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Biomedical subjects

Claudia Lange

Publications and source records attributed to Claudia Lange.

At least 19 recordsLinked to original sources

Sensitive balance of suppressing and activating effects of mesenchymal stem cells on T-cell proliferation.

The human leukocyte antigen-independent immune-modulatory potential of human mesenchymal stem cells (hMSC) makes them a promising candidate for clinical cell therapy. A better understanding of their "immune-privileged" status is therefore of high priority. Here we used Ki-67-antigen staining to estimate T-cell alloreactivity in mixed lymphocyte cultures in the presence of hMSC as second or third party. We found that the allostimulatory activity of mesenchymal stem cells (MSC) leading to an increased T-cell proliferation and interleukin-2 secretion is measurable only at low MSC/effector ratios (< or =0.1:1). Moreover, this stimulating effect could be efficiently suppressed by MSC-conditioned medium. This suggests that the "immune-privileged" status of MSC exists only when MSC-mediated downregulation of immune cell activation can overrule their own allostimulatory potential. Thus the "immune-privileged" state of MSC represents a sensitive balance of suppressing and activating effects, which should be considered in a clinical setting with limited cell amounts.

Humans↗

Abnormal size of the amygdala predicts impaired emotional memory in major depressive disorder.

BACKGROUND: Amygdala and hippocampus show significant structural abnormalities in major depressive disorder (MDD). Individuals with MDD have difficulties in emotional memory. A relationship between emotional memory deficits and structural abnormalities of amygdala and hippocampus in MDD has been proposed but not shown, yet. METHODS: The current study assessed memory for emotional faces in 21 young women with recent-onset MDD and 23 matched control subjects. All subjects underwent structural magnetic resonance imaging (3D-MRI) and a clinical and neuropsychological assessment. RESULTS: Depressive subjects had significantly enlarged amygdala size and significantly reduced hippocampal size compared with controls. Depressive subjects were significantly impaired in learning emotional facial expressions, with deficits being most pronounced for fearful, surprised and disgusted faces. Depressive subjects with amygdala volumes 1 SD or more above the mean of control subjects showed the strongest impairments. Correlation analyses revealed that larger left amygdala volumes were significantly related to worse memory performance and to higher anxiety scores of depressive subjects. Smaller left hippocampal volumes of depressive subjects were related to higher anxiety scores as well. LIMITATIONS: All MDD subjects were taking antidepressant medication at the time of the study. Longitudinal studies are needed to clarify whether the behavioral and/or volumetric abnormalities of MDD subjects can be attributed to medication or MDD or both. CONCLUSIONS: It might be speculated that amygdala enlargement in young MDD subjects is correlated with amygdalar over-activation and resolves with antidepressant treatment, as was shown for amygdalar over-activation.

Adult↗

Bovine herpesvirus 4 based vector interaction with liver cells in vitro and in vivo.

Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Bovine herpesvirus 4 (BoHV-4) based vector capability to transduce rat liver cells in vitro and in vivo was assessed. For the in vitro study, a buffalo rat liver cell line was successfully transduced by BoHV-4 and although did not show toxicity, the immediate early two viral gene was transcribed and cells harboring the intact viral genome could be pharmacologically selected, but no viral replication took place. For the in vivo study, adult male rats were inoculated intraportally and intraparenchimally with a BoHV-4 expressing enhanced green fluorescent protein and liver sections were analyzed through fluorescent microscopy. Although the liver parenchyma could not be transduced, the endothelial layer of the liver vasculature showed a robust transgene expression without toxicity. Successful BoHV-4 based vector transduction of primary cultures of rat hepatocytes suggests that extrinsic factors, and not hepatocytes per se, are the cause of such lack of transducibility. The present study serves as a starting point for study of the use of BoHV-4 based vectors to target gene delivery to vascular endothelial cells.

Animals↗

Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells.

AIM: To investigate the hepatocytic differentiation of mesenchymal stem cells (MSCs) in co-cultures with fetal liver cells (FLC) and the possibility to expand differentiated hepatocytic cells. METHODS: MSCs were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor (SCF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor 4 (FGF-4) alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction (RT-PCR) for the liver specific markers cytokeratin-18 (CK-18), albumin, and alpha-fetoprotein (AFP) was performed in different cell populations. RESULTS: Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. CONCLUSION: The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also provides a beneficial environment for expansion and differentiation of FLC.

Albumins↗

Liver-specific gene expression in mesenchymal stem cells is induced by liver cells.

AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.

Animals↗

Reduced glucose metabolism in temporo-parietal cortices of women with borderline personality disorder.

Individuals with borderline personality disorder (BPD) and posttraumatic stress disorder (PTSD) often experience dissociative symptoms. Evidence is increasing that stress-related hyperglutamatergic states may contribute to dissociative symptoms and neurodegeneration in temporo-parietal cortical areas. Seventeen young women with BPD who had been exposed to severe childhood physical/sexual abuse and presented with pronounced dissociative symptoms underwent (18)fluoro-2-deoxyglucose positron emission tomography (FDG-PET). Nine healthy, matched volunteers served as comparison subjects. Borderline subjects displayed reduced FDG uptake (as analyzed by SPM) in the right temporal pole/anterior fusiform gyrus and in the left precuneus and posterior cingulate cortex. Impaired memory performance among borderline subjects was significantly correlated with metabolic activity in ventromedial and lateral temporal cortices. Our results demonstrate regional hypometabolism in temporal and medial parietal cortical regions known to be involved in episodic memory consolidation and retrieval. Currently, the precuneus/posterior cingulate cortex is modeled as part of a network of tonically active brain regions that continuously gather information about the world around and within us. Decreased resting metabolic rate of these regions may reflect dissociative symptoms and possibly also identity disturbances and interpersonal difficulties of individuals with BPD.

Adult↗

Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.

Severe acute renal failure (ARF) remains a common, largely treatment-resistant clinical problem with disturbingly high mortality rates. Therefore, we tested whether administration of multipotent mesenchymal stem cells (MSC) to anesthetized rats with ischemia-reperfusion-induced ARF (40-min bilateral renal pedicle clamping) could improve the outcome through amelioration of inflammatory, vascular, and apoptotic/necrotic manifestations of ischemic kidney injury. Accordingly, intracarotid administration of MSC (approximately 10(6)/animal) either immediately or 24 h after renal ischemia resulted in significantly improved renal function, higher proliferative and lower apoptotic indexes, as well as lower renal injury and unchanged leukocyte infiltration scores. Such renoprotection was not obtained with syngeneic fibroblasts. Using in vivo two-photon laser confocal microscopy, fluorescence-labeled MSC were detected early after injection in glomeruli, and low numbers attached at microvasculature sites. However, within 3 days of administration, none of the administered MSC had differentiated into a tubular or endothelial cell phenotype. At 24 h after injury, expression of proinflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma, and inducible nitric oxide synthase was significantly reduced and that of anti-inflammatory IL-10 and bFGF, TGF-alpha, and Bcl-2 was highly upregulated in treated kidneys. We conclude that the early, highly significant renoprotection obtained with MSC is of considerable therapeutic promise for the cell-based management of clinical ARF. The beneficial effects of MSC are primarily mediated via complex paracrine actions and not by their differentiation into target cells, which, as such, appears to be a more protracted response that may become important in late-stage organ repair.

Acute Kidney Injury↗

Reduced size and abnormal asymmetry of parietal cortex in women with borderline personality disorder.

BACKGROUND: Evidence is accumulating that suggests borderline personality disorder (BPD) and posttraumatic stress disorder (PTSD) are related to small hippocampal size. Psychotic symptoms are frequent in both disorders. Psychotic spectrum disorders are known to be related to abnormalities of temporoparietal cortices. METHODS: Using structural magnetic resonance imaging (3D-MRI), parietal cortex and hippocampal volumes were assessed in 30 young women with BPD who had been exposed to severe childhood sexual and physical abuse and in 25 healthy control subjects. RESULTS: Compared with control subjects, BPD subjects had significantly smaller right parietal cortex (-11%) and hippocampal (-17%) volumes. The parietal cortex of borderline subjects showed a significantly stronger leftward asymmetry when compared with control subjects. Stronger psychotic symptoms and schizoid personality traits in borderline subjects were significantly related to reduced leftward asymmetry. Stronger trauma-related clinical symptoms and neuropsychologic deficits were significantly related to smaller hippocampal size. CONCLUSIONS: Our results are consistent with previous findings of small hippocampal size in BPD and PTSD. Reduced right parietal cortex size in individuals with BPD may reflect a neurodevelopmental deficit of the right hemisphere.

Adult↗

Reduction of myocardial scar size after implantation of mesenchymal stem cells in rats: what is the mechanism?

The use of a cellular therapy offers a promising approach for the treatment of heart disease. Besides other precursor cells, bone marrow (BM)-derived stem cells were discovered that migrate into ischemic myocardium and participate in myogenesis as well as angiogenesis. A subpopulation of those are the mesenchymal stem cells (MSC), which may be potential candidates for repairing ischemic heart tissue. MSC are easy to prepare and can be used in an autologous strategy. Here we demonstrate the effect of transplanted MSC in our autologous rat model of myocardial injury. BM was isolated from tibiae and femurs of Wistar rats. After 24 h, the adhering MSC were separated, expanded, retrovirally transduced using green fluorescent protein (GFP), and cloned. A cryo-infarct was generated in the rat hearts, and immediately after this the cells were injected into the border zone of the lesion. After a 10-week follow up, the hearts were excised and the myocardial scar areas were measured using computer-guided morphometry. When comparing transplanted rats (n = 8) with control animals (n = 5) treated rats demonstrated a significant reduction in the width (p < 0.05) of the myocardial scar area. The depth of the scars of the cell therapy rats was less extended (p > 0.05) and the myocardium of these animals was thicker than in the controls (p > 0.05). Immunohistochemical analyses revealed neither evidence of MSC transdifferentiation into cardiomyocytes, nor could an increased neovascularization be found. In conclusion, MSC are responsible for a remarkable reduction of the myocardial scar size in the treated animals. But, whether this strategy is directly transferable to the patient suffering from heart disease has to be determined. In addition, the mechanism by which MSC act in the ischemic heart remains to be determined.

Animals↗

High-potential human mesenchymal stem cells.

Bone marrow-derived stromal mesenchymal stem cells (MSCs) have been characterized in vitro by their growth characteristics, the expression of a panel of surface antigens, and their potential to differentiate into mesenchymal lineages. They can be separated by physical methods as well as by immunological or chemical separation or cultivation. Different protocols are used in different laboratories, making the comparison of various reported MSC populations difficult. Here we describe a population of bone marrow-derived adult stem cells that has been separated on a Percoll gradient with low density. It is characterized by an extraordinary high proliferative potential and a conserved phenotype characteristic of MSCs that retain their plutipotentiality in culture, as evidenced by their ability to differentiate into osteo-, chondro-, and adipogenic lineages. Separation of these cells provide an effective and convenient method for rapid expansion of pluripotential human MSCs for clinical use where large amounts of stem cells are needed.

Adipocytes↗

Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats.

BACKGROUND: Adult stem cells are promising for the development of novel therapies in regenerative medicine. Acute renal failure (ARF) remains a frequent clinical complication, associated with an unacceptably high mortality rate, in large part due to the ineffectiveness of currently available therapies. The aim of this study was, therefore, to evaluate the therapeutic effectiveness of bone marrow-derived mesenchymal stem cells in a rat model of ischemia/reperfusion (I/R) ARF. METHODS: We used a common I/R model in rats to induce ARF by clamping both renal pedicles for 40 minutes. Mesenchymal stem cells were iron-dextran-labeled for in vivo tracking studies by magnetic resonance imaging (MRI) and kidneys were imaged for mesenchymal stem cells immediately after infusion and at day 3 after ARF. Renal injury was scored on day 3 and cells were additionally tracked by Prussian blue staining. RESULTS: We show in I/R-induced ARF in rats, modeling the most common form of clinical ARF, that infusion of mesenchymal stem cells enhances recovery of renal function. Mesenchymal stem cells were found to be located in the kidney cortex after injection, as demonstrated by MRI. Mesenchymal stem cells-treated animals had both significantly better renal function on days 2 and 3 and better injury scores at day 3 after ARF. Histologically, mesenchymal stem cells were predominantly located in glomerular capillaries, while tubules showed no iron labeling, indicating absent tubular transdifferentiation. CONCLUSION: We conclude that the highly renoprotective capacity of mesenchymal stem cells opens the possibility for a cell-based paradigm shift in the treatment of I/R ARF.

Acute Kidney Injury↗

Differential impairments of facial affect recognition in schizophrenia subtypes and major depression.

The goal of this study was to assess facial affect recognition abilities in subjects with various schizophrenia subtypes and subjects with major depression. A total of six disorganized, 21 paranoid and 18 residual subjects with schizophrenia (DSM-IV criteria) were compared with 21 subjects with major depression (DSM-IV criteria) and 30 matched healthy control subjects. Two experimental tasks requiring the sorting and rating of emotional facial expressions were applied. Disorganized and paranoid subjects showed strong impairments in the sorting of emotional facial expressions. Depressive subjects displayed only minor deficits, and residual subjects were unimpaired. Subjects with disorganized schizophrenia rated emotional facial expressions as more aroused, and depressive subjects rated them as less aroused, than the other study groups. Our study demonstrates strong deficits in facial affect recognition in subjects with schizophrenia and pronounced disorganized or psychotic symptoms. Deficits in facial affect recognition are specific to schizophrenia. They may be considered as a state marker of schizophrenia.

Adult↗

Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.

OBJECTIVE: Mesenchymal stem cells (MSC) are promising candidates for cell-based therapies. One major obstacle for their clinical use is the biosafety of fetal calf serum (FCS), which is a crucial part of all media currently used for the culture of MSC. METHODS: Nine donors each contributed 5 mL of bone marrow aspirate. Isolation of MSC was conducted according to Caplan et al., although for expansion we used low-density seeding with 20 MSC/cm2. Four different media A, B, C, and D were tested, containing 1%, 3%, or 10% autologous serum (AS), or 10% selected FCS, respectively. MSC were cultured on 24-well plates until passage 2 and counted under the microscope at regular intervals. Osteogenic and adipogenic differentiation were induced in vitro by using a modified standard cocktail and were evaluated semi-quantitatively through a microscope. RESULTS: Isolation of MSC after 3 days appeared best in media C with almost always C>D congruent with B>A. Proliferation was exponential with generally C>D>B>A. Morphologically, MSC isolated and expanded in medium C were indistinguishable from those in medium D. Phenotypic markers of MSC grown in medium C were: CD34-, CD45-, CD90+, CD105+, MHC class I+, MHC class II-, similar to MSC isolated and grown in medium D. Moreover, MSC grown in medium C showed more osteogenic potential than those from medium D in all cases: C+++, D++, B+, A 0. Cells retained their immaturity as shown by adipogenic differentiation and it always was: D+++, C++, B+, A 0. CONCLUSIONS: Growth of MSC in a FCS-free medium is feasible without addition of growth factors. Ten percent AS appears at least as good as 10% FCS with regard to both isolation and expansion of human MSC, while 1% and 3% AS appear inferior. With respect to osteogenic differentiation, 10% AS proved superior to the other serum conditions.

Adipocytes↗

Minor histocompatibility antigens on canine hemopoietic progenitor cells.

Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs.

Animals↗

Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose.

OBJECTIVE: Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods. MATERIALS AND METHODS: HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice. RESULTS: After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin(-)c-Kit(+)) were 15 to 85% (53.7%+/-21.7%, n=23) and 30 to 95% (69.8%+/-20.4%, n=17), respectively. Even after one round of infection, gene transfer was efficient (31.2%+/-15.1%, n=12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells. CONCLUSION: The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research.

Animals↗

Liver-specific gene expression in cultured human hematopoietic stem cells.

Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34(+) bone marrow cells to differentiate into hepatocytic cells in vitro. CD34(+) and CD34(-) human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34(+) cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34(+) cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34(-) cells did not show liver-specific gene expression. The results indicate that CD34(+) adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.

Adult↗

Interleukin-10 modulation of alloreactivity and graft-versus-host reactions.

BACKGROUND: The biological properties of interleukin (IL)-10 in tolerance induction and inhibition of alloreactivity have suggested a therapeutic use of this cytokine as an additional or alternative prophylaxis for graft-versus-host disease (GvHD). However, the effects of exogenous IL-10 on GvHD are mainly studied in animal models, and the results remain conflicting. This study aims to demonstrate, for the first time, whether the addition of exogenous IL-10 can reduce the severity of graft-versus-host reactions (GvHR) in humans. METHODS: The regulatory role of exogenous IL-10 in GvHR was investigated using an in vitro human skin explant model. The effects of IL-10 on skin GvHR were tested in parallel with allo-antigen induced T-cell proliferation, cytolytic reactivity, and cytokine production. RESULTS: In the presence of IL-10, the mixed lymphocyte reaction (MLR) primed responder cells showed significantly lower proliferative and cytolytic responses compared with the responder cells from the control MLR carried out in the absence of IL-10. The responder cells from IL-10 containing MLR induced significantly less severe skin GvHR and displayed a significantly reduced T-cell activation and cytokine production. A significant correlation was observed between the levels of TNF-alpha production and the sensitivity to IL-10 modulation of GvHR. CONCLUSIONS: The addition of exogenous IL-10 strongly inhibited the broad alloreactivity initiated by primary MLR and significantly reduced the overall severity of skin GvHR induced by MLR primed responder cells. Responder cells producing high TNF-alpha following allogeneic stimulation appeared to be less sensitive to IL-10 modulation of GvHR.

Cytokines↗

Neuropsychological performance in frontal lobe epilepsy.

The search for a special neuropsychological profile of frontal lobe epilepsy subjects (FLE) has so far led to inconclusive results. In this paper we compared the preoperative neuropsychological performance of FLE and temporal lobe epilepsy (TLE) subjects. We further investigated whether frontal lobe lesions of epileptogenic cause produce the same type of cognitive dysfunction as do tumours of the frontal lobe. Sixteen FLE subjects were compared to 16 TLE subjects as well as to a group of 10 subjects after the removal of frontal lobe tumors (TUM) and a healthy control group. A set of neuropsychological test measures routinely used for presurgical evaluation, an emotional conceptualization task and two associative learning tasks were administered. We found that subjects with frontal lobe damage were significantly impaired relative to controls on a wide range of cognitive functions independent of neurological cause. FLE subjects could hardly be discriminated from TLE subjects as both groups showed a similarly reduced level of neuropsychological performance. Our results demonstrate the devastating effect that frontal lobe epilepsy can have on cognitive functioning. Routinely used neuropsychological test measures lack the specificity to distinguish between frontal and temporal lobe epilepsy. Highly specialized measures are necessary to reveal differences.

Adult↗