Biomedical subjects

D Wion

Publications and source records attributed to D Wion.

At least 37 records · Page 2Linked to original sources

PubMed1996

1,25-Dihydroxyvitamin D3 induces programmed cell death in a rat glioma cell line.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a seco-steroid hormone with potential antitumoral activities, has been recently reported to exert cytotoxic effects on C6 glioma cells. However, the molecular mechanisms which trigger this cell death remain unknown. We show here that this 1,25(OH)2D3-induced cell death is dependent upon protein synthesis and is accompanied by the expression of c-myc, p53, and gadd45 genes. Two other genes, coding for interleukin-6 and vaso-endothelial growth factor, are also upregulated after addition of 1,25(OH)2D3. This programmed cell death can be suppressed when cells are treated with forskolin, a drug which increases intracellular cAMP concentration, or with genistein, an inhibitor of tyrosine protein kinases. However, in spite of the demonstration of fragmented DNA in 1,25(OH)2D3-treated cells, the C6.9 cells used in this study do not show the classical morphological features of apoptosis. These results provide the first evidence for the existence of a programmed cell death triggered by 1,25(OH)2D3 in glioma cells and may provide a basis for the development of new therapeutic strategies. In addition, these data also suggest that the treatment of C6.9 cells with 1,25(OH)2D3 may be a useful model to study the molecular mechanisms involved in the programmed cell death of a cell of glial origin.

Animals↗

PubMed1996

1,25-Dihydroxyvitamin D3 regulates the expression of the low-affinity neurotrophin receptor.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is known to regulate the expression of neurotrophins [45,46]. Here, we report that 1,25-(OH)2D3 does not influence the expression of truncated or full-length forms of trkB and trkC receptors mRNAs in primary cultures of astrocytes and in C6 glioma cells. In contrast, low concentrations of 1,25-(OH)2D3 increased low-affinity neurotrophin receptor (P75NTR) mRNA and protein levels in C6 glioma cells. Putative vitamin D responsive elements (VDRE) in the P75NTR promoter have been investigated by transfecting plasmids containing sequences from P75NTR promoter fused to a cat reporter gene. A region between -610 and -860 bp upstream from the translation start codon was found to respond to 1,25-(OH)2D3. Interestingly, 1,25-(OH)2D3 does not regulate P75NTR in primary cultures of astrocytes even at concentration as high as 10(-7) M. Since long-term treatment of 1,25-(OH)2D3 induces cell death in C6 glioma cells but not in primary astrocytes [41], the possible involvement of P75NTR in 1,25-(OH)2D3-induced cell death is discussed. Finally, in-vivo studies show that treatment of 15-day-old and adult rats with 1,25-(OH)2D3 leads to a decrease in the level of P75NTR mRNA in the spinal cord but does not influence its expression in dorsal root ganglion or sciatic nerve. These results suggest that 1,25-(OH)2D3 may have a role in the specific regulation of P75NTR in vivo.

Animals↗

PubMed1996

1,25-Dihydroxyvitamin D3, an inducer of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) has significant therapeutic potentials, in particular for neurodegenerative disorders. To determine factors that would enhance GDNF expression, we analysed the effect of 1,25-(OH)2 D3 in C6 glioma cells. Treatment of C6 cells with 10(-7) M, 1,25-(OH)2 D3 for 48 h elicited an 18.5-fold increase in the level of GDNF mRNA. In addition, our results indicate that 1,25-(OH)2 D3 is effective at concentrations as low as 10(-10) M and that retinoic acid has additive effects. These data indicate that 1,25-(OH)2 D3 is a potent inducer of GDNF expression and suggest that 1,25-(OH)2 D3 may contribute to the regulation of GDNF in vivo.

Animals↗

PubMed1996

Cytotoxic effects of 1 alpha,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues on a glioma cell line.

1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3) has recently been reported to exert a toxic effect on both rat and human glioma cell lines. However the potential clinical use of 1 alpha,25(OH)2D3 in the treatment of glioma is impaired by its potent hypercalcemic effects. We have therefore investigated the effects on glioma cell growth of several vitamin D3 analogues which have previously been shown to be less calcemic in vivo than 1 alpha,25(OH)2D3. The present study shows that several analogues are able to induce, in vitro, the death of rat glioma cells (C6.9). The compound KH 1060 appears to be the most effective in the induction of cell death, while MC 1288 and CB 1093 are as potent as 1 alpha,25(OH)2D3. EB 1089 was somewhat less effective than 1 alpha,25(OH)2D3 and MC 903, which is currently used in the treatment of psoriasis, has only a weak activity on C6.9 cells. The effective doses used are around 10(-9) M for 1 alpha,25(OH)2D3 and 10(-10) M for KH 1060. Interestingly, the toxic effect exerted by 1 alpha,25(OH)2D3 and its analogues is accompanied by several of the biochemical features of apoptosis, such as DNA fragmentation and induction of the c-myc protooncogene. These findings, together with the fact that the therapies currently available for glioma are only palliative, suggest that 1 alpha,25(OH)2D3 analogues such as KH 1060, EB 1089 or CB 1093, alone or in combination with other therapeutic approaches, could be of potential interest in the treatment of brain glial tumors.

Animals↗

PubMed1996

Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells.

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

Animals↗

PubMed1995

Expression of the nerve growth factor gene is controlled by the microtubule network.

Colchicine, nocodazol, and vinblastine, three microtubule-disrupting drugs, were shown to increase the levels of both nerve growth factor (NGF) mRNA and cell-secreted NGF protein in L929 cells, with levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or amyloid precursor protein (APP) mRNAs remaining unaffected. Northern blot analysis demonstrated that colchicine also increased NGF mRNA levels in rat primary astrocytes and mouse skin fibroblasts. The specificity of the effects observed was assessed by the fact that the microtubule-stabilizing agent Taxotere, a semisynthetic compound structurally related to taxol, suppressed the effects of colchicine, whereas lumicolchicine, a colchicine derivative that has no action on the microtubule network, had no influence on NGF expression. Likewise, the disruption of the microfilament network by cytochalasin B did not increase NGF mRNA levels in L929 cells. Furthermore, the increase in NGF gene expression observed following microtubule disruption depended on a cascade of events involving at least one protein kinase, which is not down-regulated by phorbol ester, and on a pertussis toxin sensitive step. These results support the concept that tubulin and/or the microtubule cytoskeleton play an active role in the regulation of the NGF gene.

Amyloid beta-Protein Precursor↗

PubMed1995

Interactions between second messenger pathways influence NGF synthesis in mouse primary astrocytes.

Primary mouse brain astrocytes were stimulated with phorbol 12-myristate 13-acetate (PMA), serum, forskolin and ionophore A23187, in order to investigate the effect of distinct signalling pathways on the expression of the nerve growth factor (NGF) gene and of proto-oncogenes encoding transcription factors of the Fos and Jun families. PMA, and to a lesser extent serum, induced a marked accumulation of NGF transcripts, in agreement with published observations [Brain Res., 570 (1992) 316-322]. The effect of A23187 was less pronounced and that of forskolin barely detectable. No relationship was observed between the expression of NGF gene and that of c-fos, fos-B, fra-1, jun-B proto-oncogenes. In contrast, changes in the levels of NGF transcripts were associated with corresponding modifications of the levels of c-jun transcripts, a fact which suggests that the c-Jun protein exerts a regulatory role on the expression of the NGF gene. In these cells, however, the regulation of NGF synthesis appears complex, since a pretreatment with forskolin or ionophore A23187 interfered with the promoting effect elicited by PMA or serum in inducing an early decline of the levels of NGF transcripts. This phenomenon was accompanied by a corresponding decrease in the amounts of cell-secreted NGF in cells treated with forskolin and PMA. A23187 had a much more striking effect on the production of mature NGF since this compound maintained the level of cell-secreted NGF to basal values, irrespective of the presence of PMA. A similar inhibitory effect was observed with thapsigargin, another compound able to increase the cytosolic concentration of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Synthesis of 1,25-dihydroxyvitamin D3 by rat brain macrophages in vitro.

Cultured microglial cells were examined for their ability to metabolize 25-hydroxyvitamin D3 (25-(OH) D3). Upon exposure to lipopolysaccharide, microglial cells produced a vitamin D metabolite which comigrated with synthetic 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in two different systems of high performance liquid chromatography. This metabolite had the same affinity as synthetic 1,25-(OH)2D3 for the chick intestinal 1,25-(OH)2D3 receptor. Lipopolysaccharide-stimulated microglial cells incubated with 3 nM of 25-(OH) D3 synthesized up to 5.76 fmol 1,25-(OH)2D3/8 x 10(5) cells/2 hr. Microglial cells stimulated for 48 hr with interferon-gamma also produced a significant amount of 1,25-(OH)2D3 (4.17 fmol/8 x 10(5) cells/2 hr). In contrast, levels of 1,25-(OH)2D3 produced by resting microglial cells were barely detectable. It is concluded that activated brain macrophages may be committed to synthesize 1,25-(OH)2D3 in vitro. This raises the possibility that activation of microglial cells in vivo may be followed by an increase in the level of 1,25-(OH)2D3 in the central nervous system (CNS). These results support the emerging concept that the brain constitutes a target tissue for vitamin D metabolites.

Animals↗

PubMed1994

Induction of glioma cell death by 1,25(OH)2 vitamin D3: towards an endocrine therapy of brain tumors?

The secosteroid 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) is the major biologically active metabolite of vitamin D. Antitumor activity of this hormone has been observed on several cell lines and on breast cancer in vivo. The purpose of this in vitro study was to determine the possible effect of 1,25(OH)2D3 on glioma cells. Two glioma cell lines from rat (C6) or human (GHD) origin were cultured in the presence of 1,25(OH)2D3. The sensitivity of these cells to 1,25 (OH)2D3 was assessed with a colorimetric MTT assay. A cytotoxic effect of 1,25(OH)2D3 was detected at concentrations around 10(-8) M. A lag period of 3 days was required between the onset of the treatment and the observation of the effects. However, the continuous presence of 1,25(OH)2D3 is not required since cell death occurred even when C6 cells were challenged for 24 hr with 1,25(OH)2D3 and then cultured in the absence of the hormone. In addition, 1,25(OH)2D3 regulates the expression of its own receptors in C6 glioma. These results provide to our knowledge the first evidence for a cytotoxic effect of 1,25(OH)2D3 on rat and human glioma cells and could offer both an experimental model to study a programmed cell death in a brain-derived cell line and a new strategy for the inhibition of glioma growth in vivo.

Animals↗

PubMed1994

A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

Base Sequence↗

PubMed1994

1,25-dihydroxyvitamin D3 regulates the synthesis of nerve growth factor in primary cultures of glial cells.

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) on nerve growth factor (NGF) synthesis was investigated in primary cultures of astrocytes prepared from brain of neonatal rats. 1,25-(OH)2 D3 elicited a dose-dependent increase of NGF mRNA with a maximal effect at 10(-7) M, which persisted for at least 48 h. Northern blot analysis revealed an expression of the vitamin D3 receptor (VDR) gene in primary glial cells. Treatment of cells with 1,25-(OH)2 D3 led to an increase in the VDR mRNA levels. Similar results were obtained in C6 glioma cells. Exposure of primary glial cells to 10(-8) M 1,25-(OH)2 D3 caused only a 2-fold increase of the levels of cell-secreted NGF after 3 days of treatment. However, a 5-fold increase was observed three days after a second addition of vitamin D3. Likewise, a pretreatment with lower doses of hormone such as 10(-10) M or 10(-9) M enhanced the responsiveness of the cells to a 24 h treatment with 10(-8) M hormone. It appears, therefore, that the duration of the treatment influences the level of synthesis of NGF, possibly as a consequence of the increase of the VDR gene expression. The specificity of 1,25-(OH)2 D3 is supported by the fact that a concentration of 10(-7) M of an another vitamin D3 metabolite, 24,25-(OH)2 D3, had no effect on NGF synthesis. Several lines of evidence indicate that astrocytes constitute the major cell type responsive to 1,25-(OH)2 D3 in primary cultures of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

24,25-Dihydroxyvitamin D 3↗

PubMed1994

Reactive oxygen species influence nerve growth factor synthesis in primary rat astrocytes.

Newborn rat brain astrocytes, cultured in a serum-free medium, were exposed for 30 min to two types of reactive oxygen species. Cells were either treated with the xanthine/xanthine oxidase (X/XOD) system, which generates both H2O2 and the O2.- radical, or to H2O2 alone. Both treatments induced a dose-dependent accumulation of nerve growth factor (NGF) transcripts, 6 h after the exposure. Maximal effect was obtained with 6 mU/ml XOD, or 10(-4) M H2O2. A rapid expression of protooncogenes of the jun and fos families was also noticed in X/XOD- or H2O2-treated cells. This phenomenon was transient in cells exposed to X/XOD. However, in the case of H2O2-treated cells, the accumulation of c-fos or c-jun mRNAs was still pronounced 6 h after the end of the treatment and the levels of cell-secreted NGF appeared relatively reduced, when compared with those obtained after a shock with the X/XOD system. This raised the possibility that H2O2 at 10(-4) M could depress protein synthesis. Measurements of the incorporation of radiolabeled amino acids into trichloroacetic acid-precipitable material supported this assumption. Level of radioactivity associated with cellular material was dramatically reduced in H2O2-treated cells, when it was compared with control or X/XOD-treated cells. Furthermore, treatment of cells with the protein synthesis inhibitor anisomycin had an effect similar to that of H2O2 because it caused an accumulation of c-fos, c-jun, and NGF transcripts after 6 h of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Expression of 25(OH) vitamin D3 24-hydroxylase gene in glial cells.

The expression of the 25(OH) vitamin D3 24-hydroxylase gene was studied in C6 glioma and rat primary glial cell culture. The expression of the 25(OH)D3 24-hydroxylase gene was not detected in C6 glioma or glial cells cultured in a serum-free medium. However, the 25(OH)D3 24-hydroxylase mRNA was induced in a dose-dependent manner in cells treated with 1,25(OH)2D3. These findings provide further evidence for an involvement of vitamin D3 metabolites in brain function.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1993

Enhancement of the synthesis and secretion of nerve growth factor in primary cultures of glial cells by proteases: a possible involvement of thrombin.

Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as alpha-thrombin or collagenase induce a continuous, dose-dependent enhancement of the levels of cell-secreted NGF. Incubation of astrocytes for a 24-h period with 300 ng/ml of alpha-thrombin (approximately 9 nM, or 1 U/ml) results in an increase of the levels of cell-secreted NGF by a factor of three- to fourfold, and at doses 10 times higher, stimulation by a factor of up to four- to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP-1 family, such as c-fos, c-jun or junB. Trypsin, plasmin, alpha-chymotrypsin, or elastase also enhanced, to different extents, the levels of cell-secreted NGF. However, unlike alpha-thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose-response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell-substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of alpha-thrombin indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of alpha-thrombin with D-phenyl-alanyl-L-propyl-L-arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of alpha-thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of alpha-thrombin require binding to the cell surface. We found that gamma-thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as alpha-thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Complex interactions among second messenger pathways, steroid hormones, and protooncogenes of the Fos and Jun families converge in the regulation of the nerve growth factor gene.

Regulation of the expression of the nerve growth factor (NGF) gene has been reported previously to be mediated via the protooncogene c-fos. Activation of the protein kinase C pathway and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has also been reported to increase the pool of NGF transcripts in L929 fibroblasts. Here we show that activation of the cyclic AMP second messenger pathway antagonized the effect of phorbol 12-myristate 13-acetate (PMA) or serum on NGF synthesis, whereas it enhanced that of 1,25(OH)2D3. A positive effect was also observed when serum, PMA, and 1,25(OH)2D3 were added together, but dexamethasone reduced this enhancement. There was no close correlation between the increase in c-fos mRNA and that in NGF mRNA, suggesting that expression of the c-fos protooncogene is not necessarily followed by induction of the NGF gene. Rather, these two genes are simultaneously, and not sequentially, induced after forskolin treatment. It appears that regulation of the NGF gene depends on a repertoire of multiple regulatory AP-1 complexes arising from activation of the second messenger pathways. This suggests that NGF gene expression is under the control of a complex interplay among second messenger pathways, protooncogenes, and steroid hormones such as 1,25(OH)2D3 and glucocorticoids.

Animals↗

PubMed1992

Activation of nerve growth factor synthesis in primary glial cells by phorbol 12-myristate 13-acetate: role of protein kinase C.

Phorbol 12-myristate 13-acetate (PMA) induces a dramatic production of nerve growth factor (NGF) in primary cultures of newborn mouse astrocytes maintained in a serum-free medium. This stimulation is dose-dependent and a maximal effect on the levels of cell-secreted factor was observed at a concentration of 10 nM. At this concentration, the promoting effect of PMA appears much more important than that elicited by 10% fetal calf serum (FCS) under the same culture conditions. PMA acts primarily on the accumulation of NGF mRNA, which was detected by northern blot analysis after 6 h of treatment. This accumulation may be totally or partially prevented when PMA-treated glial cells are concomitantly exposed to the protein kinase inhibitors H-7, H-9, and to a lesser degree, HA-1004. The known specificity of these inhibitors agrees with the possibility that protein kinase C (PKC), which constitutes so far the sole known target of PMA, represents a key element involved in the stimulation of NGF gene. The role of PKC is further supported by the observation that alpha phorbol didecanoate, which has no activity on PKC, is depleted of effect on the synthesis of NGF. Likewise, 1,2-dioctanoylglycerol (1,2-DOG) has a weak, but significant promoting action on the production of NGF, unlike the 1,3-isomer which is not active on PKC. Finally, a treatment of 15 min with 100 nM PMA is sufficient to stimulate the cells, suggesting that the activation phase of PKC, rather than its down regulation, constitutes an important trigger leading to an increased expression of the NGF gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Pertussis toxin provides evidence for two independent signalling pathways leading to the activation of the nerve growth factor gene.

Increased expression of the nerve growth factor (NGF) gene may be obtained by treating L929 fibroblasts with serum, phorbol 12-myristate 13-acetate (PMA), or 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The possible involvement of GTP-binding proteins (G proteins) in these regulatory events was monitored by exposing the cells to pertussis toxin (PT), a compound known to inactivate several types of G proteins by ADP ribosylation. Measurements of the pool of NGF mRNA by Northern blot analysis, and quantification of the factor secreted by the cells with a double-site ELISA assay, indicate that pretreatment with PT decreases by about 60% the effect of serum on the levels of NGF transcript and secreted factor. This effect is accompanied by a corresponding decrease of the expression of c-fos gene, which takes place soon after the addition of serum to the cells. In contrast, PT had no effect on the basal level of NGF mRNA found in cells maintained in serum-free medium or in cells stimulated with PMA or 1,25-(OH)2D3. These results indicate that some serum factor(s) acts via plasma membrane receptors able to interact with PT-sensitive G proteins to modulate NGF gene expression. In contrast, 1,25-(OH)2D3 appears to mediate its action through a different signalling pathway, which is likely to require its cytosolic receptor, and is independent of PT-sensitive G protein and c-fos induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗

PubMed1992

Alteration in the levels of 1,25-(OH)2D3 and corticosterone found in experimental diabetes reduces nerve growth factor (NGF) gene expression in vitro.

Circulating concentrations of corticosterone and 1,25-(OH)2D3 have been reported to be respectively increased and decreased in the streptozotocin-treated rats. Using the cell line L929 cultured in a steroid-free medium, we show that the alteration in the levels of corticosterone and 1,25-(OH)2D3 found in vivo in experimental diabetes is able to decrease the synthesis of NGF by these cells. This finding raises a possible relationship between the balance in the concentration of these steroids and some aspects of the neuropathic complications found in experimental diabetes.

Animals↗