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R G Bretzel

Publications and source records attributed to R G Bretzel.

At least 73 records · Page 4Linked to original sources
PubMed1999

Cytokine mRNA expression in peripheral blood cells of immunosuppressed human islet transplant recipients.

The macrophage derived cytokines interleukin-beta (IL-1beta), and tumor necrosis factor alpha (TNFalpha), and the T-cell derived cytokine interferon gamma (IFNgamma) have been implicated to play an important role in early attack on islet cells during human islet transplantation (ITx). Therefore, the aim of this study was to investigate the influence of the current immunosuppressive induction therapy in clinical islet transplantation on mRNA expression of these cytokines in blood cells, compared to lipopolysaccharide (LPS) induced cytokine release in vitro and to plasma levels. The cytokine release correlated to lymphocyte counts and significantly decreased after ATG, and partially recovered 2 weeks after ITx. Unexpectedly, there was no correlation between mRNA expression for IL-1beta in total blood and the number of lymphocytes and monocytes remaining after anti thymocyte globulin (ATG)-therapy. Even when the blood was nearly totally depleted from mononuclear cells, high amounts of IL-1beta mRNA could be detected. However, IL-1beta secretion could not be stimulated in vitro. Our results show that application of ATG during ITx might contribute to graft survival during the early posttransplant period by suppression of the synthesis of monocyte derived cytokines IL-1beta and TNFalpha.

Antilymphocyte Serum↗
PubMed1999

Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll.

Endotoxin-induced early inflammatory reactions may inhibit the function and survival of isolated cells or cell aggregates after transplantation. By the chromogenic Limulus amebocyte lysate assay we found rather high but variable endotoxin concentrations in the chemicals used for islet isolation, i.e. collagenase and Ficoll. Liberase, a special collagenase preparation from Boehringer, was nearly endotoxin-free. Correlating to the endotoxin content, collagenase and Ficoll had the capacity to induce interleukin-1beta release from human peripheral blood mononuclear cells. Because collagenase and density gradient media are needed in most cell isolation procedures from solid organs, each lot of these chemicals should be tested for endotoxin contamination. In pancreatic islet transplantation, the use of endotoxin-free chemicals may diminish early local inflammatory reactions at the graft site and thereby reduce the number of islets needed for successful islet transplantation.

Collagenases↗
PubMed1999

Oxygen radical production in human mononuclear blood cells is not suppressed by drugs used in clinical islet transplantation.

Inflammatory islet damage mediated by cytokines and oxygen radicals may limit the success of clinical islet transplantation for treatment of insulin-dependent diabetes mellitus. In this study, we investigated whether drugs such as currently used in islet-transplanted patients inhibit the release of IL-1beta, TNFalpha, and superoxide from mononuclear blood cells in vitro. Methylprednisolone (10 microg/ml) inhibited the release of IL-1beta and TNFalpha, but had no effect on superoxide generation. Both pentoxifylline (66 microg/ml) and cyclosporin A (300 ng/ml) slightly inhibited TNFalpha release without affecting IL-1beta or superoxide generation. Nicotinamide (0.25 mM) did not interfere with the generation TNFalpha or superoxide and only slightly inhibited IL-1beta production. A combination of methylprednisolone, pentoxifylline, cyclosporin A, and nicotinamide (concentrations for each substance as described above) inhibited TNFalpha generation by 74+/-6% (mean value+/-SEM, mononuclear blood cells from seven diabetic patients) without affecting IL-1beta or superoxide generation. These data show that standard immunosuppressive therapy in islet transplanted patients may partially inhibit cytokine release but does not affect the generation of potentially islet-toxic superoxide from mononuclear cells.

Diabetes Mellitus, Type 1↗
PubMed1999

Endotoxin impairs the engraftment of rat islets transplanted beneath the kidney capsule of C57BL/6-mice.

The primary objective of this investigation was to determine the effect of endotoxin on islet xenograft survival within the first three days after transplantation. Pancreatic islets from Lewis rats were prepared under endotoxin-free conditions with Liberase (Boehringer) and purified by centrifugation on endotoxin-free Ficoll/Histopaque. After overnight incubation, with or without 10 microg/ml endotoxin, the islets were transplanted beneath the kidney capsule of normoglycemic C57Bl/6-mice. Three days later, kidneys were removed and their insulin content were measured. We could demonstrate significant differences (P<0.01) in insulin recovery between lipopolysaccharide-free and lipopolysaccharide-containing grafts. In case of endotoxin contaminated islets, we found only 13+/-2% (n=9) of the original insulin content, in contrast to 53+/-7% (n=9) when endotoxin-free islets where grafted. In experiments with islets isolated by use of conventional (lipopolysaccharide-containing) collagenase, and then cultured in endotoxin-free medium, insulin recovery three days after transplantation was 36+/-1% (n=13).

Animals↗
PubMed1999

Critical islet mass for successful porcine islet autotransplantation.

A major reason for the failure of clinical islet transplantations may be a limited islet mass. The aim of this study was to determine the critical islet mass necessary for normalization of glucose metabolism in a porcine model. Diabetes was induced by total pancreatectomy. The splenic lobe of the pancreas was intraductally distended with UW-solution containing 2.67-3.33 mg/ml collagenase, and the distended pancreas was digested in a continuous digestion filtration device. The islets were purified on a isoosmotic Ficoll-sodium-diatrizoate gradient. The survival period of the diabetic recipients in group 2 and 3 receiving, respectively, a low (2.14+/-0.39 microL/kg body weight) and a high (4.99+/-0.83 microL/kg body weight) islet mass was significantly prolonged compared to that of diabetic recipients in group 1 receiving no islet transplantation. However, the survival period of the recipients in group 2 was not significantly different to that in group 3. Three recipients of an islet mass of >5 microl/kg body weight became normoglycemic (fasting blood glucose <100 mg/dl) for more than two months. Furthermore, the glucose and insulin release reactions to the glucose challenge were comparable to that before pancreatectomy. Contrarily, another five diabetic recipients of an islet mass of <4 microL/kg body weight became a fasting blood glucose level of <200 mg/dl. The glucose and insulin release reactions to the glucose challenge were improved only, but not normalized compared to that before pancreatectomy. The data presented in this study demonstrate that metabolic normalization in pancreatectomized diabetic minipigs can be established by autotransplantation of an islet mass of >5 microl/kg body weight.

Animals↗
PubMed1999

Viability and recovery of frozen-thawed human islets and in vivo quality control by xenotransplantation.

Cryopreservation of islets of Langerhans offers advantages for the transplantation into diabetic patients. In this study two different methods of cryopreservation were compared with respect to islet viability and recovery after cryostorage. It was also investigated whether human islet survival in mice was affected by cryopreservation. Aliquots of human islets were cryopreserved conventionally or vitrified, respectively. After rapid thawing, islet viability and islet equivalent (IEQ) recovery rate were determined. Aliquots of freshly isolated or conventionally cryopreserved islets were transplanted beneath the kidney capsule of non-diabetic C57BL/6 mice. After three days renal insulin content was determined. Islet cell viability was 17.3+/-8.0% for vitrified and 51.8+/-3.0% for conventionally cryopreserved islets; the recovery rate was 84.8+/-12.2% and 92.8+/-12.4%, respectively. Insulin recovery after transplantation was 25.6+/-7.3% for fresh and 24.1+/-7.4% for cryopreserved islets. This study suggests that the conventional method of cryopreservation is superior to vitrification with respect to islet viability after thawing. We found no significant difference between fresh and cryopreserved islets with respect to insulin recovery after transplantation into mice.

Animals↗
PubMed1999

Isokinetic gradient centrifugation prolongs survival of pig islets xenografted into mice.

Highly purified porcine islets were prepared by isokinetic gradients performed subsequently to isopycnic gradients. This additional purification step separates ductal, vascular, and lymphoid tissue effectively from endocrine tissue. Although ductal, vascular, and lymphoid tissue comprises only a minor contamination of the islet suspensions, a significant prolongation of the survival of porcine islets xenografted into streptozotocin diabetic C57BL/6 mice can be achieved by the elimination of the non-endocrine tissue. Rejection after islet transplantation is delayed from 2.2+/-0.4 days (n=27) to 13.1+/-2.1 days (n=36), respectively, when conventionally purified and highly purified islets are compared. Irrespective of the purification state, pretreatment of islets by low temperature culture had no effect on xenograft survival.

Animals↗
PubMed1999

Synergistic effect of microencapsulation and immunoalteration on islet allograft survival in bioartificial pancreas.

Recently, we reported successful transplantation (Tx) of microencapsulated (mc) islets. However, graft failure observed in several cases was associated with an increased foreign body reaction compared to long-term functioning grafts. This study was performed to investigate the impact of an immunoalterating islet pretreatment (12-14 days culture at 22 degrees C) on graft function. After microencapsulation in barium alginate beads the islets were cultured for another day. Diabetic LEWIS rats (blood glucose >19 mM) were transplanted with 3500 immunoaltered mc-Wistar islets intraperitoneally. Controls were transplanted with 3500 non-cultured syngeneic or allogeneic mc-islets. Additional syngeneic and allogeneic controls were transplanted with 6000 non-cultured, non-encapsulated islets intraperitoneally. Seventy percent of the recipients of microencapsulated, long-term low temperature cultured islets maintained normoglycemia at least for 15 weeks, while this was true in only 17% of those animals receiving microencapsulated non-pretreated allogeneic islets. Islets in non-encapsulated controls were rejected within several days. Graft function correlated with histologically proven viable islets within the capsules. Microencapsulation of islets markedly prolonged allograft survival compared to non-encapsulated islets; application of an immunoaltering low-temperature culture further improved graft function significantly. These data may support the hypothesis of induction of a reaction against microcapsules by the antigen release from the graft which may be avoided by immunoaltering islet pretreatment.

Alginates↗
PubMed1999

Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation.

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4+/-30.1x10(6)) compared with empty capsules (41.4+/-19.7x10(6)) and non-encapsulated porcine islets (18.1+/-3.3x10(6)). The percentage of CD 3 positive T-lymphocytes rose to 44.5+/-11.5% in case of microencapsulated xenografts compared with 19.2+/-8.2% for non-encapsulated xenografts and 4.9+/-2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2+/-8.9% vs 15.2+/-7.0% for free islets and 4.9+/-1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.

Alginates↗
PubMed1999

Diabetic retinopathy risk correlates with intracellular concentrations of the glycoxidation product Nepsilon-(carboxymethyl) lysine independently of glycohaemoglobin concentrations.

AIMS/HYPOTHESIS: We investigated whether either the amount of diabetes-induced intracellular oxidative stress or the concentration of hyperglycaemia-induced advanced glycation endproducts is associated with the risk of diabetic retinopathy. METHODS: We measured concentrations of the glycoxidation product Nepsilon-(carboxymethyl)lysine and two non-oxidation-dependent advanced glycation end-products (methylglyoxal-derived and 3-deoxyglucosone-derived) in CD45RA+ T-cells from 21 Type I (insulin-dependent) diabetic patients with and without diabetic retinopathy and from age-matched nondiabetic control subjects. RESULTS: Intracellular concentrations of both oxidation-dependent Nepsilon-(carboxymethyl)lysine and oxidation-independent advanced glycation endproducts were increased in memory T-cells from diabetic patients. Nepsilon-(carboxymethyl)lysine: diabetic median-24176 arbitrary units/mg protein (95% confidence interval 18690-34099 arbitrary units/mg protein); nondiabetic-9088 arbitrary units/mg protein (confidence interval 6994-10696 arbitrary units/mg protein; p < 0.0001). Methylglyoxal-derived advanced glycation end products: diabetic-5430 arbitrary units/ mg protein (confidence interval 3458-13610); nondiabetic-271 arbitrary units/mg protein (confidence interval 61-760 arbitrary units/mg protein; p< 0.0001). 3-Deoxyglucosone-derived advanced glycation end products: diabetic-8070 arbitrary units/mg protein (confidence interval 7049-16551 arbitrary units/mg protein); nondiabetic-1479 arbitrary units/ mg protein (confidence interval 1169-3170; p< 0.0001). Only Nepsilon-(carboxymethyl)lysine concentrations, however, inversely correlated with the duration of retinopathy-free diabetes (r = -0.51; p < 0.02). Diabetes-dependent Nepsilon-(carboxymethyl)lysine accumulation did not correlate with age, diabetes duration, or averaged glycohaemoglobin concentrations. In vitro experiments wih menadione and lymphocytes confirmed that Nepsilon-(carboxymethyl)lysine concentrations reflect intracellular oxidative stress. CONCLUSION/INTERPRETATION: Monitoring intracellular concentrations of increased oxidative stress in long-lived CD45RA+ lymphocytes by markers such as Nepsilon-(carboxymethyl)lysine possibly identifies a subgroup of patients at high risk for microvascular complications.

Adult↗
PubMed1999

Differential accumulation of advanced glycation end products in the course of diabetic retinopathy.

AIMS/HYPOTHESIS: Glycated proteins, formed by reaction of glucose and protein, react further yielding numerous, mostly undefined advanced glycation end products (AGE). The recently characterized imidazolone-type AGE (AG-1) is non-oxidatively formed involving 3-deoxyglucosone whereas some AGEs, particularly N(epsilon)-(carboxymethyl)lysine (CML), are formed only in the presence of oxygen. METHODS: To study the possible contribution of oxidative and non-oxidative AGE formation in the development of diabetic retinopathy antibodies directed against CML-type and imidazolone-type AGEs were characterized by dot blot analysis and used to localize these well-characterized epitops in the retinas from diabetic rats (early course) and from human Type I (insulin-dependent) diabetes mellitus with laser-treated proliferative diabetic retinopathy (late course). RESULTS: In non-diabetic rats CML was moderately positive in neuroglial and vascular structures of non-diabetic rat retinas and increased strongly in diabetic retinas. Anti-imidiazolone antibody staining was strongly positive only in diabetic capillaries. Advanced human diabetic retinopathy showed strong CML-immunolabelling of the entire retina whereas control samples showed moderate staining of neuroglial structures only with the polyclonal CML-antibody. Anti-imidiazolone antibody staining was faint in the inner retina of control sections but were strong throughout the entire diabetic retina. Immunolabelling for the AGE-receptor was congruent with a marker of Müller cells. CONCLUSION/INTERPRETATION: Our data indicate that the oxidatively formed CML is present in non-diabetic retinas as a regular constituent but increases in diabetes both in neuroglial and vascular components. Imidazolone-type AGE are restricted to microvessels and spread during later stages over the entire retina, co-localizing with the expression of AGE-receptor.

Animals↗
PubMed1999

Bone metabolism and bone mineral density of systemic lupus erythematosus at the time of diagnosis.

Recent studies have shown that systemic lupus erythematosus (SLE) is associated with a loss of trabecular bone. However, these changes have not been not described in patients with SLE at the time of diagnosis. To investigate the markers of bone metabolism 20 female patients with a recently manifested clinical picture of SLE were selected. All patients included in this study met the ARA criteria (for classification) of SLE. For comparison, 35 female patients with SLE, which had previously manifested itself and which had been treated with glucocorticoids, were included in a second group. A control group (III) consisting of 20 healthy individuals of the same age was formed to compare the results obtained. Test parameters comprised both serum levels of osteocalcin (OC) as the marker for bone formation and crosslinks excretion (CE) in urine as a specific marker for bone resorption. The bone density (BMD) was examined by dual energy X-ray absorption (DEXA) of the vertebral column (L2-L4), femoral neck, Ward's triangle and trochanter. The patients under study received either no medication or nonsteroidal antirheumatic drugs. The BMD of the vertebral column was significantly lower than expected in SLE-afflicted subjects of group II when compared with the age-matched normal female controls. The reduction of BMD in female patients with SLE was related to the significantly increased excretion of urinary pyridinoline, to hypoparathyroidism, and to the decrease in serum OC. Bone loss in women with fresh manifestation of SLE (I) increases to a degree similar to that of patients in group II. Lowered BMD predicts an increased risk for bone fractures. Therefore, female premenopausal SLE patients should be monitored for osteoporosis.

Absorptiometry, Photon↗
PubMed1999

The Giessen-Bad Oeynhausen family study: improved prediction of type I diabetes in a low incidence population of relatives using combinations of islet autoantibodies in a dual step model.

To determine the value of a combined antibody screening for prediction of type I diabetes in a low incidence cohort, we prospectively studied 882 first-degree relatives (485 parents, 382 siblings and 15 offsprings) for up to 11 years who were not preselected for islet cell antibody (ICA) status. During the observation period, 16 individuals developed diabetes. The first serum sample obtained at study entry was analyzed for ICA and antibodies to insulin (IAA), glutamic acid decarboxylase (GADA) and anti-IA-2ic. A multivariate analysis, according to the Cox proportional hazard model considering the joint effects of all baseline variables, selected the four antibodies and the specific family history as significant risk confounding factors (p < 0.05). Further analysis by Kaplan-Meier Life-table methods confirmed a significantly increasing risk of diabetes with the number of autoantibodies present (p < 0.001). In accordance with the Cox model, relatives with more than one affected family member (a multiplex pedigree) and siblings and offsprings vs. parents were at increased risk of IDDM (p < 0.05). In addition to technical problems, a screening strategy based on initial ICA testing has the potential of missing ICA negative subjects among future cases of type I diabetes (19% were ICA negative in the present study) and we therefore set out to evaluate an alternative approach using a dual step strategy with a combination of GADA and anti-IA-2ic for initial screening followed by retesting of positive individuals for ICA and IAA. The combination of GADA and anti-IA-2ic for primary screening (step 1) proved to be more sensitive, identifying 94% of future cases of type I diabetes compared to 81% using ICA as initial test and this antibody combination identified 93% of those individuals with ICA of 20 JDF or more. Retesting of positive individuals for ICA and IAA (step 2) significantly improved the positive predictive value confering a risk of diabetes for siblings and offsprings with more than 2 antibodies within 5 years of 67% (95%CI: 39-90). We conclude that the prognosis of contracting IDDM in relatives is strongly related to the number of autoantibodies present, but the family history should be additionally considered for individual risk assessment. The proposed screening strategy could overcome the inherent problems of the ICA and IAA assays for large-scale screening. In the present study it allows 5-year risk estimates of up to 67% identifying 94% of future cases of type I diabetes.

Adult↗
PubMed1999

The combination of antibodies to GAD-65 and IA-2ic can replace the islet-cell antibody assay to identify subjects at risk of type 1 diabetes mellitus.

First-degree relatives of type 1 diabetic patients are at increased risk of developing diabetes and, until recently, islet cell antibodies (ICA) have represented the major risk marker used for identification of individuals at increased risk for subsequent progression to diabetes. In order to determine the value of antibodies to GAD-65 and IA-2ic to identify individuals at high risk for type 1 diabetes mellitus, we measured both autoantibodies and ICA in 1436 first-degree relatives of patients with type 1 diabetes. In addition, the sera were analyzed for thyroid, adrenal and gastric-parietal cell autoantibodies as markers for possible polyendocrine involvement. GAD-65 Abs were found in 135 out of 1436 (9.4%) first-degree relatives and in 57 of 98 (58.2%) ICA-positive subjects. IA-2ic were detected in 52 of 1436 (3.6%) first-degree relatives and in 44 of 98 (44.8%) ICA-positive relatives. IA-2ic and/or GAD-65 were detected in 73 of 98 (74.5%) ICA-positive relatives. Interestingly, antibodies to GAD-65 and/or IA-2ic were present in 91.2% of individuals with more than 20JDF-units. Anti-IA-2ic and GAD-65 were positively correlated with high levels of ICA. Anti-IA-2ic and GAD-65 were found in 19% and 48.5% of subjects with ICA levels of 5-20JDF-u but in 68.8% and 76.5% of individuals with ICA of 40JDF-u or more, respectively (p < 0.001), compared to subjects with ICA levels less than 5 JDF-u. When autoantibody frequencies among the relatives were analyzed according to relationship to the proband, the offspring and siblings had a higher frequency of ICA and IA-2ic (p<0.05) than the subgroup of parents. A significant association was observed between IA-2ic and thyroid antibodies. In addition, higher levels of IA-2ic were found in relatives with positive TPO antibodies (p < 0.001); this correlation was particularly strong in offspring and siblings (p < 0.01). Determination of GAD-65 and IA-2ic antibodies may be considered as an alternative to primary ICA-screening, enabling the screening of large populations.

Adolescent↗
PubMed1999

Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation?

The remarkable difference in success rates between clinical pancreas transplantation and islet transplantation is poorly understood. Despite the same histocompatibility barrier and similar immunosuppressive treatments in both transplantation procedures, human intraportal islet transplantation has a much inferior success rate than does vascularized pancreas transplantation. Thus far, little attention has been directed to the possibility that islets transplanted into the blood stream may elicit an injurious incompatibility reaction. We have tested this hypothesis in vitro with human islets and in vivo with porcine islets. Human islets were exposed to nonanticoagulated human ABO-compatible blood in surface-heparinized polyvinyl chloride tubing loops. Heparin and/or the soluble complement receptor 1 (sCR1) TP10 were tested as additives. Adult porcine islets were transplanted intraportally into pigs, and the liver was recovered after 60 min for immunohistochemical staining. Human islets induced a rapid consumption and activation of platelets. Neutrophils and monocytes were also consumed, and the coagulation and complement systems were activated. Upon histological examination, islets were found to be embedded in clots and infiltrated with CD11+ leukocytes. Furthermore, the cellular morphology was disrupted. When heparin and sCR1 were added to the blood, these events were avoided. Porcine islets retrieved in liver biopsies after intraportal islet allotransplantation showed a morphology similar to that of human islets perifused in vitro. Thus, exposure of isolated islets of Langerhans to allogenic blood resulted in significant damage to the islets, a finding that could explain the unsatisfactory clinical results obtained with intraportal islet transplantation. Because administration of heparin in combination with a soluble complement receptor abrogated these events, such treatment would presumably improve the outcome of clinical islet transplantation by reducing both initial islet loss and subsequent specific immune responses.

Adult↗
PubMed1999

N(epsilon)(carboxymethyl)lysin and the AGE receptor RAGE colocalize in age-related macular degeneration.

PURPOSE: To investigate whether glycoxidation products and the receptor for advanced glycation end products (RAGE) are present and colocalize in subfoveal membranes of patients with age-related macular degeneration (ARMD). METHODS: Surgically removed subfoveal fibrovascular membranes from 12 patients, 11 related to ARMD and 1 to an idiopathic membrane, were analyzed for the presence of the glycoxidation product N(epsilon)-(carboxymethyl)lysin (CML), one of the receptors for advanced glycation end products, RAGE, and the activation of NFkB, using immunohistochemistry. RESULTS: CML-like immunoreactivity was found in all ARMD specimens examined adjacent or colocalized with RAGE, but not in the idiopathic membrane. RAGE immunoreactive material was found in CD68-positive cells and in the fibrous matrix. CD68-positive cells and surrounding areas stained for p50, the activated form of NFkB. CONCLUSIONS: These results indicate that glycoxidation products are present in subretinal membranes of patients with ARMD. The concomitant expression of RAGE in these membranes and the finding of activated NFkB is suggestive of an implication of glycoxidation product formation in the pathogenesis of the disease.

Adolescent↗