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Efficacy of the inflammatory cell infiltration inhibitor IS-741 on colitis induced by dextran sulfate sodium in the rat.

We examined the therapeutic effects of the inflammatory cell infiltration inhibitor IS-741 (N-(2-((ethylsulfonyl)amino)-5-(trifluoromethyl)-3-pyridinyl)-cyclohexanecarboxamide monosodium salt monohydrate) on a rat colitis model. As a result of its effects on leukocyte infiltration, IS-741 inhibits cell adhesion, alleviates symptoms and signs of pancreatitis and multiple organ failure and demonstrates a life-saving effect in a model of severe acute pancreatitis. A rat model was prepared by inducing colitis with 3% dextran sodium sulfate (DSS) and maintaining pathology with 1% DSS. Repeated oral administration of IS-741 at 1, 10 or 100 mg/kg per day was conducted for 2 weeks (during treatment with 1% DSS). IS-741 at each dose decreased the area of erosion in the large intestine, thickening of the wall of the large intestine and anemia caused by melena. Some effects of IS-741 were nearly equivalent to those of the control compound salazosulfapyridine. Furthermore, IS-741 markedly alleviated inflammatory cell infiltration into the intestinal wall. IS-741 improved lesions in a rat DSS model by inhibiting leukocyte infiltration, suggesting the possibility of clinical application of this drug for IBD.

Administration, Oral↗

Immunohistological relation between DR antigen expression on melanoma cells and infiltration by CD8+ T cells.

Previous studies have suggested that class II major histocompatibility (MHC) antigen expression on melanoma cells may influence immune responses against melanoma and the nature of lymphocytic infiltrates against the tumor. This question was examined further by immunoperoxidase studies on sections from 29 primary and 30 metastatic melanoma with monoclonal antibodies (MAbs) against different lymphocyte subsets. The results indicated that expression of MHC class II DR* antigens on melanoma cells was associated with increased overall lymphocytic infiltration and that this applied particularly to the CD8+ subset of T cells. The CR3 receptor (CD 11b) was expressed predominantly on T cells and not macrophages but infiltration by CD11b+ cells did not correlate strongly with DR expression on melanoma cells. Dual staining with MAbs to CD8 and CD11b revealed that, whereas most of the CD8+ cells in DR- primary melanoma and DR+ metastatic melanoma were CD11b+, only approximately 50% of the CD8+ cells in DR+ primary melanoma were also CD11b+. Expression of CD11b on T cells was reported to define a suppressor subset of T cells. If the latter is correct the present results suggest that DR expression on primary melanoma is associated with infiltration by the cytotoxic T cell subset, whereas in the absence of DR antigens and in metastases this subset is absent and the predominant subset appears to be CD8+ CD11b+ T cells with suppressor activity. The biologic and prognostic significance of these findings remains to be determined.

Antibodies, Monoclonal↗

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

Animals↗

Estrogen supplementation failed to attenuate biochemical indices of neutrophil infiltration or damage in rat skeletal muscles following ischemia.

This study examined the effects of estrogen supplementation on markers of neutrophil infiltration and damage in skeletal muscle of rats following ischemia. Male and female gonad-intact rats, with or without 14 days of estrogen supplementation were subjected to two hours of hind-limb ischemia and sacrificed at 24, 48 or 72 hours post-ischemia. Control animals were sacrificed without ischemia. Plantaris and red and white gastrocneimus muscles were removed and assayed for myeloperoxidase (MPO), a marker of neutrophil infiltration, and glucose-6-phosphate dehydrogenase (G6PD) and beta-glucuronidase (betaGLU), as markers of muscle damage. Significant elevations of MPO, G6PD and betaGLU activities were observed at various time points post-ischemia. No systematic differences between genders were noted in any of the measures. Estrogen supplementation in both male and female animals failed to significantly attenuate post-ischemia increases in MPO, G6PD and betaGLU activities in any of the muscles studied and in some cases accentuated activities of some of these measures. Unlike previous findings following exercise in skeletal muscle, this study failed to demonstrate estrogen-induced attenuation of indices of neutrophil infiltration or damage in skeletal muscles of rats up to 72 hours following ischemia. This demonstrates that estrogen may not consistently attenuate neutrophil infiltration and that a number of variables including damage modality, tissue or estrogen level may influence this.

Animals↗

Increased apoptosis in infiltrating mononuclear cells of colorectal cancer: a mechanism for tumor escape.

CONTEXT: Disturbance in apoptosis has been proposed as one of the mechanisms involved in the immune response targeting tumor outgrowth. How colorectal cancer cells escape from attack by the immune system is not yet fully understood. OBJECTIVE: To investigate apoptotic molecules associated with colorectal cancer counterattack. DESIGN AND SETTING: Tissue samples of colon from 12 patients with colorectal cancer were collected and analyzed by immunostaining. In addition to tumorous tissues, corresponding nontumorous specimens of colon were obtained as controls. MAIN OUTCOME MEASURES: We examined the expression of Bcl-2, Bcl-xl, Bax, caspase-3, and inducible nitric oxide synthase in infiltrating mononuclear cells of colorectal cancer tissues and also in colorectal cancer tissues. The TUNEL assay was used to detect in situ apoptosis. RESULTS: Apoptosis was barely detectable in specimens of colorectal cancer, which was consistent with an increase in Bcl-2 level and a decrease in caspase-3 level. In contrast, infiltrating mononuclear cells of tumorous tissues showed a marked increase in apoptosis compared with those of nontumorous tissues. The increased apoptosis might have resulted from an imbalance of antiapoptotic and proapoptotic molecules, as reflected by reduction of Bcl-2 level and elevation of Bax level. The elevated caspase-3 levels found in this study could be a downstream effector of the Bcl-2 and Bax apoptotic pathways. A significant increase in inducible nitric oxide synthase observed in the infiltrating mononuclear cells might contribute to immunosuppression seen in colorectal cancer. CONCLUSION: It is tempting to speculate that aberrant expression of apoptotic molecules and inducible nitric oxide synthase in infiltrating mononuclear cells provides the underlying mechanism through which colorectal cancer cells escape attack by the immune system and subsequently grow without control.

Apoptosis↗

Tumor infiltration by adoptively transferred T cells is independent of immunologic specificity but requires down-regulation of L-selectin expression.

Adoptive immunotherapy with anti-CD3/IL-2 activated tumor-draining lymph node (LN) T cells is capable of eradicating tumor established at various histological sites. Tumor-specific effector lymphocytes have recently been identified to be LN T cells with down-regulated L-selectin (L-selectin-). Using fluorochrome labeling, the present study determined the early trafficking pattern of systemically transferred cells. In mice with 10-day established pulmonary 3-methylcholanthrene (MCA) 205 metastases, accumulation of cells in tumors was evident as early as 2 h after i.v. cell transfer, and, by 24 h, >50-fold higher numbers of cells were seen in metastases than in normal tissues. Similarly, transferred cells selectively infiltrated s.c. tumors, albeit at a lower rate. Analysis of the transferred cells isolated from recipient mice revealed that tumor-infiltrating cells were mostly L-selectin- (>95%). By contrast, only 24% and 58% L-selectin- cells were found in the LN and spleen, respectively. The ability of L-selectin- cells to accumulate at tumor sites was confirmed by the transfer of purified cell populations. Despite this selective tumor infiltration, the trafficking pattern did not reflect antigenetic specificity, and tumor regression occurred only after the transfer of tumor-specific effector cells. These results, thus, suggest that there are two distinct mechanisms operative in successful adoptive immunotherapy. Early infiltration of tumors by transferred cells is dictated by the physiological properties of cells and is independent on their immunologic specificity. Tumor regression, however, requires immunologically specific interactions at the site of tumor.

Adoptive Transfer↗

Recent physiopathological insights in cutaneous lymphocytic infiltrates.

The spectrum of benign cutaneous lymphocytic infiltrates (CLI) includes a variety of entities which are classified on the basis of clinicopathological findings, and of the phenotype of the predominant lymphocytic subset among the skin infiltrate. Major concern has been recelntly given to the clonality status of CLI by using highly sensitive assays based on the PCR amplification of TCR/Ig loci. These studies allowed the characterisation of clonal benighn cutaneous lymphocytic expansions. Other studies have shown evidence of oligoclonal patterns in HIV-associated CD8 cutaneous pseudolymphomas, and functionnal studies of the skin infiltrate further showed that an antigen-driven mechanism was involved in the pathogenesis of this latter entity. Finally, the knowledge in the field of CLI has been improved by the identification of antigens associated with skin-homing properties such as the so-called. Cutaneous Lymphocyte-associated Antigen (CLA) whicis expressed at the surface of most memory T cells infiltrating the dermis in inflammatory conditions.

Humans↗

Increase of tumour infiltrating lymphocytes in mice treated with antimetastatic doses of NAMI-A.

NAMI-A is a novel antitumour agent, based on ruthenium, which has proved effectiveness against lung metastases of solid mouse tumours. The study focuses on the effects of NAMI-A on leukocyte infiltration into the primary tumour of MCa mammary carcinoma, implanted subcutaneously (s.c.) or intramuscularly (i.m.) into CBA mice. NAMI-A, given with a cycle of daily treatments for six consecutive days on advanced tumours at 35 mg/kg/day, markedly reduces lung metastasis independently of the tumour type (Lewis lung carcinoma, MCa mammary carcinoma or TS/A adenocarcinoma) being treated and of the site of tumour implantation (s.c. or i.m.). The analysis of leukocyte infiltration of the primary tumour, performed on a single cell suspension of cells isolated from a Ficoll gradient on which a raw suspension of primary tumour cells was layered, showed NAMI-A to significantly increase tumour infiltrating lymphocytes. These lymphocytes are almost all CD3+ cells with a significant increase of the CD8+ over the CD4+ subpopulation that reduces the helper/suppressor ratio from 2.8 to 2.1. These data indicated the absence of toxicity of NAMI-A for tumour infiltrating lymphocytes and suggested that this compound might even synergize in combined treatments with cancer immunotherapy.

Adenocarcinoma↗

Analysis of T-cell receptor alpha/beta variability in lymphocytes infiltrating a melanoma metastasis.

Multiple experimental and clinical studies have suggested that the immune system may, to some extent, control the development of melanomas. The presence of tumor-infiltrating lymphocytes could reflect an in situ immune reaction directed to the malignant cells. The characterization of T-cell receptor (TCR) expressed by tumor-infiltrating lymphocytes is one way to precisely analyze these local T-cell responses. In this study, we have assessed the TCR alpha/beta variability in tumor-infiltrating lymphocytes from a subcutaneous metastasis of a melanoma patient. Using the anchored-polymerase chain reaction 268 TCR alpha and 266 TCR beta chain transcripts have been cloned and sequenced. Their analysis shows that the T-cell infiltrate is extremely diverse, with no preferential TCR gene segment usage.

Adult↗

A radiopharmaceutical for imaging areas of lymphocytic infiltration: 123I-interleukin-2. Labelling procedure and animal studies.

The labelling of interleukin-2 (IL-2) with 123I and its in vivo application for imaging chronic pathological lymphocytic infiltrations are described. The lactoperoxidase/glucoseoxidase technique was the labelling method of choice leading to immunoreactive IL-2 with high specific activity. Labelled IL-2 was injected in diabetes-prone non-obese diabetic (NOD) mice with pancreatic lymphocytic infiltration. As control animals, Balb/c mice were used. As specificity control, monoclonal antibodies AMT13 and UCHT1, bovine serum albumin and alpha-lactalbumin were radioiodinated and injected in mice. Eighteen NOD mice and four control Balb/c mice were used for gamma camera imaging experiments. Fifty-four NOD and 20 Balb/c mice were used for time course single organ counting and autoradiography. Gamma camera images showed that radioactivity accumulated in the pancreatic region from the 10th minute onwards in NOD mice injected with 123I-IL-2 but not in Balb/c mice, or in NOD mice injected with control radiopharmaceuticals. These findings were confirmed by counting the radioactivity present in single organs. Autoradiography of NOD pancreas, after injection of labelled IL-2, showed that radioactivity was specifically associated with infiltrating lymphocytes. In conclusion, this technique is highly specific and easy to perform and we suggest its application in humans for in vivo detection of areas of lymphocytic infiltration.

Animals↗

Immune cell infiltration of intrinsic and metastatic intracranial tumours.

BACKGROUND: Immune escape is one prerequisite for the formation of neoplasms that is reflected by the pattern of immune cell infiltration. Abundant monocytic infiltration without apparent phagocytic activity is well known in human gliomas, while other types of human intracranial tumours have not yet been investigated. MATERIALS AND METHODS: We analysed LCA-positive lymphocytes and CD68-positive macrophages/microglia by immunohistochemistry in 67 intracranial neoplasms: 18 glioblastomas (GBM), 14 primitive neuroectodermal tumours and medulloblastomas (PNET), metastases of 9 adenocarcinomas and of 8 malignant melanomas, and 18 benign meningiomas. RESULTS: Levels of monocytic infiltration in GBM and adenocarcinomas were higher than in PNET and meningiomas. Lymphocytes were rare in all tested tumours. No differences were found between all malignant neoplasms and benign meningiomas and between primary intracranial and metastatic tumours. CONCLUSION: Malignancy or primary intracranial origin seem not to be major determinants of immune cell infiltration. Different patterns of cytokine production may explain the differences in single tumour entities.

Adenocarcinoma↗

[Changes in the polymorphonuclear leukocyte infiltration, hemorrhage and plasma exudation in the pulmonary tissue in rats with smoke inhalation injury].

OBJECTIVE: To investigate the different velocity of polymorphonuclear neutrophil (PMN) infiltration, erythrocyte diapedesis and plasma exudation into the pulmonary tissue of the rats inflicted with smoke inhalation injury, so as to explore the different mechanisms of their existence in rat pulmonary tissue after inhalation injury. METHODS: The rat smoke inhalation injury model was employed in the study. Wistar rats were inflicted with smoke inhalation injury, and then sacrificed at 1, 3, 6, 12 and 24 post injury hours (PIH). 131I-BSA, 99mTc-PMN or 99mTc-erythrocytes (RBC) were injected into rat pulmonary tissue 1 hour before sacrifice. Isotonic saline was infused into blood vessel to wash out circulation blood. Then the pulmonary tissue samples were harvested for gamma-value counting and then weighed. The infiltration of 131I-BSA, 99mTc-PMN or 99mTc-RBC in pulmonary tissue per gram and per minute was calculated, and MPO content was measured by phosphate T-tolidine method. RESULTS: The amount of RBC diapedesis in rat lung tissue peaked at 1 PIH, decreased thereafter and approached to normal level at 24 PIH. The amount of PMN infiltration increased at 3 PIH, slightly decreased at 6 PIH but still higher than that in normal tissue, and increased again at 24 PIH. The pulmonary tissue content of MPO gradually increased from 1 PIH to 24 PIH. The pulmonary tissue content of 131I-BSA began to increase at 1 PIH and peaked at 6 PIH, and remained higher than that in normal tissue till 24 PIH. CONCLUSION: Even though there was remarkable postburn increase in the erythrocyte diapedesis, neutrophil infiltration and albumin exudation with different peak time points (1, 3 and 6 PIH, respectively), Inflammation seemed not to be the premise of erythrocyte diapedesis, while the secondary inflammatory reaction might be the main cause of pulmonary edema.

Animals↗

Epstein-Barr virus LMP1 status in relation to apoptosis, p53 expression and leucocyte infiltration in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection. EBV-encoded LMP1, expressed in most of NPC, has been suggested to have an important role in the pathogenesis and development of NPC and its expression correlates with poor prognosis. MATERIALS AND METHODS: Eighty-seven NPC biopsies were analyzed by immunohistochemistry for expression of markers of cell proliferation, apoptosis, infiltrating T lymphocytes and macrophages in relation to the LMP1 status. RESULTS: Our findings indicate that the p53 accumulation in NPC was significantly correlated to LMP1 and MMP9 overexpression in NPC cells. The frequency of apoptotic cells in NPC, as analyzed by TUNEL labeling, correlated to Fas-L and caspase-3 expression, and inversely to LMP1, p53 and MMP 9 expression. CD8+ T cell infiltration was predominately seen in nests of cancer cells with a high level of EBV-LMP1 expression, but these CD8+ T cells showed low expression of CD25 and TIA-1, indicating that they were not activated. CONCLUSION: Our observation suggests that the heavy infiltration by lymphocytes in LMP1-positive NPC tumors does not appear to counteract tumor growth by cytoxicity as indicated by the low apoptotic index. Thus, LMP1 seems to enhance survival- and proliferation-related signals in NPC. In analogy with other tumors, both the infiltrating T cells and the accumulated p53 may be inactive.

Adolescent↗

[Effect of Panax notoginseng saponins on intercellular adhesion molecule-1 expression and neutrophil infiltration in cerebral infarction tissue of rats].

OBJECTIVE: To investigate the effect of Panax notoginseng saponins (Pns) on the expression of intercellular adhesion molecule-1 (ICAM-1) and brain neutrophil infiltration induced by transient focal cerberal ischemia and reperfusion in rats. METHODS: Focal cerebral ischemia-reperfusion model in rats (male) were made by transient occlusion of the middle cerebral artery (MCA) for 2 h followed by 24 h reperfusion. Pns 25 and 50 mg/kg were administered ip at 2 h after reperfusion. At 24 h after reperfusion, neutrophil infiltration and the expression of intercellular adhesion molecule-1 (ICAM-1) were measured with myeloperoxidase (MPO) activity, and immunohistochemistry, respectively. RESULTS: After ischemia reperfusion, the expression of ICAM-1 and MPO activity in the ischemic hemisphere cortex of MCA area and caudate putamen were markedly increased. Treatment with 50 mg/kg Pns inhibited neutrophil infiltration and the expression of ICAM-1. CONCLUSION: Pns inhibited neutrophil infiltration and the expression of ICAM-1 following focal cerebral ischemia-reperfusion in rats, attenuated the ischemic brain inflammation.

Animals↗

Postoperative adjuvant immunochemotherapy and infiltration of dendritic cells for patients with advanced gastric cancer.

To determine the group of patients for whom a biological response modifier is useful, the efficacy of postoperative adjuvant immunotherapy was investigated with regard to infiltration of dendritic cells (DC) in patients with advanced gastric cancer who underwent absolute curative resection. The infiltration of DC was classified into marked and slight. The chemotherapy group was prescribed mitomycin C and tegafur, while for the immunochemotherapy group a biological response modifier, PSK, was also prescribed in survival between the chemotherapy and immunochemotherapy groups. In patients with slight infiltration, however, the 5 - year survival rates were 0% in the chemotherapy and 15% in the immunochemotherapy group. Patients given PSK survived longer than those given chemotherapy only (P less than 0.05). Therefore, adjuvant immunotherapy may be considered for patients with advanced gastric cancer with slight infiltration of DC.

Adjuvants, Immunologic↗

DNA ploidy, cyclin D1, bcl-2 and lymphocytic infiltration of the tumor microenvironment as prognostic factors in laryngeal cancer patients.

The aim of this study was to evaluate correlations between DNA ploidy type, the immunostaining of cyclin D1, bcl-2 and the intensity of lymphocytic infiltration of the tumor microenvironment in relation to the histopathological G differentiation and pTNM classification. Thirty two patients were treated surgically for laryngeal cancer with total or partial laryngectomy in the Department of Otolaryngology Zabrze. The percentage of bcl-2 immunostaining was showed in 53% of the cases and was found to correlate with G differentiation and the patients' age. Cyclin D1 antigen stained positively in 24 cases (75%). Expression of cyclin D1 correlated with cancer stage. Cyclin D1 negative stain was found in T4-stage. DNA ploidy was examined in 19 cases. Aneuploidy was found in 5 cases only, while the rest were diploid. DNA ploidy value correlated with cyclin D1 expression. All paraffin sections were found to contain lymphocytic infiltrations of CD43 and CD45RO phenotype in the tumor front. Some cases showed high intensity of lymphocytic infiltrations of CD45RO phenotype. The intensity of CD43 lymphocytic infiltrations in the tumor front was related to the expression of cyclin D1 and bcl-2.

Adult↗

[Study on the extent of peritumoral edema in glioma: its correlation with the proliferative potential and tumor-infiltrating mononuclear cells].

The correlation between the extent of peritumoral edema and the proliferative potential or the infiltration of mononuclear cells was studied in 17 gliomas. The peritumoral edema was evaluated on contrast enhanced CT scan as the ratio of the low density area around the tumor to the enhanced high density area. The proliferative potential and the infiltration of mononuclear cells into the tumor were investigated immunohistochemically using monoclonal antibody (MAb) against DNA polymerase alpha and anti-Leu MAb's respectively. There was a significant correlation between the extent of the peritumoral edema and the percentage of DNA polymerase alpha positive cells. The degree of the infiltration of mononuclear cells into the tumor tissue also correlated with the extent of peritumoral edema. In gliomas with high proliferative potential and/or severe infiltration of mononuclear cells, the peritumoral edema may be aggravated by disruption of the blood-brain-barrier and increased vascular permeability.

Brain Edema↗

[Isolation and expansion of glioma-infiltrating lymphocytes in vitro: an analysis of their surface phenotypes and antitumor activities].

The present study was conducted in order to examine the feasibility of isolating and growing glioma-infiltrating lymphocytes in vitro as possible effector cells for use in an adoptive immunotherapy. Thirty surgical specimens obtained from patients with malignant astrocytomas were studied. The glioma-infiltrating lymphocytes were separated from tumor tissue, expanded in the presence of interleukin-2, and evaluated their anti-tumor activities in vitro. Eighteen of 30 cultures of glioma-derived lymphocytes expanded with a substantial increase in cell numbers, of at least 5 x 10(8) cells up to 5 x 10(9), 4 to 8 weeks after the initiation of culture. The expanding glioma-derived lymphocytes consisted of 88 +/- 10% CD3+ T cells including both CD4+ and CD8+ subpopulations. CD16 was expressed on 4 +/- 5% of the cells and three cultures studied exhibited 14% +/- 1 of CD56+ cells. After 4 to 8 weeks of the proliferation period, the lymphocytes ceased to grow in all cultures. The glioma-derived effector lymphocytes could lyse almost all the autologous tumor targets as well as allogeneic glioma cells. The cytotoxic activity of the glioma-derived lymphocytes appeared to be similar or inferior to that of interleukin-2-activated peripheral blood lymphocytes obtained from the same patients in killing autologous glioma cells. The glioma-derived effector cells could also lyse three-dimensional glioma targets (spheroids), but this lytis activity was clearly lower than that of the activated peripheral blood lymphocytes. Ability of these effector cells to infiltrate glioma tissue was doubtful, since after 24-hours coculture of effector cells with target spheroids, the effector cells scarcely infiltrated the spheroids. In summary, glioma-derived lymphocytes expanded in bulk culture with interleukin-2 consisted predominantly of T-lymphoblasts with the ability to kill autologous glioma cells and also to lyse glioma spheroids. However a benefit of use of the glioma-derived lymphocyte as a novel effector cells in clinical trail replacing the IL-2-activated peripheral blood lymphocytes could not be found.

Adult↗