Biomedical subjects

Anders Hjerpe

Publications and source records attributed to Anders Hjerpe.

At least 19 recordsLinked to original sources

PubMed2006

Expression of p16(INK4a) in relation to histopathology and viral load of 'high-risk' HPV types in cervical neoplastic lesions.

A total of 91 cervical archival biopsy series were analysed for the presence and viral load of 'high-risk' types of human papillomavirus (HR-HPV), and p16(INK4a) expression. The women had various degrees of CIN (cervical intraepithelial neoplasia). HPV 16 was the most prevalent type found, at 47% frequency. The frequency of HPV 16 increased with increasing immunoreactivity to p16(INK4a), from 39% to 44% at cases scored low to medium, to 65% at high reactivity. Thirty (33%) of the samples had negative p16(INK4a) analysis results, but were positive for HR-HPV. There was no significant correlation between viral load and the level of p16(INK4a) expression, while the grade of CIN correlated to such expressions. Thus, p16(INK4a) expression analysis yielded information which is consistent with results from the histopathology and might complement the HPV analysis in a clinical prognostic procedure in order to find women at risk for cervical cancer.

Adult↗

PubMed2006

Serglycin constitutively secreted by myeloma plasma cells is a potent inhibitor of bone mineralization in vitro.

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n=2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.

Calcification, Physiologic↗

PubMed2006

Cartilage aggrecan undergoes significant compositional and structural alterations during laryngeal cancer.

Aggrecan is a key component of cartilage and is responsible for the integrity and function of the tissue. In this study, the content of aggrecan and its structural modifications in adjacent to cancer apparently normal cartilages (AANCs) from various stages of laryngeal squamous cell carcinoma (LSCC) were investigated. Our data demonstrated a stage-related loss of aggregable aggrecan in AANCs, compared to the healthy laryngeal cartilage (HLC), which was excessive in advanced stages of disease. On aggregable aggrecan level, AANCs were characterized by significant compositional and structural modifications, the extent of which was closely related with the stage of LSCC. Four concrete subpopulations of aggregable molecules with particular physicochemical characteristics were identified with a strong tendency to prevail subpopulations of molecules of lower hydrodynamic sizes with increasing LSCC stage. These findings demonstrated that the cleavage of aggregable aggrecan occurred in concrete peptide bonds within the CS-1 and CS-2 attachment domains. These significant alterations were closely associated with the process of cartilage destruction, indicating the crucial role of aggrecan during LSCC.

Adult↗

PubMed2006

Proteasome inhibitor PSI induces apoptosis in human mesothelioma cells.

Malignant mesothelioma is an increasingly common tumor with an almost 100% mortality rate. It is refractory to conventional treatment. We have previously shown with SSH and microarray that the mRNA expression level of proteasome is higher in epithelioid mesothelioma cell lines than in sarcomatoid ones. This study evaluates the differential apoptotic effect of proteasome inhibitors on both of these mesothelioma sub-lines. Proteasome inhibitors show substantial anti-tumor activity in some tumor cells in vitro and in vivo, but the effects on mesothelioma cells has not been studied. The viability of mesothelioma cells was reduced in a dose- and time-dependent manner by the proteasome inhibitors tested; PSI was effective with a low dose, but higher concentrations were needed for calpain inhibitor I. The epithelioid mesothelioma cells are more sensitive to the inhibitors than the sarcomatoid ones, their IC50 after 24 h of treatment with PSI being 4 and 16 microm, respectively. Other mesothelioma cell lines show similar sensitivity. PSI seemed to decrease mesothelioma viability by inducing apoptosis, as verified by cell morphology, Western blotting analysis of caspase 3 cleavage, and flow-cytometric analysis. In conclusion, PSI, a representative agent that reduces viability and induces apoptosis of mesothelioma cells, might be useful in the treatment of patients with mesothelioma, especially of epithelioid phenotype.

Apoptosis↗

PubMed2006

Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.

Detection of E6/E7 mRNA expression with real-time nucleic acid sequence-based amplification assay (NASBA) method (PreTect HPV-Proofer) from high-risk types of human papillomaviruses (HR-HPV) were compared with the presence of viral load, determined with quantitative real-time PCR in 80 cervical samples. Results regarding positivity and typing were in agreement using the two methods. However, there was no correlation between viral loads for HPV 16 or 18/45 and oncogene expression. Among 15 women with low grade atypia detected at a population-based cytology screening, and scored as 'within normal limits' according to histopathology, 14% were positive for oncogene expression, whereas 71% were HR-HPV positive. A correlation was observed between HR-HPV oncogene expression and high scores of p16(INK4a) positivity. Since HPV-Proofer detects full-length E6/E7 mRNA, a positive result should correlate with presence of integrated HPV, loss of HPV replication and stabilized E6/E7 full-length mRNA expression. Such expression from integrated HR-HPV generates a high and stable expression of full-length E6 proteins, which explains why a positive HPV-Proofer result was independent of viral load and correlate with high expression of p16(INK4a). Thus, E6/E7 oncogene expression analysis yielded information, which is consistent with and will complement the results from a real-time PCR method in a clinical prognostic procedure.

Adult↗

PubMed2005

The combination of CEA, EMA, and BerEp4 and hyaluronan analysis specifically identifies 79% of all histologically verified mesotheliomas causing an effusion.

A previously tested antibody panel identified three criteria of major importance for distinguishing between mesothelioma and adenocarcinoma (ACA): carcinoembryonic antigen (CEA), BerEp4, and epithelial membrane antigen (EMA) accentuated at the cell membrane. An extended panel, consisting of CEA, BerEp4, EMA, vimentin, mesothelioma antibody (HBME-1), thrombomodulin, Ca125, and sialyl-Tn was applied to effusions from 86 ACAs and 21 mesotheliomas. The specificities and sensitivities of the previously identified reactivity patterns were tested on the new material and the effect of the added antibodies was evaluated. Further, hyaluronan analysis was added as a parameter. The previously selected criteria remained fully predictive for mesothelioma and ACA, respectively, also in the extended material (in all, 139 ACAs and 57 mesotheliomas). With the addition of the hyaluronan value, 79% of the cases was identified with 100% specificity. Among the new antibodies sialyl-Tn seemed the most promising because it specifically identified ACAs not expressing CEA.

Adenocarcinoma↗

PubMed2005

Molecular characterization of tumour heterogeneity and malignant mesothelioma cell differentiation by gene profiling.

Malignant mesothelioma is an aggressive tumour, characterized by a variable differentiation pattern and poor prognosis. At present, the clinical outcome in patients with malignant mesothelioma is mainly predicted by the morphological phenotype of the tumour. However, this conventional clinicopathological parameter is of limited value, partly because of the biological heterogeneity of this tumour and poor understanding of the regulatory mechanisms underlying the various patterns of growth. To elucidate the intrinsic molecular programmes that determine tumour differentiation, oligonucleotide arrays were used in an in vitro model of mesothelioma differentiation. The analysis of 2059 genes detected 102 genes that were significantly deregulated. Clustering of these genes into functional categories showed distinctive patterns for the two phenotypes, namely epithelioid and sarcomatoid. The molecular fingerprint of the sarcomatoid tumour component indicates overrepresentation of growth factor receptors and growth factor binding proteins, whereas epithelioid mesothelioma cells express other tumour-promoting factors involved in differentiation, metabolism, and regulation of apoptosis. These differences in the molecular phenotype may give a better basis for diagnosis and for designing novel therapies.

Blotting, Western↗

PubMed2004

A sandwich ELISA for the estimation of human syndecan-2 and syndecan-4 in biological samples.

Syndecans are integral membrane proteoglycans containing both chondroitin and heparan sulphate side chains. Syndecans are shed from the cell surface, and may be detected in cellular supernatant or body fluids. In this study, we wished to design ELISA methods for syndecan-2 and syndecan-4, with the future aim of analyzing syndecans in large number of patient materials. Briefly, sandwich ELISAs for human syndecan-2 and syndecan-4 were designed employing monoclonal mouse antibodies as capture and commercially available polyclonal rabbit anti-syndecan as secondary antibodies. Although no quantified standard was available, relative estimation of syndecan-2 and syndecan-4 levels was possible in a small pilot material. The paper presents the ELISA method for these proteoglycans. We suggest that this may be a useful tool in the analysis of patient materials and in the detection of syndecans during proteoglycan purification.

Animals↗

PubMed2003

Mesothelial differentiation as reflected by differential gene expression.

Human mesothelial cells obtained from benign effusions retain their proliferative capacity and grow uniformly either with a fibroblastic or epithelioid morphology in vitro. These cultures therefore provide a model for the process of mesothelial differentiation in vivo. To study this differentiation, we isolated differentially expressed genes obtained by suppression subtractive hybridization. Of the nine genes found to be overexpressed in fibroblastic mesothelial cells, three are matrix-associated (integrin alpha5, collagen binding protein 2, human cartilage glycoprotein 39), whereas the others are associated with a proliferative cell type (14-3-3 epsilon, plexin B2, N33, and three genes encoding ribosomal elements). Seven of the eight genes upregulated in the epithelioid phenotype are related rather to specialized functions, such as metabolism (aldose reductase, lecithin:cholesterol acyltransferase, ATPase 6), cytoskeletal composition (cytokeratins 7 and 8), and regulation of differentiation (granulin, annexin II). Immunohistochemistry with available antibodies to six of the differentially expressed gene products confirmed the differences also in pleural tissues, where submesothelial cells displayed the fibroblastic markers, whereas surface cells displayed the epithelioid markers. In summary, this approach revealed a pattern of genes coordinately regulated during mesothelial differentiation and suggests that mesothelium may regenerate also by recruiting cells from the submesothelial layer. Some of the gene products may also be useful markers for differentiation and activation in serosal tissues.

14-3-3 Proteins↗

PubMed2003

Proteoglycans and WT1 as markers for distinguishing adenocarcinoma, epithelioid mesothelioma, and benign mesothelium.

Malignant mesothelioma is a tumour frequently accompanied by an effusion with elevated hyaluronan levels. To distinguish malignant mesothelioma, adenocarcinoma, and reactive benign mesothelium with cytological and histological methods including immunocytochemistry is a major diagnostic challenge. The Wilms' tumour susceptibility gene 1 (WT1), expressed during transition of mesenchyme to epithelial tissues, is regarded as a marker for the mesothelial lineage. Its effect on the cell phenotype may be regulated via the syndecans, i.e. proteoglycans on the cell surface. To determine how WT1, proteoglycans, and hyaluronan synthase are expressed in mesothelioma, adenocarcinoma, and reactive benign mesothelium, we studied these molecules at the mRNA and protein levels, with the additional purpose of finding diagnostic parameters. Adenocarcinoma cells produced more mRNA for syndecan-1, but cells derived from mesothelium expressed WT1, biglycan, and larger amounts of syndecan-2. The difference in gene expression of these two syndecans was best monitored by the ratio between them. Syndecan-4 was highly expressed in all malignant cell lines, this expression being 1.7-5 times greater than in benign mesothelial cells. Although hyaluronan synthase-1 and versican could not distinguish between the three conditions, versican expression was associated with a high rate of proliferation. These findings suggest that syndecan-1 and syndecan-2 together may be useful diagnostic markers, with stronger staining for the latter epitope in mesothelial tissues. The alternating appearance of these two syndecans can be shown not only by RT-PCR and FACS in vitro, but also by immunohistochemistry on clinical material.

Adenocarcinoma↗

PubMed2003

Adenocarcinoma of the uterine cervix: the presence of human papillomavirus and the method of detection.

BACKGROUND: Effective screening programs have contributed to a decrease in the incidence of cervical squamous cell carcinomas but have had a limited sensitivity in the detection of adenocarcinoma precursor lesions. The aim of our study was to analyze cervical adenocarcinoma in greater detail: symptoms preceding the detection, the method of detection and the prevalence of human papillomavirus (HPV) with respect to age at diagnosis. MATERIAL AND METHODS: Clinical data were abstracted from the medical records of 82 women with pure invasive cervical adenocarcinomas. As diagnostic tools we used polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) and/or direct DNA sequencing for HPV detection. RESULTS: Age at diagnosis predicting factors were HPV status, positive lymph nodes, histology and stage. HPV-negativity, lymph node metastases, advanced stage and poor differentiation were all associated with a high diagnostic age. In the multivariate analysis only HPV status was shown to have an independent impact on age at diagnosis, while stage showed only borderline significance. Twenty-three percent of the cancers were detected by screening and the remaining were due to different symptoms. Among the women considered, 93% had a normal Papanicolaou (Pap) smear 3 years before diagnosis and 60% within 1 year. There was no significant correlation between smoking, oral contraceptives and HPV-positivity. CONCLUSIONS: The absence of HPV was significantly associated with a high age at diagnosis. Pap screening had a limited effect in detecting adenocarcinoma at an early stage.

Adenocarcinoma↗

PubMed2003

Distribution of chondroitin 4-sulfate epitopes (2/B/6) in various zones and compartments of articular cartilage in guinea pig osteoarthrosis.

We studied changes in the morphology of tibial articular cartilage in early guinea pig osteoarthrosis (OA) at 6 and 12 months of age with quantitative light microscopy, and the distribution of chondroitin-4-sulfate with quantitative ultrastructural immunolabeling, using the 2/B/6 epitope. Labeling was correlated to previous chromatography findings concerning proteoglycan (PG) concentration in animals of the same age. The cell volume fraction had decreased at 12 months in the superficial zone of cartilage with OA (medial condyle) as well as in cartilage without OA (lateral condyle), being lower medially than laterally. The PG concentration differed between the zones and matrix compartments. Medially, a reduction in PG concentration occurred between 6 and 12 months in the interterritorial compartment of the two uppermost zones. Laterally, the concentrations increased. In general, the pericellular PG concentration was higher than the interterritorial in the two uppermost zones. The striking variation in structural and labeling responses in the various zones and compartments indicate a heterogeneous tissue response in guinea pig OA that will probably affect sampling in biochemical analyses of cartilage homogenates and synovial fluid.

Animals↗

PubMed2003

The effect of body mass and physical activity on the development of guinea pig osteoarthrosis.

We quantitatively evaluated the morphological and biochemical effects of body mass and physical activity on spontaneously developing guinea pig osteoarthrosis (OA). 6-month-old male guinea pigs were allocated to 3 groups: controls (C) living under standard laboratory conditions with food ad libitum; mobilized animals (M) allowed unrestricted motion in large rooms with food ad libitum; and a diet group (D) weight-matched with the M-group. At 9- and 12-months of age they were killed and the left proximal tibia was processed for quantitative histology and the right tibial articular cartilage for analyses of glycosaminoglycan (GAG). OA mostly occurred on the medial condyle's central part not covered by the meniscus. The thinnest cartilage was found in 12-month-old M-animals (M12), which had 60% of the central cartilage surface affected by lesions that extended down to the mineralized cartilage. C12 had 25% exposed mineralized cartilage and D12, 2%. Subchondral bone density followed the loading patterns--the highest in M12 and lowest in D12. M12 had the lowest cartilage GAG concentrations. Load appears to be a key external factor in guinea pig OA. An increase in physical activity may be chondroprotective in the early phase, but harmful when fibrillations eventually have developed. This is underscored by the extensive OA changes in M12, although these animals weighed about the same as D12 (which had the least extensive OA). Therefore, a reduction in body mass seems to retard the progression of OA in animals, which are mainly subjected to a static load (C12 and D12), but not sufficiently in animals with a more dynamic load (M12). Changes in morphological patterns are paralleled by changes in GAG concentration, which probably reflect the metabolic capacity of the cartilage.

Analysis of Variance↗

PubMed2002

Immunoreactivity to cell surface syndecans in cytoplasm and nucleus: tubulin-dependent rearrangements.

Syndecans are transmembrane proteoglycans implicated in the regulation of cell growth and differentiation, by interacting with growth factors. Although syndecans play a major role in regulating cell morphology, little is known about their subcellular distribution and in vivo association with the cytoskeleton. To address this question, we investigated the subcellular distribution and dynamic rearrangement of syndecans-1, -2, and -4, using confocal laser microscopy. Furthermore, we monitored the spatial relation of syndecans to tubulin in both mitotic and interphase cells. Initially, the reactivity to syndecans was confined to the cytoplasm, staining of the cell membranes appearing later. Syndecan-1 also seems to translocate to the nucleus in a time-dependent manner. The mitotic spindle shows unexpectedly more syndecans than that found in interphase cells. After vinblastine treatment, both syndecan-1 and tubulin were recovered as paracrystalline occlusion bodies, and the nuclear reactivity to syndecan-1 disappeared, suggesting tubulin-mediated nuclear transport of this proteoglycan. Plasma membrane staining reappeared in the postmitotic cells. Nuclear translocation predominantly affected syndecan-1, whereas syndecan-2 and -4 remained in cytoplasm and cell membrane. This is the first report on regulated nuclear translocation and the presence of syndecan-1 in the mitotic spindle, where it may stabilize the mitotic machinery. The syndecan-1/tubulin complex may also act as a vehicle for the transport of protein growth factors to the cell nucleus.

Active Transport, Cell Nucleus↗

PubMed2002

Use of human papillomavirus DNA testing to compare equivocal cervical cytologic interpretations in the United States, Scandinavia, and the United Kingdom.

BACKGROUND: Human papillomavirus (HPV) DNA testing may be useful in clarifying equivocal cervical cytologic interpretations. One application might be to standardize the meaning of equivocal interpretations from laboratories in various regions. Because international differences may be particularly marked, international comparisons of emerging data will require clear translations of "equivocal" and similar terms. METHODS: To perform a three-country comparison, the authors selected a morphologically diverse set of 188 conventional Papanicolaou tests initially classified as "squamous atypia" from a study of more than 20,000 women in Portland, Oregon (1989-1990). Previously, five U.S. expert cytopathologists independently interpreted the slides with screening cytotechnologists' marks in place. For this comparison, one British and two Scandinavian reviewers involved in HPV research reviewed the slides after original marks had been removed. The authors compared all eight reviewers' classifications of negative, equivocal, or abnormal in a series of pairwise comparisons using the kappa statistic. They then compared cytologic interpretations with HPV DNA testing. RESULTS: Oncogenic HPV DNA detection was significantly associated with increasingly abnormal interpretations for each reader. The British reader tended to rate tests as more abnormal than the American pathologists did, whereas the Scandinavians tended to rate tests as more normal. Reference to the HPV DNA standard clarified the tendency of readers to render systematically more or less severe interpretations. For example, the Scandinavian cytologists discounted subtle (often HPV-associated) changes in favor of cytologic certainty, making HPV triage of equivocal tests less applicable there. CONCLUSIONS: International research on cytopathology, particularly on the possible uses of HPV DNA testing, will require calibration of local cytologic definitions.

Adolescent↗

PubMed2002

Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection.

In quest for high sensitivities, we developed an ultrahigh capillary electrophoresis (CE) method for the structural analysis of heparin and heparan sulfate (HS) in biologic samples. Heparin and HS were digested with an equi-unit mixture of heparin lyases I, II and III and the obtained Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone. All known twelve non-, mono-, di- and trisulfated Delta-disaccharides were completely resolved in a single run, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV. Relative standard deviation in migration times and peak areas as well as day-to-day variance ranged from 0.9 to 2.4%, suggesting a reproducible and precise method. Detection of 2-aminoacridone (AMAC)-derivatives of Delta-disaccharides by UV at 255 nm showed 2.8 and 10 times higher sensitivity than that of derivatized and non-derivatized ones at 232 nm. Laser-induced fluorescence detection with an Ar-ion laser source showed an approximately 100 times higher sensitivity than that obtained at 232 nm of the non-derivatized species. Application of this method to quantitative analysis of Delta-disaccharides derived from porcine intestinal mucosa heparin and bovine kidney HS showed excellent agreement with previously published methods, suggesting an accurate method. The developed method can be easily applied for the disaccharide analysis of heparin/HS at the attomole level with high accuracy, for distinguishing between heparin and HS and may be of value for studying their interactions with matrix effective molecules.

Aminoacridines↗

PubMed2002

Compositional and structural alterations of chondroitin and dermatan sulfates during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta.

Glycosaminoglycans participate in several biological functions in the arterial wall through their specific structures. They undergo specific compositional and structural modifications during the development of vascular diseases. The present study was performed to determine the variations in the concentration and the structural characteristics of galactosaminoglycans--chondroitin sulfate (CS) and dermatan sulfate (DS)--during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta. The concentration of CS was increased 24% (p < or = 0.05) in atherosclerotic type II aortas, but it was significantly decreased (29%, p < or = 0.05) in atherosclerotic type V aortas and aneurysmal aortas (65%, p < or = 0.01). In contrast, the concentration of DS was almost constant in all stages of arterial disease examined. Significant structural alterations were detected in the disaccharide composition of galactosaminoglycans. The ratio of 6-sulfated to 4-sulfated disaccharides was increased in atherosclerotic type II aortas (4.0 instead of 3.1 in normal aortas) due to the marked increase of CS in this tissue. This ratio was significantly decreased in atherosclerotic type V and aneurysmal aortas (2.1 and 1.6, respectively) due to the significant reduction of CS in the respective tissues. In addition, significant decrease of the oversulfated disaccharides, which are mainly located in DS chains, was recorded in atherosclerotic and aneurysmal aortas. Particularly, deltadi-di(2,6)S were decreased 32% (p < or = 0.01) and 86% (p < or = 0.01) in atherosclerotic type II and V aortas and 88% (p < or = 0.01) in aneurysm. Deltadi-di(2,4)S were increased in atherosclerotic type II aortas (21%, p < or = 0.01), but significantly decreased in atherosclerotic type V (33%, p < or = 0.01) and aneurysmal aortas (56%, p < or = 0.01). The amounts of deltadi-di(4,6)S were not markedly affected in the diseased tissues. These results suggest that the concentration of galactosaminoglycans is differentially affected during the progression of atherosclerosis. Furthermore, the development of vascular disease is associated with specific structural modifications, especially with the significant reduction of particular types of oversulfated disaccharides, which may play essential biological roles in the arterial wall.

Adult↗

PubMedDate not supplied

Presence of simian virus 40 (SV40) is not frequent in Swedish malignant mesotheliomas.

UNLABELLED: Simian virus 40 (SV40), a contaminant of polio vaccines used in the United States and Europe between 1955 and 1963, has been detected with high frequency in human malignant mesotheliomas (MM). In Sweden, from 1958, due to production in Javanese macaque kidney cells and SV40 testing from 1961, only polio vaccine claimed to be free of SV40 has been used. Hence, we aimed to screen Swedish MM patients for the presence of SV40. MATERIALS AND METHODS: Forty-one paraffin-embedded pleural MM samples, obtained from patients born from 1893-1958, were examined for amplifiable DNA. Testable samples were thereafter evaluated for the presence of SV40 DNA by two types of polymerase chain reactions (PCR) followed by sequencing. RESULTS: SV40 could be confirmed by sequencing in only 3 of the 30 MM samples containing DNA that could be amplified by PCR. CONCLUSION: The presence of SV40 is not frequent in Swedish MM patients.

Adult↗