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Biomedical subjects

A L Rubin

Publications and source records attributed to A L Rubin.

At least 19 recordsLinked to original sources
PubMed1992

High rate of diversification and reversal among subclones of neoplastically transformed NIH 3T3 clones.

NIH 3T3 cells undergo neoplastic transformation when exposed to conditions of moderate physiological growth constraint. One of several characteristics of this transformation that indicates its adaptational nature is its gradual reversibility under conditions of unconstrained growth. We explored the origins of reversibility by isolating cells from each of three highly transformed foci and comparing their focus-forming capacity with that of derivative clones and subclones. A high proportion of the parental cells made dense foci. Six of the nine clones obtained from the three foci produced foci, though the percentage varied widely. The other three clones produced no foci at all. The transformed clones were subcloned and analyzed to evaluate the possibility that the negative clones were genuine revertants, rather than being derived from a small minority of nontransformed cells surrounding or underlying the original foci. In each case the subclones varied widely in the percentage of focus-forming cells and the average was much lower than the parental clone from which they were derived. Indeed, 15 of the 53 subclonal populations produced no foci. The high degree of heterogeneity, including complete reversal of focus-forming capacity, provides additional support for the hypothesis that "spontaneous" transformation is driven by an adaptive response to moderate growth constraint rather than by one or more effectively irreversible mutations.

3T3 Cells↗
PubMed1992

Hypertension is not adequately controlled in hemodialysis patients.

To examine the adequacy of hypertension control, we monitored the blood pressure (BP) of 53 hemodialysis patients who received treatment for hypertension. BP measurement using an ambulatory BP monitor began 1 hour before dialysis and continued every 30 to 60 minutes for 48 hours until the next dialysis. Diet, medications including antihypertensive drugs, and hemodialysis prescription were not changed during this study. Each patient had a mean of 68 BP measurements during the monitoring period. Mean (+/- SD) systolic and diastolic BP levels of all patients over 48 hours were 158.6 +/- 22.7 mm Hg and 88.7 +/- 16.6 mm Hg, respectively, without diurnal variations. In these, BP loads (the percentage of systolic BP exceeding 150 mm Hg and diastolic BP exceeding 90 mm Hg) were 58.4% and 39.4%, respectively, suggesting that hypertension was inadequately controlled for more than half of the study period. Eight patients (15%) maintained BP within normal ranges at all times. All patients lost weight (2.9 +/- 0.9 kg) at the end of dialysis by ultrafiltration. However, only 27 patients (51%) had a greater than 5% decrease in mean arterial BP post-dialysis, which returned to predialysis levels within 12 to 24 hours. Reduction of BP postdialysis was significantly more common among black patients (72%) than white patients (30%) (P less than 0.01). However, there was no difference in age, cause of kidney disease, amount of ultrafiltration, and BP loads between those whose BP decreased and those whose did not. BP monitoring was repeated in eight patients, 2 to 3 months after adjustment of their antihypertensive regimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Keratinocyte differentiation markers: involucrin, transglutaminase, and toxicity.

Studies of three keratinocyte differentiation markers are described. First, the involucrins of several mammals are identified, facilitating use of this marker in animal models of human disease. The rapid evolution of involucrin has prevented its routine immunochemical identification beyond the primates, but its unusual solubility and its labeling by transglutaminase have permitted detection in rats, cats, and sheep. Second, the re-expression of keratinocyte transglutaminase in carcinoma cells lacking the enzyme is demonstrated. Lack of this enzyme expression has been observed previously in squamous cell carcinomas. The present finding suggests genomic hypermethylation could contribute to this phenomenon and offers an approach to analyzing transcriptional features of the enzyme regulation. Third, the sensitivity of keratinocytes to growth suppression by aflatoxin B1 is reported. The observed toxicity appears to be mediated by aryl hydrocarbon hydroxylase, a metabolic enzyme inducible in keratinocytes by environmental agents. Such expression may be relevant to carcinogenesis in tissues subject to squamous metaplasia as well as in other exposed cell types stimulated to express this biotransformation enzyme.

Aflatoxins↗
PubMed1992

Keratinocyte transglutaminase: differentiation marker and member of an extended family.

Transglutaminases stabilize a variety of biological structures by cross-linking constituent proteins. This action appears physiologically important in stabilizing (1) keratinocyte cornified envelopes, (2) fibrin clots, (3) the copulation plug in rodents, and (4) the fertilized egg surface in aquatic species. Several transglutaminases that participate in such processes have been well characterized and found, though highly divergent, to differ in sequence primarily at the amino terminus. Comparison of their gene structures suggests a likely mechanism by which new members may arise that assume a diversity of functions. The functions of some members of this family are presently unknown, including the tissue transglutaminase found in many mammalian cell types, and those found in plants. Most of the transglutaminases identified are soluble enzymes, but several that are membrane-bound have gained recognition recently. The best characterized of the latter is keratinocyte transglutaminase, which is anchored in the membrane by acylated fatty acid. Important for proper epidermal cell maturation, expression of this enzyme is greatly altered by physiological effectors and toxic agents. In addition, it is induced by cultivation of cells from non-squamous epithelia. Thus, it is a promising marker for helping to elucidate the molecular basis by which keratinocyte differentiation is elicited or altered.

Amino Acid Sequence↗
PubMed1991

Induction of transformation in NIH3T3 cells by moderate growth constraint: evidence that neoplasia is driven by adaptational change.

The NIH3T3 cell line undergoes transformation when the cells are maintained at high densities for extended periods. In an effort to define the physiological status of the cells before and during the transformation process, they were seeded in medium containing 2, 5 or 10% calf serum (CS) and growth was monitored by measurements of cell numbers and by incorporation of [3H]thymidine into DNA. Transformed foci began to appear by 7 days in 2% CS, at least 3 days after the onset of density-dependent growth inhibition, and by 10 days in 5% CS, 6 days after the onset of growth inhibition. The rate of DNA synthesis in both serum concentrations had declined by 7 days to less than 1/15th of the initial rate measured by day 2. Transformation did not occur in cells exposed to 10% CS which, though supporting even higher saturation densities, underwent a 100-fold decline in DNA synthesis after reaching confluence. Transfer and regrowth in 2% CS of cells from all three serum concentration revealed a steep increase in capacity for focus formation of cells previously cultured in 2 and 5% CS after they had reached confluence, but no such increase in cells previously cultured in 10% CS, indicating that the lack of foci in the original 10% CS incubation was due to suppression of transformation and not to masking of foci at very high cell densities. Determination of the fraction of dividing cells by [3H]thymidine autoradiography showed a similar decline prior to the appearance of transformed cells in 2% CS. In addition, a reactivation of mitosis in non-focal cells occurred by day 24, indicating that a much higher proportion of cells escaped the moderate growth constraint than might have been expected by the total number of foci formed on transfer. Our results are consistent with the premise that transformation is an adaptive response to moderate growth constraints, but not with a mutational origin.

3T3 Cells↗
PubMed1991

Minimal sensitization and excellent renal allograft outcome following donor-specific blood transfusion with a short course of cyclosporine.

UNLABELLED: A new protocol of donor-specific blood transfusion under cyclosporine coverage was developed and examined for immunologic consequences and clinical efficacy in recipients of one- or zero-HLA-haplotype-matched renal allografts. Between 1985 and 1989, 75 recipients were transfused with 100 ml of stored whole blood at 1, 8, and 15 days of its storage from either one-HLA-haplotype-matched related donors (n = 65, 33 from their parents, 30 from siblings, and 2 from offspring) or from zero-HLA-haplotype-matched donors (n = 10, 7 from spouses and 3 from siblings). During DST, all recipients received cyclosporine, 6 mg/kg/day, starting a day before and finishing a week after DST (23 days). Recipients were monitored by donor-specific mixed lymphocyte culture responses before and after DST, and serially for antibodies by fluorescence activated cell sorter analysis and by standard complement-dependent lymphocytotoxicity assay. Following DST with CsA, only 3 of 75 patients (4%) were sensitized against the blood donor. This rate is considerably lower, albeit statistically not significantly, compared with the 10% rate found in 30 recipients who had received DST without CsA in our previous study. Repeat MLC studied one to two months after DST (the day before transplant) were significantly increased compared with pre-DST (stimulation index: mean +/- SEM; 10.3 +/- 1.4 to 15.8 +/- 2.8, P = 0.004, and relative response: 40.9 +/- 5.1% to 49.8 +/- 5.5%, P = 0.003). Since the stimulation index with controls did not change after DST (23.4 +/- 2.9 to 26.2 +/- 3.3), enhanced MLC responses appear to be donor-specific. The changes in MLC responses did not correlate with the number of blood transfusion received prior to DST, the number of rejection episodes, or graft outcome. Fifty-seven recipients underwent a kidney transplant from their one-HLA-haplotype-matched blood donors within two to three months after DST. All 10 recipients of zero-haplotype-matched donors were also successfully transplanted from their respective blood donors. The graft survival rates were at least 90% at two years in both groups. IN CONCLUSION: (1) 100 ml of stored whole-blood DST, three times at weekly intervals with a short course of CsA is minimally sensitizing but effective in enhancing graft survival; (2) this protocol could be used in donor-recipient pairs who do not share a haplotype; and (3) DST with CsA elicits augmentation of donor-specific MLC responses.

Adolescent↗
PubMed1990

Progressive state selection of cells in low serum promotes high density growth and neoplastic transformation in NIH 3T3 cells.

A subline of NIH 3T3 cells maintained by frequent passage (every 2 to 3 days) in 10% calf serum (CS) at low population density reached a low saturation density in 2% CS and produced no transformed foci on prolonged incubation at confluency in 2% CS. Within 3 frequent low density passages in 2% CS, the saturation density and focus-forming capacity in that serum concentration began an increase which was continued in subsequent passages. The saturation density and focus-forming capacities of the cells in both 2% and 1% CS were further enhanced by passage in 1% CS. The cells could then be passaged in 0.5% CS and then in 0.25% CS, which would support no multiplication of cells previously passaged only in 10% CS. The cells passaged in 0.25% CS gradually increased their saturation density and focus-forming capacity in that extremely low serum concentration during 24 low density passages, although their initial growth rate did not increase. They also attained a colony-forming efficiency in 0.25% CS of about 30%, as compared to less than 1% for cells passaged in 10% CS. Cells passaged, cloned, and passaged again in 2% CS yielded clonal populations which differed from one another in saturation density and focus-forming capacity in 2% CS. We conclude that NIH 3T3 cells diversify phenotypically at a high rate in their capacity to multiply and produce foci in limiting concentrations of serum, and we propose that progressive selection of these heterogeneous states accounts for the acquired capacity to function effectively in low concentrations of serum growth factors. Since lymph and presumably extracellular fluid in vivo contain low concentrations of growth factors which govern the multiplication of normal cells, the adaptation we observe in vitro may be related to tumor production in the animal.

Animals↗
PubMed1990

Suppression of transformation by and growth adaptation to low concentrations of glutamine in NIH-3T3 cells.

NIH-3T3 cells, commonly used as targets for oncogene-mediated neoplastic transformation, undergo high rates of spontaneous transformation. When the glutamine concentration in the medium was reduced from 5 to 1 mM or less, the transformation rate was reduced. This effect was not dependent upon a reduction in the growth rate, which remained unaffected by reduction of glutamine even to 0.6 mM. Upon trypsinization and transfer to 5 mM glutamine-containing medium, cells exposed to 0.2 mM glutamine for as little as 4 days formed fewer foci than control cells exposed over a similar period to 5 mM glutamine. This indicates that short term changes in the supply of this polyfunctional metabolite have heritable consequences in later cell generations. If populations containing highly transformed cells were passaged weekly for 1-3 weeks in 0.2 mM glutamine, resultant populations were better adapted to grow in low-glutamine medium and formed fewer transformed foci upon re-transfer to 5 mM glutamine medium, suggesting that the transformed state is at least partially reversible. If similar cell populations were exposed to low-glutamine medium but were not passaged, growth adaptation occurred but there was no reduction in focus formation, indicating that maintenance of a moderate rate of cell division may be required in addition to the lowered glutamine for reversal of transformation. Transformed and non-transformed cells originating from foci and from nonfocal areas of the same culture dishes multiplied at the same reduced rate in 0.2 mM glutamine. This indicates that suppression of spontaneous transformation in low-glutamine medium was not the result of selecting pre-existing variants but was itself an adaptive response of the population.

Adaptation, Physiological↗
PubMed1990

Systemic lupus erythematosus in patients with end-stage renal disease: long-term follow-up on the prognosis of patients and the evolution of lupus activity.

We studied the clinical course of 59 lupus patients with end-stage renal disease (ESRD) to determine their long-term prognosis and delineate the evolution of their lupus activity. The study population was predominantly female (86%) and young (mean age, 27.4 years), and they were observed for a mean of 6.5 years from the inception of dialysis. At the time dialysis was initiated, only 21 patients (35.6%) had clinically active systemic lupus erythematosus (SLE). The remaining patients progressed to ESRD despite the absence of clinical lupus activity. Lupus activity was clinically apparent in 55.4% of patients in the first year, 6.5% in the fifth, and none in the tenth year. In 45% of patients, lupus activity was clinically inactive at entry to ESRD and remained inactive throughout the observation period. Serological activity declined proportionally, but to a lesser extent than clinical activity. Cumulative patient survival was 81.1% and 74.6% at the fifth and tenth year, respectively, from the inception of dialysis treatment; similarly it was 78% at the fifth and tenth year after the transplantation. Graft survival was 60.4% at the fifth and 45.5% at the tenth year. No one had recurrence of clinical lupus nephritis in the graft for up to 16 years of follow-up. Fourteen patients died from either infectious or cardiovascular complications, but none from SLE per se. This long-term study with a large number of lupus patients confirms our previous findings that the progression of renal disease to ESRD may be mediated by nonimmunologic mechanisms, as well as immunologic insults.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1990

Humoral immune response following extracorporeal immunoadsorption therapy of patients with hypercholesterolemia.

Low-density lipoprotein apheresis (LDL-apheresis) is an extracorporeal procedure that preferentially removes LDL cholesterol from the blood. One of the primary techniques for performing this procedure uses immunoadsorption columns containing monospecific polyclonal sheep antibodies to human LDL covalently coupled to a gel filtration medium. LDL-apheresis has generally been well-tolerated, with chills, fever, or flushing occurring rarely. The possibility of an immune reaction was investigated as a basis for these reactions observed in 12 of the 1312 procedures performed. Antibodies to sheep IgG developed in 12 of the 15 patients treated with LDL-apheresis as a result of the shedding of small quantities of the sheep immunoglobulin from the columns. A column acid-washing procedure minimized the quantity of shed antibody but did not prevent immunization of the patient. The clinical reactions were probably unrelated to shedding and immunization, as the reactions occurred even in patients who were not immunized to the sheep IgG. Immunization to ethylene oxide was not the cause, as determined by a radioallergosorbent test. The reactions were more likely related to the activation of complement, as indicated by the generation of C3a des Arg by the columns and an increase in C3a des Arg levels systemically.

Antibody Formation↗
PubMed1990

Relation of spontaneous transformation in cell culture to adaptive growth and clonal heterogeneity.

Cell transformation in culture is marked by the appearance of morphologically altered cells that continue to multiply to form discrete foci in confluent sheets when the surrounding cells are inhibited. These foci occur spontaneously in early-passage NIH 3T3 cells grown to confluency in 10% calf serum (CS) but are not seen in cultures grown to confluency in 2% CS. However, repeated passage of the cells at low density in 2% CS gives rise to an adapted population that grows to increasingly higher saturation densities and produces large numbers of foci in 2% CS. The increased saturation density of the adapted population in 2% CS is retained upon repeated passage in 10% CS, but the number and size of the foci produced in 2% CS gradually decrease under this regime. Clonal analysis confirms that the focus-forming potential of most if not all of the cells in a population increases in response to a continuously applied growth constraint, although only a small fraction of the population may actually form foci in a given assay. The acquired capacity for focus formation varies widely in clones derived from the adapted population and changes in diverse ways upon further passage of the clones. We propose that the adaptive changes result from progressive selection of successive phenotypic variations in growth capacity that occur spontaneously. The process designated progressive state selection resolves the apparent dichotomy between spontaneous mutation with selection on the one hand and induction on the other, by introducing selection among fluctuating states or metabolic patterns rather than among genetically altered cells.

Animals↗
PubMed1990

Physiological induction and reversal of focus formation and tumorigenicity in NIH 3T3 cells.

NIH 3T3 cells undergo morphological transformation in response to conditions of constrained growth, such as occur in low serum concentrations or at confluence. Transformation is expressed in a small fraction of the cells by the appearance of discrete foci of multiplying cells on a confluent monolayer of quiescent cells. We isolated and expanded cell populations from three dense and three light foci. Cells from each of these populations efficiently reproduced foci of the same morphotype when grown on a background of nontransformed NIH 3T3 cells. Using cultures derived from one of the dense foci (subline D/2), we found that the number of focus-forming units was stable and the cells remained tumorigenic when they were subjected to repeated thrice-weekly passage in 2% calf serum. However, equivalent passage in 10% calf serum eventually rendered the cells incapable of both focus production and tumor formation. The results show that the capacity to produce tumors as well as morphological transformation are produced as a response to physiological constraints of growth and/or metabolism in the absence of carcinogens and that both properties can be reversed by lifting the constraints. This behavior is typical of an adaptational response and, taken together with other supporting evidence, shows that tumorigenesis does not require conventional genetic alteration.

Animals↗
PubMed1989

Coordination of keratinocyte programming in human SCC-13 squamous carcinoma and normal epidermal cells.

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC-13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56-58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5- to 10-fold in high calcium medium. However, they were also increased 5- to 30-fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.

Calcium↗
PubMed1989

Modulation of 3-methylcholanthrene toxicity in cultured neoplastic keratinocytes by glucocorticoids and retinoids is not accounted for by macromolecular adduct formation.

3-Methylcholanthrene (3-MC) greatly inhibits the growth of two lines of human squamous carcinoma cells, SCC-9 and SCC-12B2. Exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin alone was much less effective and, in the presence of 3-MC, did not alter the sensitivity (EC50 = 0.3 microM) or extent of growth inhibition by the latter. The degree of 3-MC-mediated inhibition, however, was markedly alleviated by inclusion of retinoic acid (EC50 greater than or equal to 0.7 microM) and hydrocortisone (EC50 = 40 nM) or dexamethasone (EC50 = 3 nM) in the culture medium. These physiological effectors, which are known to have opposing actions on keratinocyte character in SCC cells, did not significantly alter either aryl hydrocarbon hydroxylase activity or macromolecular adduct formation. Further analysis of the cellular responses indicated that hydrocortisone and, in some experiments, retinoids increased the growth rate in 3-MC-exposed cultures, while 3-MC increased the saturation density in retinoic acid-exposed cultures, an example of interference with a physiological response of the cells. These results indicate that alteration of the differentiated state, regardless of the direction of the change, can alter the sensitivity of the cells to toxic stimuli. Further investigation of the bases of such toxic responses and their modulation by the microenvironment may enhance our understanding of the target cell specificity of polycyclic aromatic hydrocarbons.

Aryl Hydrocarbon Hydroxylases↗
PubMed1989

Hypertension in kidney transplant recipients. Effect on long-term renal allograft survival.

To examine the effects of hypertension on renal graft function, we studied the clinical course of 144 kidney transplant recipients who had functioning grafts for three to 13 years. The patients were divided into three groups: normotensive (n = 32), controlled hypertensive (n = 49) and uncontrolled hypertensive group (n = 63). In addition to the difference in their blood pressure status, the three groups had significantly different levels of serum creatinine at entry to the study (mean +/- SE in mg/dL: 1.41 +/- 0.02, 8.89 +/- 0.02 and 2.30 +/- 0.03, respectively, P = .0002). Cumulative graft survival (CGS) at ten years for normotensive patients was 81%, whereas it was 58% for controlled hypertensive patients and 50% for uncontrolled hypertensive patients. The difference of CGS between normotensive and hypertensive patients was significant (P = .01), whereas the difference between the two hypertensive groups, controlled v. uncontrolled, was not. If serum creatinine levels at entry to the study were adjusted and the CGS of hypertensive patients was compared to normotensive patients with comparable levels of serum creatinine, the differences in CGS between the two groups were no longer significant. Regression analyses for potential prognostic factors revealed that serum creatinine levels were of more primary importance as a prognostic variable than blood pressure status. We conclude that hypertension is an important risk factor for renal graft survival, but control of hypertension alone does not appear to improve it. Graft survival appears to be influenced more by the severity of graft dysfunction at entry to the study irrespective of blood pressure control.

Adolescent↗
PubMed1989

A phase II clinical trial of adoptive immunotherapy for advanced renal cell carcinoma using mitogen-activated autologous leukocytes and continuous infusion interleukin-2.

Forty patients with metastatic, recurrent, or unresectable renal cell carcinoma were entered into a study of the therapeutic efficacy of adoptive immunotherapy using periodate (IO4-) and interleukin-2 (IL2)-activated autologous leukocytes and continuous infusion low-dose IL2. Patient survival was also examined. The first 15 consecutive patients were enrolled in protocol A without an IL2 priming phase and the following 25 patients were entered in protocol B where a 5-day priming phase was initiated before leukapheresis. A maintenance regimen consisted of either 3 x 10(6) units of recombinant interferon-alpha (rIFN-alpha), three times per week only or together with leukapheresis and infusion of IO4-/IL2-activated cells and 2 days of continuous infusion IL2 per month. Thirty-four patients completed the protocol treatment. Four patients were removed from the study owing to rapid tumor progression and two patients died while receiving treatment. The clinical response rate was 22%: two patients had a complete response and five patients had a partial response. Among the 25 patients who had no clinical response, 11 patients had either a mixed response or stabilization. Neither response, response duration, nor site response correlated with the total dose of IL2 administered or the number of activated killer cells infused. Patients who received maintenance therapy had longer survival times than patients who did not receive such therapy. All toxicity and side effects associated with IL2 treatment were transient and resolved after discontinuation of the drug. Patients on maintenance therapy tolerated both rIFN-alpha and monthly infusions of activated killer cells and IL2 well. This study confirms the concept of adoptive immunotherapy as a new treatment approach for advanced renal cell carcinoma and suggests that maintenance therapy may prolong survival time.

Adult↗
PubMed1989

Extracorporeal LDL cholesterol removal: role of LDL-pheresis in combination with other hypolipidemic therapy to regress vascular disease.

The direct relationship between hypercholesterolemia and atherosclerosis has resulted in formal cholesterol-lowering recommendations for patients at increased risk. The incomplete response to therapy of some forms of hypercholesterolemia as well as not uncommon drug intolerance prompted the development of extracorporeal techniques to reduce serum cholesterol levels. Nonhuman primate data and an analysis of human cholesterol epidemiology and reduction trials were used to establish guidelines that would maximize the likelihood of stabilizing or regressing established coronary artery atherosclerosis. These goals are a total cholesterol (TC) level of less than or equal to 150 mg/dL (3.9 mmol/L) and a ratio of TC to high-density lipoprotein cholesterol (HDL) of less than 2.8. Selective, extracorporeal removal of LDL cholesterol (LDL-pheresis) was combined with diet and hypolipidemic drugs in a pilot study at The Rogosin Institute to achieve these lipid end-points. Technical aspects of LDL-pheresis, the background rationale for its use as part of a combined hypolipidemic therapy, the initial experience at The Rogosin Institute, and plans for future studies and applications are presented.

Animals↗