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Results for “Complement Hemolytic Activity Assay”

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PubMed2001

Immunological and clinical follow up of hepatitis C virus associated cryoglobulinaemic vasculitis.

OBJECTIVE: To study immunological markers and compare these markers with standard measures for the clinical and immunological follow up of vasculitis activity in hepatitis C virus (HCV) associated cryoglobulinaemic vasculitis (CV). METHODS: Serial serum samples from eight patients with newly diagnosed HCV associated CV were followed during interferon alpha treatment induced remission of the CV. Vasculitis activity and disease extent were evaluated with the Birmingham vasculitis activity score (BVAS) and disease extent index (DEI). Cryoglobulinaemia, complement levels (C3c, C4, and CH50), rheumatoid factor (RF), autoantibodies such as antinuclear antibodies, soluble interleukin 2 receptor (sIL2r), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble CD30 (sCD30) were determined. RESULTS: All patients achieved either complete or partial remission of their CV during interferon alpha treatment. There was a significant reduction in vasculitis activity and disease extent (BVAS, DEI), cryoglobulinaemia, RF, sIL2r, sICAM-1, and sCD30. Complement C3c levels increased significantly during this period. Erythrocyte sedimentation rate and levels of complement C4 and CH50 did not change significantly. Both clinical measures (BVAS and DEI) correlated significantly only with C3c and sCD30. CONCLUSIONS: Although this study was of only a small group of patients, it shows that BVAS and DEI as clinical measures and C3c and sCD30 as immunological markers may be useful in the follow up of disease activity of HCV associated CV. The data indicate that activity of the humoral (cryoglobulinaemia, RF, autoantibodies) and cellular (sIL2r, sICAM-1, sCD30) immune response and endothelial damage (sICAM-1) are found in HCV associated CV.

Antibodies, Antinuclear↗

PubMed2000

Clinical, histological and immunopathological features of 58 patients with subacute cutaneous lupus erythematosus. A review by the Italian group of immunodermatology.

BACKGROUND: Subacute cutaneous lupus erythematosus (SCLE) is a distinct subset of cutaneous lupus erythematosus clinically characterized by psoriasiform and/or annular lesions and by a mild or absent systemic involvement. OBJECTIVE: The Italian Group of Immunodermatology of the Italian Society of Dermatology and Venereology reviewed the cases of SCLE seen in 10 years (1987-1996). PATIENTS: Forty-six women and 12 men have been retrospectively studied, 42% had annular lesions, 39% psoriasiform ones and 16% both. RESULTS: Lesions were mainly localized on the neck and face and relapsed in spring and autumn. Seventeen patients had 4 or more American College of Rheumatology criteria and could be classified as having systemic lupus erythematosus. The most frequent histopathological alterations were epidermal atrophy, hydropic degeneration of the basal layer and perivascular lymphocytic infiltrate. Deposits of immunoglobulins and C3 at the dermo-epidermal junction on the clinically involved skin were present in 86% of the patients. Dust-like particles in the epidermis were only found in 3% of cases. Anti-Ro/SSA antibodies were found in 71% of the cases and anti-dsDNA only in 5% of cases. CONCLUSIONS: SCLE is a particular subset of cutaneous lupus erythematosus with peculiar clinical and immunopathological features.

Adolescent↗

PubMedDate not supplied

Proinflammatory effects in experimental mesangial proliferative glomerulonephritis of the immunosuppressive agent SDZ RAD, a rapamycin derivative.

BACKGROUND/AIM: The new immunosuppressant SDZ RAD, a rapamycin derivative, inhibits growth factor driven cell proliferation. SDZ RAD designed for transplantation may also be a candidate agent to treat inflammatory kidney diseases. Therefore, we investigated the effects of SDZ RAD in two different animal models of glomerulonephritis, in anti- Thy1.1 nephritis and in acute puromycin aminonucleoside (PAN) nephrosis. METHODS: Eighty-seven male Wistar rats were investigated. Anti-Thy1.1 nephritis: healthy rats (n = 9), SDZ RAD-treated healthy rats (n = 6), nephritic rats (n = 9), SDZ RAD placebo treated nephritic rats (n = 6), SDZ RAD-pretreated nephritic rats (n = 9), and early (n = 6) as well as delayed (n = 6) SDZ RAD-posttreated nephritic rats. PAN nephrosis: healthy rats (n = 6), SDZ RAD-treated healthy rats (n = 6), nephritic rats (n = 12), and SDZ RAD-pretreated nephritic rats (n = 12). In a separate study, 12 male Sprague-Dawley rats were analyzed in anti-Thy1.1 nephritis: healthy rats (n = 3), nephritic rats (n = 3) and pretreated nephritic rats (n = 6). SDZ RAD and SDZ RAD placebo were given at single doses of 2.5 mg/kg body weight per day by gavage. The experiments lasted until days +2 and +9 after induction of anti-Thy1. 1 nephritis and until day +13 in the case of PAN nephrosis. RESULTS: In anti-Thy1.1 nephritis, SDZ RAD demonstrated marked proinflammatory effects in a time-dependent manner, as reflected by severe focal damage to glomerular histology including inhibition of mesangial cell proliferation, reduction of creatinine clearance, and increase in plasma creatinine levels as well as proteinuria. Almost identical results were obtained in both rat strains. In contrary, SDZ RAD ameliorated significantly the development of PAN nephrosis. Animals pretreated by this agent showed a significant reduction of proteinuria and of glomerular invasion of monocytes/macrophages. CONCLUSION: Some caution is warranted for the use of SDZ RAD in inflammatory glomerular diseases, since it accentuated glomerular damage induced by anti-Thy1.1 antibodies.

Animals↗

PubMed2003

Two-year follow-up study after human parvovirus B19 infection.

BACKGROUND: Acute human parvovirus B19 (B19) infection induces systemic lupus erythematosus (SLE)-like symptoms. It has been controversial whether B19 infection causes SLE and rheumatoid arthritis (RA). OBJECTIVES: This study prospectively investigated whether symptoms of B19 infection persisted for more than 2 years and whether persistent symptoms contributed to the development of SLE and RA. METHODS: In this prospective study, clinical findings were examined and laboratory examinations were performed in 42 adult patients 1, 2, 6, 12 and 24 months after the first consultation. RESULTS: Most acute symptoms disappeared within 2 weeks. However, arthralgia continued for 2 months in 2 women, 6 months in 1 woman and more than 2 years in 1 woman with positive antinuclear antibody and hypocomplementemia. The clinical findings of the patient with persistent arthralgia did not fulfill the criteria for SLE and RA. CONCLUSION: The symptoms caused by B19 infection were transient in most cases but persisted in 1 case in our series.

Adolescent↗

PubMed1994

Effect of 15-deoxyspergualin on accelerated rejection in rat heart transplantation.

The effect of 15-deoxyspergualin (DSG) on accelerated rejection was evaluated using a rat heart transplantation model. Lewis rats (LEW, RT1l) served as the organ recipient and Brown Norway rats (BN, RT1n) as the donor. In the accelerated rejection model, the LEW recipient was sensitized with BN skin and BN heart was transplanted 7 days later; the heart graft was rejected within 2 days (n = 7). Histologically, the graft showed coagulation necrosis and hemorrhage throughout the myocardium. DSG (2.5 mg/kg/day) was administered to the recipient under the following three protocols: group 1: during the sensitization period (7 days); group 2: from 3 days after the sensitization to 2 days after grafting (7 days), and group 3: immediately after heart transplantation. The mean graft survival period in groups 1, 2, and 3 was 4.3 +/- 0.8 days (n = 7, p < 0.01 vs untreated host), 11.7 +/- 2.1 days (n = 7, p < 0.001, vs. untreated host), and 2.0 +/- 0 days (n = 6), respectively. The rejected grafts in groups 1 and 3 histologically showed coagulation necrosis and hemorrhage. By contrast, in group 2, the major histological change was interstitial lymphocyte infiltration and there were few findings such as coagulation necrosis or hemorrhage. In the complement-dependent cytotoxicity test, serum obtained at the second posttransplant day from the recipients treated in group 1 showed a high cytotoxicity level, although the cytotoxicity level of serum obtained from the recipients treated in group 2 was consistently low.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Antiphospholipid antibodies reactive with bromelain-treated mouse erythrocytes in mice, rats and rabbits.

It has been reported that normal mice have a large number of B cells against bromelain-treated mouse erythrocytes (BrMRBC) and the anti-BrMRBC antibodies are reactive with liposomes of phosphatidylcholine and a phospholipid moiety of low-density lipoproteins from chicken egg yolk (EyLDL). In this study, we detected anti-BrMRBC hemolytic antibodies in the normal sera of rats and rabbits as well as mice. The antibodies in all the three species of animals bound to the columns of EyLDL. The EyLDL-binding antibodies could react with both liposomes of phosphatidylcholine and BrMRBC. The immunization with EyLDL evoked a vast production of anti-BrMRBC antibodies in rats and rabbits as well as mice. The anti-BrMRBC antibodies in the immunized animals could also react with liposomes of phosphatidylcholine. These findings suggest that rats and rabbits have the equivalent of mouse anti-BrMRBC B cells.

Animals↗

PubMed1989

Transfected cDNA directs expression of hemolytically active murine C4 in cultured mouse and monkey cells.

Previously cloned and sequenced full-length cDNAs for murine C4 and the closely related sex-limited protein (Slp) have been placed into an eucaryotic expression vector. Transfer of these DNA constructs transiently into monkey COS cells or stably into mouse L cells results in the expression and secretion of hemolytically active mouse C4 and mature Slp. We estimate from hemolytic activities that COS and L cells secrete 0.04 and 3%, respectively, of the C4 level found in mouse plasma. Slp expression is consistently only 10-20% that of C4 although the identical expression system is used for both. Our results show subtle but reproducible cell-type-specific differences in C4 maturation; they also indicate that surprisingly large shifts in electrophoretic mobility on SDS-polyacrylamide gels are induced by a small number of amino acid substitutions. The expression of C4 from cDNAs of known sequence provides a starting point for studies of structure/function relationships in C4 employing site-specific mutagenesis and gene transfer.

Animals↗

PubMed2004

Enzymatic modification of low-density lipoprotein in the arterial wall: a new role for plasmin and matrix metalloproteinases in atherogenesis.

OBJECTIVE: Functionally interactive proteases of the plasminogen/plasmin and the matrix metalloproteinase (MMP) system degrade and reorganize the extracellular matrix of the vessel wall in atherosclerosis. Here we investigated whether such proteases are able to confer atherogenic properties onto low density lipoprotein by nonoxidative modification. METHODS AND RESULTS: Similar to the recently described enzymatically-modified low-density lipoprotein (E-LDL), native LDL exposed to plasmin or matrix MMP-2 or MMP-9 and cholesterylester-hydrolase (CEH) showed extensive deesterification, with ratios of free cholesterol to total cholesterol rising to 0.8 compared with 0.2 in native LDL. When the ratio exceeded 0.6, both plasmin/CEH-LDL and MMP/CEH-LDL fused into larger particles. In parallel, they gained C-reactive protein-dependent complement-activating capacity. E-LDL produced with any protease/CEH combination was efficiently taken up by human macrophages, whereby marked induction of MMP-2 expression by E-LDL was observed. These in vitro findings had their in vivo correlates: urokinase-type plasminogen activator, MMP-2, and MMP-9 were detectable in both early and advanced human atherosclerotic lesions in colocalization with E-LDL. CONCLUSIONS: Plasmin and MMP-2/MMP-9 may not only be involved in remodeling of the extracellular matrix in progressing plaques, but they may also be involved in lipoprotein modification during genesis and progression of atherosclerotic lesions.

Adolescent↗

PubMed1992

A new apheresis system using a copolymer (polyvinyl alcohol triethylene glycol diacrylate) for removal of LDL from dog whole blood.

Studies were undertaken to determine the effectiveness of a copolymer composed of PVA-TEGDA (Poly Vinyl Alcohol TriEthylene Glycol) as a plasma-cholesterol lowering procedure. For a comparative study, five dogs underwent plasmapheresis including the transfusion bag containing gel in the plasma line, while three control dogs underwent the same plasmapheresis without gel. Numerous biological determinations were performed firstly in whole blood circulation before and after treatment over 10 days, and secondly in plasma before and after LDL binding on the gel. In the whole blood circulation, the average significant depletion of cholesterol levels was 31-51% for treated, 0-16% for control dogs and the average significant depletion of LDL cholesterol was 26-75% for treated and 0-3% for control dogs. Gel was therefore able to bind 121-217 mg of total cholesterol and 34-70 mg of LDL cholesterol per gram of gel. Lipid and lipoprotein levels rebounded 3-4 days after treatment. Adverse effects were not observed during all plasmapheresis. No significant differences between control plasmapheresis and gel-plasmapheresis were obtained for blood cell counts while lengthened coagulation times were observed during 24 h. Complement was not significantly activated by the copolymer as shown by a same decreased activity in the blood stream of all dogs: in fact, CH 50 depletion in the gel incubated plasma was due to a protein adsorption on the hydrogel. This new approach for LDL apheresis appears to be a promising new technique.

Acrylic Resins↗

PubMed1991

Lipopolysaccharide-induced pulmonary vascular sequestration of polymorphonuclear leukocytes is complement independent.

Lipopolysaccharide (LPS) injected intravenously produces leukopenia and sequestration of polymorphonuclear leukocytes (PMN) in the pulmonary vascular bed. To evaluate the role of complement in this process, we used C5-sufficient (B10.D2/nSn) and C5-deficient (B10.D2/oSn) mice and Sprague-Dawley rats depleted of complement with Naja naja cobra venom factor (CVF). We found a comparable increase in the number of PMN in lung tissue of C5-sufficient and C5-deficient mice given Escherichia coli LPS (0127:B8, 3 mg/kg), revealing that LPS acts independently of C5 and its biologically active fragments. Intravenous injection of LPS (3 mg/kg) into rats caused significant intravascular complement activation as assessed by serum CH50 and resulted in an almost 10-fold increase in numbers of PMN in lung tissue. Pretreatment of rats with CVF (50 U) did not reduce LPS-induced PMN sequestration, suggesting that the process is independent of C3. As reported previously, we found large numbers of PMN in bronchoalveolar lavage samples of 24 h after injection of LPS (3 mg/kg). Complement depletion did not prevent LPS-induced migration of PMN. No PMN migration occurred 2, 6, 12, 24, or 48 h after injection of CVF alone, indicating that complement activation is not sufficient to cause PMN migration. In contrast to our findings in rats, no PMN migrated into airspaces of C5-sufficient and C5-deficient mice 24 or 48 h after injection of LPS (3 to 20 mg/kg).

Animals↗

PubMed2001

A family with complement factor D deficiency.

A complement factor D deficiency was found in a young woman who had experienced a serious Neisseria meningitidis infection, in a deceased family member with a history of meningitis, and in three relatives without a history of serious infections. The patient and these three relatives showed a normal activity of the classical complement pathway, but a very low activity of the alternative complement pathway and a very low capacity to opsonize Escherichia coli and N. meningitidis (isolated from the patient) for phagocytosis by normal human neutrophils. The alternative pathway-dependent hemolytic activity and the opsonizing capacity of these sera were restored by addition of purified factor D. The family had a high degree of consanguinity, and several other family members exhibited decreased levels of factor D. The gene encoding factor D was found to contain a point mutation that changed the TCG codon for serine 42 into a TAG stop codon. This mutation was found in both alleles of the five completely factor D-deficient family members and in one allele of 21 other members of the same family who had decreased or low-normal factor D levels in their serum. The gene sequence of the signal peptide of human factor D was also identified. Our report is the first, to our knowledge, to document a Factor D gene mutation. The mode of inheritance of factor D deficiency is autosomal recessive, in accordance with the localization of the Factor D gene on chromosome 19. Increased susceptibility for infections in individuals with a partial factor D deficiency is unlikely.

Adult↗

PubMed1994

Suicide in patients with systemic lupus erythematosus: a clinical analysis of seven suicidal patients.

Despite many suicidal cases in patients with systemic lupus erythematosus (SLE), literature on this subject is lacking. To elucidate and prevent this phenomenon, we re-evaluated the clinical records of seven suicidal patients with SLE. Six patients had photosensitivity and insomnia. At the time of the suicide attempt, hypocomplementemia was observed in five of six patients. Diffuse slowing on electroencephalograms were observed in four of five patients. One patient successfully committed suicide while on no therapy while five patients made their attempts under the tapering courses of steroids. Five patients manifested psychoses whereas two patients displayed no psychotic findings. All patients attempted suicide shortly after admission (mean time 20 days). The subsequent courses of the survivors who received more medication were favorable. Therefore, it appears that disease activity was not fully controlled in these patients. Furthermore, signs of an imminent suicide attempt were missed in some cases. Psychosis, insomnia, history of photosensitivity, an incompletely controlled disease state, receiving tapering steroid dose, diffuse slowings on electroencephalograms and the presence of hypocomplementemia appeared to be risks for attempting suicide in SLE. We would recommend that such patients be under psychiatric care for at least 2 months to prevent suicide. When the patient is still psychotic or unstable, further medical care will be required.

Adolescent↗

PubMed2003

Association of the porcine C3 gene with haemolytic complement activity in the pig.

The complement component C3 plays an essential role in the activated complement system, which is involved in phagocytosis, inflammation and immunoregulation to destroy infectious microorganisms. The C3 molecule has more implications in the general defence mechanisms. In this study, the porcine C3 cDNA sequences including 5'- and 3'- flanking regions were determined and the polymorphisms in this gene were identified to carry out an association analysis between C3 and complement activity traits. Porcine C3 gene has high homology with human C3. Five single nucleotide polymorphisms (SNPs) and one microsatellite were detected in the porcine C3 gene. Haemolytic complement activity of alternative and classical pathways (ACH, CCP) was measured in 416 F2 animals of a crossbred of Duroc x Berlin Miniature Pig, which were immunized with Mycoplasma, Aujeszky and PRRS vaccines. C3 markers were found to be significantly associated (P <0.05) with both ACP and CCP. Animals with the more frequent haplotype present in Duroc and other commercial breeds exhibit higher ACP and CCP levels than the animals with haplotype specific to some Berlin Miniature Pigs. The association of C3 with complement activity reinforces the importance of C3 as a candidate gene for natural resistance to microorganisms.

Animals↗

PubMed1998

Phase I trial of a human-mouse chimeric anti-disialoganglioside monoclonal antibody ch14.18 in patients with refractory neuroblastoma and osteosarcoma.

PURPOSE: To evaluate the toxicity, immunogenicity, and pharmacokinetics of a human-mouse chimeric monoclonal antibody (mAb) ch 14.18 directed against disialoganglioside (GD2) and to obtain preliminary information on its clinical efficacy, we conducted a phase I trial in 10 patients with refractory neuroblastoma and one patient with osteosarcoma. PATIENTS AND METHODS: Eleven patients were entered onto this phase I trial. They received 20 courses of mAb ch 14.18 at dose levels of 10, 20, 50, 100, and 200 mg/m2. Dose escalation was performed in cohorts of three patients; intrapatient dose escalation was also permitted. RESULTS: The most prevalent toxicities were pain, tachycardia, hypertension, fever, and urticaria. Most of these toxicities were dose-dependent and rarely noted at dosages of 20 mg/m2 and less. Although the maximum-tolerated dose was not reached in this study, clinical responses were observed. These included one partial (PR) and four mixed responses (MRs) and one stable disease (SD) among 10 assessable patients. Biologic activity of ch 14.18 in vivo was shown by binding of ch 14.18 to tumor cells and complement-dependent cytotoxicity of posttreatment sera against tumor target cells. An anti-ch 14.18 immune response was detectable in seven of 10 patients studied. CONCLUSION: In summary, with the dose schedule used, ch 14.18 appears to be clinically safe and effective, and repeated mAb administration was not associated with increased toxicities. Further clinical trials of mAb ch 14.18 in patients with neuroblastoma are warranted.

Adult↗

PubMed1994

Preparation and antigen specificity of an anti-(1-->3)-beta-D-glucan antibody.

Antibody for (1-->6)-branched (1-->3)-beta-D-glucan was prepared using rodents. An antitumor (1-->6)-beta-monoglucosyl branched (1-->3)-beta-D-glucan (GRN: grifolan) was conjugated with bovine serum albumin and used as an immunogen. The antibody titer in serum was determined by ELISA using biotin-conjugated GRN. Administration of the antigen raised the antibody titer only in the rabbit, with mouse and rat showing no significant antibody titer for the glucan. The antigen specificity of the anti-GRN antibody was determined by competitive ELISA. The rabbit anti-GRN antibody bound to structurally related antitumor (1-->6)-branched (1-->3)-beta-D-glucans such as lentinan, schizophyllan and SSG, whereas it did not react with linear (1-->3)-beta-D-glucan, curdlan or GRN-derivatives obtained by periodate-oxidation and Smith degradation. These facts strongly suggest that the hapten site of the antibody was the monoglucosyl branched moiety of (1-->3)-beta-D-glucan. These results indicate that this antibody would be a useful probe for the detection of (1-->6)-branched antitumor glucans administered to the host.

Adjuvants, Immunologic↗

PubMed1989

[Pharmacological study on citrus fruits. I. Anti-allergic effect of fruit of Citrus unshiu Markovich (1)].

A study was carried out to examine the effect of 50% ethanolic extract from green fruit of Citrus unshiu Markovich on type I, II and IV allergic reactions. Forty eight-hour homologous passive cutaneous anaphylaxis (PCA) in rats as a typical model of the type I reaction was significantly inhibited by the oral administration of the extract. Complement-dependent cytolysis of sheep red blood cell as a typical model of the type II reaction was significantly inhibited by incubation with it. Contact dermatitis in mice as a model of the type IV reaction caused by picryl chloride or sheep red blood cell was significantly inhibited by the oral application of it. The anti-allergic actions decreased with the growth of fruit of Citrus unshiu. These results suggested that the 50% ethanolic extract from green fruit of Citrus unshiu have anti-allergic actions against the type I, II and IV reactions.

Animals↗

PubMed1994

Acetoside, a component of Stachys sieboldii MIQ, may be a promising antinephritic agent: effect of acteoside on crescentic-type anti-GBM nephritis in rats.

Effects of acetoside (ACT) on crescentic-type anti-GBM nephritis in rats were investigated. When rats were treated with ACT from the 1st day after i.v. injection of anti-GBM serum, ACT inhibited the elevation of protein excretion into urine. In the ACT-treated rats, cholesterol and creatinine contents and antibody production against rabbit gamma-globulin in the plasmas were lower than those of the nephritic control rats. Histological observation demonstrated that this agent suppressed hypercellularity and the incidence of crescent formation, adhesion of capillary wall to Bowman's capsule and fibrinoid necrosis in the glomeruli. Furthermore, rat-IgG and C3 deposits on the GBM were significantly less in the ACT-treated group than in the control nephritic group. When the treatment was started from the 20th day after i.v. injection of anti-GBM serum, by which the disease had been established, ACT resulted in a similar effect on the nephritic rats as stated above. These results suggest that ACT may be a useful medicine against rapidly progressive glomerulonephritis, which is characterized by severe glomerular lesions with diffuse crescents.

Analysis of Variance↗

PubMed2001

Vaccine-induced antibody responses as parameters of the influence of endogenous and environmental factors.

In laboratory animals, an adequate way to assess effects of environmental exposures on the immune system is to study effects on antigen-specific immune responses, such as after sensitization to T-cell-dependent antigens. This probably also applies to testing effects in the human population. It has thus been suggested that antibody responses to vaccination might be useful in this context. Vaccination responses may be influenced by a variety of factors other than environmental ones. One factor is the vaccine itself; a second is the vaccination procedure used. In addition, the intrinsic capacity of the recipient to respond to a vaccine, which is determined by sex, genetic factors, and age, is important. Psychological stress, nutrition, and (infectious) diseases are also likely to have an impact. We reviewed the literature on vaccine response. With regard to exogenous factors, there is good evidence that smoking, diet, psychological stress, and certain infectious diseases affect vaccination titers, although it is difficult to determine to what extent. Genetic factors render certain individuals nonresponsive to vaccination. In general, in epidemiologic studies of adverse effects of exposure to agents in the environment in which vaccination titers are used, these additional factors need to be taken into consideration. Provided that these factors are corrected for, a study that shows an association of exposure to a given agent with diminished vaccination responses may indicate suboptimal function of the immune system and clinically relevant diminished immune response. It is quite unlikely that environmental exposures that affect responses to vaccination may in fact abrogate protection to the specific pathogen for which vaccination was performed. Only in those cases where individuals have a poor response to the vaccine may exogenous factors perhaps have a clinically significant influence on resistance to the specific pathogen. An exposure-associated inhibition of a vaccination response may, however, signify a decreased host resistance to pathogens against which no vaccination had been performed.

Aged↗