are best suited for all assays.

Several speakers at the workshop discussed T cell assays that reflect new accomplishments in the field, as well as highlighting Selleck Tamoxifen areas of click here active assay development and potential roadblocks. Topics included: Successful generation of CD4+- and CD8+-specific multimers that allow for higher numbers of low-affinity autoreactive cells to be detected from the peripheral blood [7]. Application of class II tetramer assays for direct detection of autoreactive

CD4+ cells without culture or in-vitro expansion [8]. Functional assays [e.g. cytokine enzyme-linked immunospot assay (ELISPOT)] that use naturally processed and presented epitopes of putative islet autoantigens validated in blinded studies [9]. Molecular engineering efforts using structure–function studies to improve T cell detection with better MHC binding peptides [10]. Quantum (Q-) dot assay, for multiplex, sensitive detection of MHC class I-restricted T cell receptors (TCRs), allowing for T cell-based immune signatures of remission and relapse of autoimmunity in the islet transplantation setting; correlative studies of T1D clinical trials; and discovery of new autoreactive T cell epitopes [11, 12]. High-throughput TCR sequence analysis including TCR-β chain

deep sequencing within functional populations in T1D subjects [13]. These assays potentially define intermediate immunological phenotypes associated with clinical prognosis. Workshop highlights included ADP ribosylation factor the following: T cell proliferation assays coupled with phenotypic characterization of surface markers that may be used to align appearance of T cell memory with appearance of autoantibodies in the at-risk populations (unpublished). Functional interrogation of disease-specific pathogenic or beneficial T cells as a gauge of T cell ‘health’, including assays for requisite signalling pathways and other intracellular events downstream of TCR and cytokine receptor engagement [14]. At the development stage, both improvements in existing technologies as well as exploration of new technologies are needed. Miniaturizing – most assays still utilize larger than desirable sample volumes – and the limiting factors of procuring, handling and storing of human samples are barriers to rapid evaluation.

Data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 as compared to control. Figure S3. (A) Fleshly isolated CD4+CD25- and CD4+CD25+ T cells were stimulated with anti-CD3 mAb (0.5 ìg / mL) and IL-12 receptor (IL-12R) ® chain expression was analyzed with flowcytometry. Bold line : CD4+CD25- T cells, Thin line: CD4+CD25+ T cells, filled grey : isotype control. (B) Expression level of IL-12R®2

after siRNA treatment was confirmed. 1 × 106 siRNA transfected or untreated CD4+CD25- T cells were cultured with 1 × 105 irradiated autologous CD4-depleted PBMCs and anti-CD3 mAb. Three days later, cells were harvested and RNA was extracted to confirm knockdown of IL-12R®2 expression by real-time RT-PCR. The relative differences in gene expression were calculated using threshold

cycle (Ct) values that were normalized to those of ICG-001 in vivo TATA-box-binding protein gene, and compared with the relative Ct value of untreated CD4+CD25- T cells by the 2-ddCt. (C)) 1 × 104 CD4+CD25- T cells with/without siRNA treatment were cultured with 1 × 105 irradiated autologous CD4-depleted PBMCs and anti-CD3 mAb in the presence or absence of 5 × 103 CD4+CD25high Tregs with OK-432 (1 ìg / mL). Proliferation was evaluated as described in Materials and Methods. These experiments were performed independently at least twice with similar results. Data are expressed as mean ± SD. “
“Citation Noronha LE, Antczak DF. Maternal immune responses to trophoblast: BMS-777607 cost the contribution of the horse to pregnancy immunology. Am J Reprod Immunol 2010 The horse has proven to be a distinctively informative species in the study of pregnancy immunology for several reasons. First, unique aspects of the anatomy and physiology of the equine conceptus facilitate approaches that are not possible in other model organisms, such as non-surgical SB-3CT recovery of early stage embryos and conceptuses and isolation of pure trophoblast cell populations. Second, pregnant mares make strong cytotoxic antibody responses to paternal major histocompatibility complex class I antigens expressed by the chorionic girdle cells, permitting detailed evaluation of the antigenicity of

these invasive trophoblasts and how they affect the maternal immune system. Third, there is abundant evidence for local maternal cellular immune responses to the invading trophoblasts in the pregnant mare. The survival of the equine fetus in the face of strong maternal immune responses highlights the complex immunoregulatory mechanisms that result in materno–fetal tolerance. Finally, the parallels between human and horse trophoblast cell types, their gene expression, and function make the study of equine pregnancy highly relevant to human health. Here, we review the most pertinent aspects of equine reproductive immunology and how studies of the pregnant mare have contributed to our understanding of maternal acceptance of the allogeneic fetus.

Readout of a neo-epitope on the TCC, which is dependent of hydrophobic interactions with the target, could thus be influenced by other factors than the upstream complement components. The complement activity levels were analysed

in patients with defined complement deficiencies. Serum samples deficient in C2 showed normal AP activity but undetectable CP Wnt inhibitor and LP activity. This is due to a lack of formation of a functional CP and LP C3-convertase. Factor I and factor H deficiency leads to sustained consumption of complement, which explains the reduced complement activity in all the pathways. Sera from patients diagnosed with HAE, due to lack of the inhibitor of C1r/s (C1INH) were also tested. Lack of C1INH leads to consumption of components of the classical pathway [24], and consistent with this, a decreased functional activity of the classical pathway was demonstrated

in nine of 10 of these sera. These results demonstrate that by analysing and comparing the functional capacity of each complement AP24534 molecular weight pathway, it is possible to obtain reliable clues to which component(s) of the complement system that are functionally defect or present in insufficient amount to activate complement. In summary, we have developed and analysed ELISA-based assays for the measurement of the functional activation capacity of the three complement pathways. These methods are applicable at high serum concentrations, which minimize false negative and represent – especially for the assessment of the MBL activity – an improvement on the existing assays. This work was supported by the University of Southern Denmark and the John and Birthe Meyer Foundation. A PCT application (PCT/DK2008/050221) has been submitted

for the use of SPS in complement assays. “
“Infections that occur early in life may have a beneficial effect Acetophenone on the immune system and thereby reduce the risk of allergen sensitization and/or allergic disease. It is not yet clear to what extent specific virus and/or bacteria can mediate this effect. The purpose of this study was to assess the role of virus and bacteria in CD4+ T cell-derived cytokine production in newborns. We compared the effects of five bacteria (Staphlococcus aureus, Escherichia coli, Clostridium difficile, Lactobacillus rhamnosus and Bifidobacterium bifidus) and seven virus (adenovirus, coronavirus, cytomegalovirus, herpes simplex virus, influenza virus, morbillivirus and poliovirus) on the Th1/Th2 cytokine production in mixed lymphocyte reactions using CD4+ T cells from cord blood cocultured with allogenic myeloid or plasmacytoid dendritic cells. When comparing the baseline cytokine production prior to microbial stimulation, we observed that cord plasmacytoid DC were stronger inducers of Th2 cytokines (IL-5 and IL-13) compared with cord myeloid DC and to adult DC.

In the next subsections, we will illustrate the protective effects mediated by Ab–FcR interactions in the context of a selection of infections with intracellular bacteria and parasites. Legionella pneumophila are Gram-negative bacteria that, when inhaled, can infect and replicate within alveolar macrophages and cause a severe form of pneumonia known as Legionnaire’s disease. Upon contact with the macrophage, L. pneumophila uses its Icm/Dot type IV secretion system (T4SS)

to inject a large number of effector proteins into the cytosol of the NSC 683864 molecular weight host cell 63. This promotes phagocytosis and modulates trafficking within the host cell, resulting in the evasion of phagolysosomal fusion and the establishment of a replication-permissive vacuole 64. We have recently shown that this Icm/Dot T4SS-mediated subversion of trafficking within the host cell does not take place in the presence of specific Abs as, in such circumstances, L. pneumophila is targeted to lysosomes and can no longer replicate intracellularly 65. Thus, opsonized L. pneumophila are targeted Ruxolitinib datasheet into

degradative pathways, indicating that specific Abs can effectively oppose the events initiated by the T4SS. The opsonization of L. pneumophila with specific Abs does not interfere with the function of the T4SS itself but it is actually the cross-linking of activating FcRs on the surface of macrophages that renders these host cells nonpermissive for intracellular replication of L. pneumophila. The importance of FcR triggering for this protective effect was demonstrated both in vitro and in vivo; however, the lysosomal targeting of L. pneumophila is not simply a direct consequence of FcR-mediated endocytosis and subsequent phagolysosomal fusion since macrophages, which had received an FcR trigger before infection with nonopsonized bacteria, also effectively targeted L. pneumophila to lysosomal compartments and hence did not permit

their intracellular replication 65. These Rho results suggest that FcR cross-linking induces a signaling cascade that effectively counteracts the modulation of host cell trafficking by Legionella effectors and redirects the bacteria to lysosomes where they are degraded. By arresting phagosome maturation M. tuberculosis survives and replicates in membrane-bound compartments in macrophages 66. Ab responses have long been believed to play a negligible or even detrimental role in protection against this intracellular bacterium, whereas cell-mediated immunity was assigned to be crucial in resolving infections. Nevertheless, newer findings implicate a role for Abs in protection against mycobacterial infections 67, 68. mAbs of the IgG3 or IgG1 subclass recognizing surface Ags of M. tuberculosis such as the carbohydrate lipoarabinomannan (LAM) have been shown to prolong survival of intratracheally or intravenously M.

All experiments were repeated more than three times and representative results

are shown. Data are expressed as mean ± 2 standard errors (s.e.). Statistical analyses were performed using Student’s unpaired t-test (specifically for immunoblotting determination, we compared with each respective control) and analysis of variance (anova). P-values of less than 0·05 indicated a statistically significant difference. https://www.selleckchem.com/products/icg-001.html A potent inhibitory ITAM (iITAM) signalling triggered by monovalent targeting of FcαRI requires an associated FcRγ chain. Transfectants expressing a R209L transmembrane FcαRI mutant that cannot associate with the FcRγ chain elicited neither inhibitory nor activating responses. To evaluate the precise role of FcαRI/FcRγ, we generated three Tg mouse lines with C57BL/6J backgrounds and designated them as 503, 505 and 604 using a construct containing human full-length FcαRIR209L cDNA, mouse FcRγ subunit and FLAG-tag under the control of the CAG promoter [18] (Fig. 1a). Macrophages isolated from the peripheral blood of C57BL/6J-Tg mice expressed FcαRIR209L/FcRγ (Fig. 1b). Macrophage FcαRIR209L/FcRγ expression was stable in 6–24-week-old mice (data not shown). The level of transgene expression was ∼10-fold higher in macrophages from line 604 than from the other two lines (Fig. 1b).

An example of a PCR assay demonstrating the simultaneous presence of human FcαRI DNA is shown in Fig. 1c. Analysis of protein extracts and sections from the peripheral blood in FcαRIR209L/FcRγ Tg mice by Western blotting PD0325901 datasheet and staining with anti-FLAG antibody demonstrated the presence of a full-length 74-kDa human FcαRIR209L/mouse FcRγ chimeric protein in FcαRIR209L/FcRγ Tg mouse serum (Fig. 1c). The existence of soluble FcαRI was analysed using serum from aged FcαRIR209L/FcRγ Tg because soluble FcαRI formed an immune complex with mouse IgA that led to IgA deposition in the

glomeruli and nephropathy. As shown in Fig. 1d,e, there was no particularly soluble FcαRI band in FcαRIR209L/FcRγ Tg mouse serum. Figure 1f,g shows that polymeric mouse IgA binds weakly to FcαRI and is sufficient to induce strong negative signals, whereas huge complexes such as soluble FcαRI/ mouse polymeric IgA Chloroambucil induced aggregation of the receptor, which led to activation signals in the FcαRIR209L/FcRγ transfectants (I3D). To determine whether monovalent targeting of anti-FcαRI (MIP8a Fab) might have therapeutic implications for HAF-CpG-GN, we analysed the effect of MIP8a Fab treatment in HAF-CpG-GN mouse models of kidney disease. Mice treated with PBS or an unrelated control IgG developed elevated proteinuria, BUN and creatinine levels (Fig. 2a,b and not shown). Albuminuria was significantly attenuated in mice treated with MIP8a Fab (Fig. 2a). There were no significant differences in BUN and creatinine levels (Fig. 2b, not shown).