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K Motoyoshi

Publications and source records attributed to K Motoyoshi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Expression of dystroglycan complex in satellite cells of dorsal root ganglia.

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan composes the dystroglycan complex together with the extracellular glycoprotein alpha-dystroglycan, which binds laminin-2 (alpha2/beta1/gamma1), a major component of the Schwann cell basal lamina. In the Schwann cell cytoplasm, beta-dystroglycan is anchored to a dystrophin isoform, Dp116. In this study, we investigated the expression of beta-dystroglycan, Dp116 and the laminin-alpha2 chain in satellite cells of rat dorsal root ganglia (DRGs). Immunohistochemical study showed that immunoreactivities for beta-dystroglycan and Dp116 were both localized to the outer rim of neuron-satellite cell and axon-Schwann cell units, indicating that both satellite and Schwann cells expressed these proteins in DRGs. Immunoreactivity for the laminin-alpha2 chain was detected in a similar location, indicating that the basal lamina surrounding satellite and Schwann cells in DRGs contained laminin-2. Ultrastructurally, immunoreactivity for the cytoplasmic domain of beta-dystroglycan as well as that for Dp116 was most intense in the cytoplasm just underlying the outer membrane of satellite cells. The immunoreactivity for laminin was associated with the outer surface of those cells, suggesting that it was localized in the surrounding basal lamina. These results indicate that the dystroglycan complex is expressed in the satellite cell outer membrane and involved in the adhesion with the basal lamina through the interaction with laminin-2.

Animals↗
PubMed2001

Expression of Jagged1 gene in macrophages and its regulation by hematopoietic growth factors.

OBJECTIVE: Serrate/Jagged and Delta are cell surface ligands for Notch receptors that may influence hematopoietic cell fate decisions and are known to be expressed in bone marrow stromal cells. In a series of screenings of cDNAs constructed by a cDNA library subtraction technique, we identified Jagged1, one of the Notch ligands, as a gene up-regulated by macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Therefore, we compared stromal cells and macrophages for expression of Notch ligands including Jagged1 and analyzed the regulation of their expression by cytokines. MATERIALS AND METHODS: Murine bone marrow macrophages were prepared by culturing femoral bone marrow cells with M-CSF. Primary bone marrow fibroblastic stromal cells were prepared by a culture system that we recently developed. The expression of Notch ligands was analyzed by either Northern blot analysis or reverse transcriptase polymerase chain reaction. RESULTS: The bone marrow macrophages expressed Jagged1 but not Jagged2 and Delta1 at a level that was detectable by Northern blot analysis. Expression of the Jagged1 gene was markedly up-regulated by growth factors for the cells, i.e., M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. Expression of Jagged2 and Delta1 seldom was affected by the stimuli. The primary bone marrow fibroblastic stromal cells, and murine stromal cell lines, such as PA6 and ST2, also expressed Jagged1 transcript, at levels comparable to the steady-state level in macrophages. However, expression of the Jagged1 gene was little affected when these cells were stimulated with fibroblastic growth factor and platelet-derived growth factor. CONCLUSIONS: We demonstrated that bone marrow macrophages as well as stromal cells constitutively produced Jagged1 and that the expression was markedly up-regulated by hematopoietic growth factors, M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. The results highlight the involvement of macrophages and these growth factors in hematopoietic cell fate decisions via the production of Jagged1.

Animals↗
PubMed2001

Somatosensory evoked potential recovery in myotonic dystrophy.

OBJECTIVE: To evaluate recovery functions of the sensory cortex using somatosensory evoked potentials (SEPs) elicited by paired stimuli of the median nerve in patients with myotonic dystrophy (MD). SUBJECTS/METHODS: Twelve MD patients were enrolled in the present investigation. Five patients with facioscapulohumeral muscular dystrophy (FSH) and 12 healthy volunteers were studied as control groups. SEP was recorded from the hand sensory area contralateral to the median nerve stimulated at the wrist. Single pulse or paired-pulse stimuli at various interstimulus intervals (ISIs) (10, 20, 40, 60, 80, 100, 150, 200 and 300 ms) were given. Recovery functions of N9, N20onset-N20peak, N20-P25 and P25-N33 components were studied. RESULTS: Conventional SEPs to a single stimulus were normal in the latency and amplitude in all the patients. Recovery functions of both N9 and N20o-N20p components were normal in the patients. In contrast, in MD patients, disinhibited or hyperexcitable recovery pattern was observed in recovery curves of the N20-P25 or P25-N33 components, whereas those were normal in FSH patients. CONCLUSIONS: Disinhibited cortical excitability (or hyperexcitability) is present in the sensory cortex in patients with myotonic dystrophy. This may reflect cortical pathology or functional alteration of the sensory cortex in MD.

Adult↗
PubMed2001

A new matrix metalloproteinase inhibitor SI-27 induces apoptosis in several human myeloid leukemia cell lines and enhances sensitivity to TNF alpha-induced apoptosis.

MMP inhibitors are used clinically for the stabilization of tumor growth, thus prolonging survival in cancer patients. However, their role in the treatment of hematopoietic malignancies remains unclear. In the present study, we investigated the effects of a new MMP inhibitor, SI-27, in hematopoietic malignancies. SI-27 alone induces apoptosis in several human myeloid leukemia cell lines such as U937, NB4, and HL60 cells by activating caspase 8, 9, and 3. Apoptosis was measured with annexin V positive staining, a drop in mitochondrial transmembrane potential (deltapsim), presence of hypodiploid DNA, and cleavage of PARP and IkappaBalpha. Furthermore, at lowered concentrations, which did not directly induce apoptosis, SI-27 acted to sensitize U937 cells and other cells to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. The accumulation of membrane Fas, the Fas ligand, and TNFR1 were not apparent due to exposure to SI-27, and antagonistic anti-Fas or anti-Fas ligand antibodies did not block SI-27-induced apoptosis. Thus, SI-27-induced apoptosis is not mediated by the Fas pathway. These results suggest that MMP inhibitors, alone or in combination with other cytotoxic agents, can provide a unique method for treating acute myeloid leukemia, refractory to classical anti-cancer drugs, and may thus suppress recurrence.

Apoptosis↗
PubMed2001

Burkitt's acute lymphoblastic leukaemia transformation after myelodysplastic syndrome.

We describe a patient with myelodysplastic syndrome (MDS) that transformed to Burkitt's acute lymphoblastic leukaemia (ALL). The leukaemic blasts were negative for peroxidase staining, and expressed CD10, CD19, CD22, CD38, human leucocyte antigen (HLA)-DR and surface immunoglobulin (sIg) M, but neither sIgD nor sIgG were expressed. Chromosomal study during the ALL phase showed t(8;22)(q24;q11) in addition to the karyotypes determined during the MDS phase. Furthermore, overexpression of c-myc mRNA was confirmed in ALL blasts. These findings indicate that MDS transformed to Burkitt's ALL through multiple cytogenetic evolutions, the final event of which seems to be overexpression of the c-myc gene.

Acute Disease↗
PubMed2001

Differential regulations between adenosine triphosphate (ATP)- and uridine triphosphate-induced Cl(-) secretion in bovine tracheal epithelium. Direct stimulation of P1-like receptor by ATP.

Adenosine 5'-triphosphate (ATP) stimulates airway epithelial Cl(-) secretion in a complicated manner. We examined the difference between ATP- and uridine 5'-triphosphate (UTP)-induced responses of short-circuit current (Isc) in bovine tracheal epithelium treated with amiloride. Each nucleotide caused an increase in Isc composed of the first and second peaks, where the second peak induced by ATP was higher compared with UTP. The ATP-induced second peak was inhibited by the protein kinase (PK) A inhibitor H89, saturation of P1 receptor with adenosine, and the P1 receptor antagonist 8-p-sulfophenyltheophylline, but not by the Ca(2+) chelator ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin, the adenosine breakdown enzyme adenosine deaminase, the ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate, or saturation of P2Y2 receptor with UTP. Thus, the response is associated with PKA-dependent pathway via P1-like receptor but not with Ca(2+)-dependent pathway via P2Y2 receptor, and ATP degradation products do not contribute to this response. Further, stimulation of cells with ATP increased PKA activity. In addition, pretreatment with glybenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator, reduced the second peak of Isc induced by ATP but was without effect on that induced by UTP. Therefore, ATP stimulates glybenclamide-sensitive Cl(-) secretion, and this action is partly mediated by PKA-dependent pathway via P1-like receptor.

Adenosine↗
PubMed2001

Hemophagocytic syndrome and adult Still's disease associated with meningoencephalitis and unconsciousness.

We describe a 19-year-old woman with hemophagocytic syndrome and adult Still's disease who showed rare features of central neurological involvement, including cerebellar symptoms and the sudden onset of unconsciousness with pleocytosis in the cerebrospinal fluid during the early course of the illness. As this patient's serum showed a high level of interferon-gamma and soluble interleukin 2 receptor, this might play a pathologic role in the development of central nervous system symptoms. Intensive treatment consisting of methylprednisolone pulse therapy followed by the oral administration of methylprednisolone and cyclosporine, as well as plasma exchange, was found to achieve good results.

Adult↗
PubMed2001

Nodular cutaneous amyloidosis and carpal tunnel syndrome due to the amyloidogenic transthyretin His 114 variant.

This is the second report of transthyretin (TTR) amyloidosis in a patient who had ATTR Tyr114His diagnosed by mass spectrometry and gene analysis. This case had some clinical features that differed from those of the first reported cases. The patient, 73-year-old man, complained of generalized cutaneous tubercula that had started at age 68. These tubercula gradually increased in size and became generalized. He felt a slight numbness in his extremities. Clinical and electrophysiological examinations revealed that he had bilateral carpal tunnel syndrome (CTS), whereas there was no clear evidence of sensory and/or motor polyneuropathy. Autonomic symptoms were not present. Biopsy studies revealed that both his tuberculum and his sural nerve contained TTR-related amyloid. In his sural nerve, amyloid deposits were observed mainly in the perineurium, not in the endoneurium, and there was no significant depletion of myelinated fibers. The features of this patient were clinically characterized by generalized cutaneous amyloid deposits and late-onset CTS with a lack of overt polyneuropathy and autonomic dysfunction. The unique clinical features in this case seemed to be consistent with the distribution of amyloid deposits.

Aged↗
PubMed2000

p56(dok-2) as a cytokine-inducible inhibitor of cell proliferation and signal transduction.

p56(dok-2) acts as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. However, the role of p56(dok-2) in biological functions of cells is not clear. We found that transcription of the p56(dok-2) gene in macrophages was increased markedly in response to cytokines such as macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF and interleukin-3 (IL-3). Forced expression of p56(dok-2) inhibited M-CSF-, granulocyte-CSF-, IL-3- and stem cell factor-induced proliferation of myeloid leukemia cells, M-NFS-60. The p56(dok-2)-overexpressing cells showed an impaired induction of c-myc but not of c-jun, junB or c-fos when stimulated with M-CSF. Consistent with these results, the peritoneal cavity of the hairless (hr/hr) strain of mutant mice, whose cells expressed less p56(dok-2) than wild-type mice, contained more macrophages than that of +/hr mice. Moreover, the inhibition of endogenous p56(dok-2) expression in macrophage-like tumor cells, J774A.1, by stable expression of antisense p56(dok-2) mRNA accelerated cell proliferation. The study identifies a novel role for p56(dok-2) as a molecule that negatively regulates signal transduction and cell proliferation mediated by cytokines in a feedback loop.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Plasma levels of the differentiation inhibitory factor nm23-H1 protein and their clinical implications in acute myelogenous leukemia.

A previous study reported that a nondifferentiating myeloid leukemia cell line produced differentiation-inhibiting factors. One of the factors was purified as a homologue of the nm23 genes. The nm23 genes were overexpressed in acute myelogenous leukemia (AML) cells, and a higher level of nm23 gene expression was correlated with a poor prognosis in AML. The present study determined the plasma levels of nm23-H1 protein by enzyme-linked immunosorbent assay and assessed the association between this level and the clinical outcome in 102 patients with AML. The plasma concentration of nm23-H1 was higher in patients with AML than in normal controls (P =.0001). Plasma nm23-H1 levels were correlated with the product of the intracellular nm23 messenger RNA (mRNA) level and the white blood cell count, but not with the mRNA level alone. Therefore, nm23-H1 plasma levels probably depend on the total mass of leukemic cells overexpressing the nm23-H1 gene. Overall survival was lower in patients with higher plasma nm23-H1 levels than in those with lower levels. Multivariate analysis using the Cox proportional hazard model showed that elevated plasma nm23-H1 levels significantly contributed to the prognosis of AML patients. Furthermore, the plasma nm23-H1 levels were investigated in 70 patients with other hematologic neoplasms, including 6 with acute lymphoblastic leukemia, 13 with chronic myelogenous leukemia, and 12 with myelodysplastic syndrome. Plasma nm23-H1 levels were significantly higher in all of these hematologic neoplasms than in normal controls. Increased plasma levels of nm23-H1 may have prognostic value in these hematologic malignancies as well as in AML.

Adult↗
PubMed2000

Association of Cbl with Fms and p85 in response to macrophage colony-stimulating factor.

Tyrosine phosphorylation of Cbl and its association with signal-transducing molecules in response to macrophage colony-stimulating factor (M-CSF) were analyzed by using cell lines which express the wild-type and a mutant M-CSF receptor, Fms. We found that in a clone, F723 TF-1 cells expressing mutant Fms in which tyrosine 723 had been substituted with phenylalanine, the M-CSF stimulation-dependent association between Cbl and Fms was markedly impaired. However, phosphorylation of Cbl and its association with the p85 subunit of phosphatidylinositol 3-kinase were induced in these mutant cells as seen in the wild-type fms transfectant. These results suggest that phosphorylation of tyrosine 723 is particularly important for the recruitment of Cbl to the M-CSF receptor, but is not required for the phosphorylation and binding of Cbl to signal-transducing molecules such as p85.

Amidohydrolases↗
PubMed2000

IL-4, but not vitamin D(3), induces monoblastic cell line UG3 to differentiate into multinucleated giant cells on osteoclast lineage.

The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)-4 and 1alpha,25(OH)(2) vitamin D(3) (D(3)) are known to induce MGC formation from monocytes/macrophages. D(3) is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL-4 as an inhibitor. Previously, we showed that IL-4-induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite-resorptive activity in the presence of M-CSF without stroma cells. In this study, we examined the effects of D(3) and/or IL-4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D(3)-induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal-R) expression, hydroxyapatite-resorptive activity, and bone-resorptive activity. A low concentration of D(3) synergistically stimulated IL-4-induced TRAP-positive MGC formation, whereas a high concentration of D(3) inhibited it. When IL-4 was added on day 7 of the 2-week culture with D(3), TRAP positivity reached maximum. On the other hand, delayed addition of D(3) on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2-week culture in the presence of D(3), it increased further in the second week following the addition of IL-4 on day 7. Furthermore, IL-4-induced, or IL-4- and D(3)-induced MGCs differentiated into functional osteoclasts with bone-resorptive activity following coculture with osteoblastic cells, whereas D(3)-induced MGCs did not acquire bone-resorptive activity even after coculture with osteoblastic cells in the presence of D(3). These findings suggest that IL-4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D(3) had only a "supportive" effect on this differentiation. IL-4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells.

3T3 Cells↗
PubMed2000

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells.

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.

Animals↗
PubMed2000

Expression of dystroglycan and laminin-2 in peripheral nerve under axonal degeneration and regeneration.

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan composes the dystroglycan complex, together with the extracellular glycoprotein alpha-dystroglycan which binds laminin-2, a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerve, the expression of beta-dystroglycan and laminin-alpha2 chain was studied in rat sciatic nerves undergoing axonal degeneration and regeneration as well as in normal condition. In normal sciatic nerve, immunoreactivity for the cytoplasmic domain of beta-dystroglycan was consistently and selectively localized in the Schwann cell cytoplasm underlying the outer (abaxonal) membrane apposing the basal lamina. While beta-dystroglycan expression was gradually down-regulated in Schwann cells losing contact with axons during axonal degeneration, it was progressively up-regulated as the regenerating process of ensheathment and myelination proceeded during regeneration. Interestingly, beta-dystroglycan expression, when detectable, was always restricted to the Schwann cell cytoplasm beneath the outer membrane apposing the basal lamina during both axonal degeneration and regeneration. Furthermore, laminin-alpha2 immunoreactivity roughly paralleled that of beta-dystroglycan during both axonal degeneration and regeneration, indicating that the expression of beta-dystroglycan and laminin-alpha2 is induced and maintained by the Schwann cell contact with axons. Our results indicate that the dystroglycan complex is involved in the adhesion of the Schwann cell outer membrane with the basal lamina and suggest that the dystroglycan complex may play a role in the process of Schwann cell ensheathment and myelination through the interaction with laminin-2.

Animals↗
PubMed2000

Effects of macrophage-colony-stimulating factor on cyclophosphamide-injected mouse NK1.1+ cell activity.

We injected cyclophosphamide into mice and examined their natural killer (NK) activity both in vitro and in vivo. Cyclophosphamide injection temporarily abrogated the lung clearance activity of Yac-1 lymphoma cells, which is considered to be an index of NK activity in vivo. However, administration of recombinant human macrophage-colony-stimulating-factor (rhM-CSF) to cyclophosphamide-injected mice restored the lung clearance activity. To clarify whether the administration of rhM-CSF activated NK cells, we purified NK1.1+ cells from mice treated with cyclophosphamide and/or rhM-CSF and examined their functions (cytotoxicity, proliferation, and interferon gamma production) in vitro. Cyclophosphamide injection decreased the number, but did not suppress the functions of NK1.1+ cells. The numbers of NK1.1+ cells in cyclophosphamide-injected mice restored by rhM-CSF administration. And the functions of NK1.1+ cells from both saline-injected and cyclophosphamide-injected mice were accelerated by rhM-CSF administration. These results suggested that the temporary abrogation of NK activity in vivo caused by cyclophosphamide injection was due to a decrease in the number and not to suppression of the functions of NK1.1+ cells. The injection of cyclophosphamide into mice increased the number of tumor (B16 melanoma) nodules formed in the lungs and liver. However, treatment with rhM-CSF recovered the anti-metastatic activity in the lungs of cyclophosphamide-injected mice. These results show that administration of rhM-CSF restores NK activity suppressed by cyclophosphamide injection in vivo.

Animals↗
PubMed2000

Effects of macrophage colony-stimulating factor and interleukin-2 administration on NK1.1(+) cells in mice.

We studied the effects of M-CSF and IL-2 on NK1.1(+) cell activity in vivo and in vitro. Administration of M-CSF increased the number of splenic NK1.1(+) cells (vs. saline: P<0.01). Moreover, the combination of M-CSF and IL-2 (M-CSF+IL-2) produced a synergistic expansion of the number of NK1.1(+) cells compared with each single treatment (vs. saline: P<0.001). The NK1.1(+) cells were isolated from the spleen of each treated mouse (four treatment groups: saline, IL-2 alone, M-CSF alone, M-CSF+IL-2) and their functions (IL-2-induced proliferation, IFN-gamma production and cytostatic activity) were evaluated in vitro. The NK1.1(+) cells from M-CSF alone and M-CSF+IL-2 treated mice showed greater responsiveness in terms of IL-2-induced proliferation, production of IFN-gamma and cytostatic activity than the cells from saline and IL-2 alone treated mice. The NK activity in vivo was enhanced by the administration of M-CSF and IL-2, as assessed by the 'Lung clearance assay' (clearance of Yac-1 cells in lung). And the M-CSF+IL-2 treatment induced the highest NK activity of the four treatments. To show a practical effect of upregulation of NK activity in vivo by M-CSF and IL-2 administration, the effect of the four treatments on an experimental tumor metastasis model was examined. The IL-2 alone, M-CSF alone and M-CSF+IL-2 treatment reduced the metastasis of B16 melanoma. And the M-CSF+IL-2 treatment proved of greater benefit to the antimetastatic activity than each single treatment. Our results demonstrated that the administration of M-CSF increases the number of NK1.1(+) cells, which have good responsiveness to IL-2. Furthermore, the combination treatment of M-CSF and IL-2 in vivo augments the increase of NK1.1(+) cells. And these effects can contribute to the antimetastatic activity in vivo.

Animals↗
PubMed2000

Competitive reverse transcription-polymerase chain reaction assay for quantification of human multidrug resistance 1 (MDR1) gene expression in fresh leukemic cells.

We have analyzed MDR1 gene expression in 69 clinical samples obtained from 64 patients with leukemic hematologic malignancies by using a competitive reverse transcription-polymerase chain reaction assay with a heterologous competitor RNA. To exclude a false-positive result caused by concomitant normal lymphocytes that physiologically express MDR1, in samples we determined a cut-off value of 8 amol MDR1 transcript per microgram of RNA by simultaneous measurement of rhodamine 123 dye efflux either in lymphocyte or gated leukemic cell populations. Consequently, 23 of 69 samples were concluded to be MDR1-positive in leukemic cells per se. The MDR1 expression rate was significantly correlated with factors such as a history of preceding chemotherapy, elder age of the patient, and certain disease types (eg, leukemia progressed from myelodysplastic syndrome). Moreover, the complete response rate after chemotherapy was significantly higher in MDR1-negative patients than in MDR1-positive patients (52% vs 17%, respectively; P = .01). The assay established will enable the quantification of MDR1 gene expression in blood samples from patients with leukemic hematologic malignancies and will be applicable to clinical laboratories as a routine test.

Adolescent↗