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Biomedical subjects

M Silver

Publications and source records attributed to M Silver.

At least 55 records · Page 3Linked to original sources
PubMed1997

Histochemical staining following LacZ gene transfer underestimates transfection efficiency.

Analysis of LacZ gene expression is conventionally inferred from blue staining that results from exposure of the transfected cells or tissue to the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). Such histochemical staining reports not whether the gene product is present or absent, but where it is active. We investigated the hypothesis that identification of activity, as opposed to presence, of the enzyme underestimates gene expression following LacZ gene transfer. Under conditions optimized for in vitro histochemistry, up to 20% of cells stably transfected with nls-LacZ remained unstained by X-Gal. In contrast, immunostaining with a monoclonal or a polyclonal anti-beta-galactosidase (beta-Gal) antibody positively stained 99% of the cell nuclei. Following in vivo transfection of naked DNA encoding for nls-LacZ, X-Gal staining disclosed 2.7 +/- 1.7 positive nuclei per LacZ-transfected animal, or a transfection efficiency of 0.015%. In contrast, immunohistochemical staining disclosed 118 +/- 32.7 positive nuclei per transfected animal, yielding a transfection efficiency of 0.64% (p < 0.0001 versus X-Gal staining). Thus, 42.9 times more positive cells were detected by antibody than X-Gal staining. Finally, LacZ gene expression following intramuscular gene transfer with an adenoviral vector was observed in 7.6% of skeletal muscle cells assessed with X-Gal; anti-beta-Gal antibody identified 21.8% of cells as being successfully transfected (p < 0.0001). Thus, X-Gal histochemistry following gene transfer of constructs encoding LacZ may underestimate the anatomic extent of gene expression. The superior sensitivity of immunostaining suggests that anti-beta-Gal antibody represents the preferred analytical tool for light microscopic evaluation of LacZ gene transfer.

Adenoviridae↗
PubMed1997

Isolation of putative progenitor endothelial cells for angiogenesis.

Putative endothelial cell (EC) progenitors or angioblasts were isolated from human peripheral blood by magnetic bead selection on the basis of cell surface antigen expression. In vitro, these cells differentiated into ECs. In animal models of ischemia, heterologous, homologous, and autologous EC progenitors incorporated into sites of active angiogenesis. These findings suggest that EC progenitors may be useful for augmenting collateral vessel growth to ischemic tissues (therapeutic angiogenesis) and for delivering anti- or pro-angiogenic agents, respectively, to sites of pathologic or utilitarian angiogenesis.

Animals↗
PubMed1997

Augmentation of impaired tumoricidal function in alveolar macrophages from lung cancer patients by cocultivation with allogeneic, but not autologous lymphocytes.

It has been reported that the in vitro development of tumoricidal function in alveolar macrophages from lung cancer patients is reduced significantly when compared to that in peripheral blood monocytes from the same patients or alveolar macrophages from control patients. In the present investigation, a method for potentiating the development of tumoricidal function in alveolar macrophages from lung cancer patients is described. This method, which relies on priming the macrophages with purified, allogeneic peripheral blood lymphocytes from normal donors, could not be demonstrated when autologous lymphocytes from lung cancer patients were used in the priming coculture. The augmentation of tumoricidal function appears to be mediated by one or more soluble factors, since supernatants from cocultures of alveolar macrophages and allogeneic peripheral blood lymphocytes could enhance the cytotoxic function of freshly obtained alveolar macrophages. Furthermore, it appears that NK cells are necessary for this effect, since depletion of CD56+/CD57+ cells from allogeneic lymphocytes eliminated their capacity to enhance alveolar macrophage cytotoxic function. The augmentation of cytotoxic function elicited in alveolar macrophages by this method was not associated with changes in the secretion of tumor necrosis factor alpha, or interleukin 1 beta.

CD3 Complex↗
PubMed1997

Modulation of tumoricidal function in alveolar macrophages from lung cancer patients by interleukin-6.

Previous studies have demonstrated that alveolar macrophages from lung cancer patients are impaired in their ability to develop tumoricidal function when stimulated by activators such as interferon gamma + lipopolysaccharide. However, these same macrophages have been shown to develop significant tumoricidal function when precultured with macrophage-depleted allogeneic peripheral blood lymphocytes from normal donors, an effect that was lost by the elimination of natural killer cells from the allogeneic lymphocyte population. In the present study, the effect of each activation condition on the expression of mRNA for interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF alpha) and IL-6 was determined using reverse transcription/polymerase chain reaction. The results show that the non-permissive activation condition is associated with the expression of mRNA for IL-6 while the permissive activation condition is not. Antibodies against IL-6 were subsequently shown to permit the development of tumoricidal function in alveolar macrophages stimulated with interferon gamma + lipopolysaccharide while IL-6 protein was shown to inhibit the stimulatory action of allogeneic lymphocytes on the development of tumoricidal function in the same alveolar macrophages. Neither the permissive (i.e. allogeneic lymphocyte stimulation) nor the non-permissive (i.e. interferon gamma + lipopolysaccharide) activation condition had any effect on the capacity of alveolar macrophages from lung cancer patients to express mRNA for IL-1 alpha, IL-1 beta or TNF alpha. These results show that IL-6 can regulate the ability of alveolar macrophages from lung cancer patients to be stimulated by interferon gamma + lipopolysaccharide to develop significant tumoricidal function. They also show that allogeneic lymphocytes have the capacity to down-regulate IL-6 mRNA synthesis by alveolar macrophages thereby permitting the development and/or expression of macrophage tumoricidal function.

Antibodies↗
PubMed1997

Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA.

OBJECTIVE: Previous studies have demonstrated that arterial gene transfer of naked DNA encoding for a secreted protein may permit modulation of the host phenotype despite a low transfection efficiency. Acidic fibroblast growth factor (aFGF) is an angiogenic growth factor, but is not secreted by intact cells. In the current study, we investigated the hypothesis that addition of a hydrophobic leader sequence to achieve active secretion of the gene product would permit therapeutic angiogenesis following arterial gene transfer of naked DNA encoding for aFGF. METHODS: Ten days following surgical induction of unilateral hindlimb ischemia, New Zealand white rabbits were randomized to intra-arterial gene transfer with one of three plasmids: p267 (encoding non-secreted aFGF, n = 10), pMJ35 (encoding secreted aFGF) (n = 10), or 500 micrograms of pGSVLacZ (control, n = 10) (500 micrograms each). All animals were studied at 30 days post-gene transfer for evidence of therapeutic angiogenesis. RESULTS: pMJ35 transfectants had more angiographically visible collaterals (angiographic score = 0.76 +/- 0.02) than either p267 (0.55 +/- 0.02, p < 0.01) or LacZ (0.47 +/- 0.02, p < 0.001). Limb blood pressure ratio for pMJ35 was 0.88 +/- 0.02 vs. 0.68 +/- 0.04 for p267 (p < 0.01) and 0.57 +/- 0.04 for LacZ (p < 0.001). Vascular resistance was significantly lower in the pMJ35 group, compared with that in pGSVLacZ group, both in resting state (3.2 +/- 0.4 vs. 7.4 +/- 1.4 respectively, p < 0.05) and after the administration of nitroprusside. Capillary density (per mm2) was also superior in pMJ35 group (274 +/- 10) vs. p267 (204 +/- 9, p < 0.01) and LacZ (177 +/- 6, p < 0.001). CONCLUSION: The paracrine effects of a secreted gene product may obviate the need for adjunctive vectors in strategies of arterial gene therapy.

Analysis of Variance↗
PubMed1997

Glucose, lactate and oxygen metabolism in the fetal pig during late gestation.

Using [U-14C]glucose tracer, rates of umbilical uptake, utilization and production of glucose, and of CO2 production from glucose carbon, were measured in seven chronically catheterized fetal pigs, when the sow was in the fed state, between 100 and 113 days of gestation (term, 114 +/- 2 days). At the same time, rates of umbilical O2 and lactate uptake were determined in all seven fetuses by the Fick principle. The mean fetal rates of umbilical glucose uptake, glucose utilization and CO2 production from glucose carbon were 38.4 +/- 4.2, 41.3 +/- 5.2 and 126.9 +/- 12.6 mumol min-1 (kg fetal body weight)-1, respectively (n = 7), No glucose production was therefore detected in the fetuses. Production of CO2 from glucose carbon accounted for 37.3 +/- 3.4% (n = 7) of the umbilical O2 uptake, which averaged 340 +/- 13 mumol min-1 kg-1 (n = 7). There was also significant umbilical lactate uptake in the fetal piglets when the sow was in the fed state (32.6 +/- 10.4 mumol min-1 kg-1, n = 7, P < 0.05). No significant changes in fetal glucose, O2 or lactate metabolism were observed with increasing age towards term. The fetal rates of glucose metabolism and of umbilical uptake of O2 and lactate were not correlated with fetal blood glucose level. Hence, glucose is used for both oxidative and non-oxidative metabolism in utero and is an important, although not the sole, source of carbon for metabolic processes in the fetal pig during late gestation.

Animals↗
PubMed1996

A model of Ca(2+)-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch.

BACKGROUND: In contrast to conventional muscle myosins, where two different light chains (LCs) stabilize the elongated regulatory domain (RD) region of the head portion of the molecule, unconventional myosins are a diverse group of motors in which from one to six calmodulin (CaM) subunits are bound tandemly to the RD. In both cases, the heavy chains of the RDs have special sequences called "IQ motifs' to which the LCs or CaM bind. A previously puzzling aspect of certain unconventional myosins is their unusual mode of regulation, where activation of motility occurs at low levels of Ca2+. Although the atomic structure of the conventional muscle myosin RD has been determined, no crystallographic structure of the RD of an unconventional myosin is yet available. RESULTS: We have constructed a model of vertebrate CaM bound to the first IQ motif present in the neck region of an unconventional myosin (chicken brush border myosin I), using strict binding rules derived from the crystal structure of the scallop RD. The model accounts for aspects of the regulation of many unconventional myosins where CaM is bound at low levels of Ca2+ and released or changed in conformation at high levels of Ca2+. The conformational changes as a function of Ca2+ depend not only on the precise sequence of the IQ motifs but also on the interactions between CaM molecules bound to adjacent sites on the myosin heavy chain. CONCLUSIONS: According to our model, the full versatility of CaM binding to target peptides is displayed in the regulation of unconventional myosins. At low concentrations of Ca2+, CaM binds in a manner similar to the LCs of conventional myosins. At higher Ca2+ concentrations, CaM changes conformation and acts as a switch to regulate the activity of the unconventional myosin molecules.

Amino Acid Sequence↗
PubMed1996

Proliferative response of human prostate tumour xenografts to surgical trauma and the transurethral resection of the prostate controversy.

Transurethral resection of the prostate (TURP) as an excisional procedure involving multiple incisions into the prostate does not differentiate between palpably benign prostate tissue and microscopic foci of well-differentiated adenocarcinoma. The impact of TURP on the progression of such 'latent' or 'incidental' tumours unique to the prostate gland has been a focal point of a continuing controversy. In studies designed to develop preclinical evidence that would lend support to, or detract from, either side of the TURP controversy, surgical trauma-induced stimulation of in situ tumour growth was extended to include human prostate tumour tissue PC-3, DU-145 and H-1579, albeit as xenografts in athymic nude males. A significant proliferative response of prostate tumours implanted directly in, adjacent to, or distant from, a freshly induced surgical wound, could be inhibited by a somatostatin analogue (Lanreotide) applied topically to the surgical site. This preclinical model supports TURP as a risk factor for biopsy or therapeutic surgical intervention procedures in benign prostatic hypertrophy (BPH), a risk factor that increases with the stage of disease in undetected cancers. It also suggests a potential clinical benefit that might be derived by applying Lanreotide directly to the surgically traumatised genitourinary area by simple irrigation of the urethra and bladder during or shortly post TURP.

Adenocarcinoma↗
PubMed1995

The effects of thyroid hormones on oxygen and glucose metabolism in the sheep fetus during late gestation.

1. The effects of thyroid hormones on fetal metabolism during late gestation were examined by measuring the rates of glucose and oxygen utilization rates in chronically catheterized sheep fetuses made hypothyroid by either fetal thyroidectomy (TX) or hypophysectomy (HX). The values were compared with those in intact fetuses and in thyroxine (T4)-treated TX and HX fetuses. 2. Umbilical O2 uptake expressed on a weight-specific basis was reduced by 20-30% in the hypothyroid fetuses and was restored to normal values when plasma T4 levels were maintained in the TX and HX fetuses by exogenous T4 administration. 3. The low O2 consumption rates of the untreated hypothyroid fetuses were accompanied by fetal growth retardation, an abnormal blood gas status, and in the TX fetuses, by significant reductions in the rates of glucose oxidation, CO2 production from glucose carbon and O2 utilization for glucose oxidation. 4. When T4 levels were maintained in the TX fetuses, these metabolic rates and fetal blood gas status were restored to their normal values. Replacement of T4 also sustained growth in TX but not in HX fetuses. 5. When the data from all fetuses were combined irrespective of treatment, there were significant positive correlations between plasma levels of T4 (but not triiodothyronine (T3)) and the rates of umbilical O2 uptake, glucose oxidation, CO2 production from glucose carbon and O2 utilization for glucose oxidation in the individual fetuses. 6. These findings demonstrate that T4 is a physiological regulator of O2 utilization by the sheep fetus close to term.

Animals↗
PubMed1995

The role of psychiatrists in community mental health centers: a survey of job descriptions.

There is little data about the role of psychiatrists within CMHCs. To gain perspective on this issue, job descriptions for medical directors and staff psychiatrists were collected from 214 CMHCs. The data demonstrated that most CMHCs want fully trained psychiatrists involved in a variety of activities in addition to prescribing medication. Policy development was specifically included as part of the medical director's job for 69% of the CMHCs, and 50% mentioned training as part of the staff psychiatrist's job. Although job descriptions may not accurately reflect the actual roles of the psychiatrist in all cases, these data suggest that CMHCs support a multifaceted role for their psychiatrists.

Community Mental Health Centers↗
PubMed1995

Maturational effects of cortisol on the exocrine abomasum and pancreas in fetal sheep.

The role of cortisol in the prenatal development of digestive enzymes in the abomasum (prochymosin and pepsinogen) and pancreas (amylase, trypsin, chymotrypsin) has been investigated in the fetal lamb during late gestation. The abomasum and pancreas were collected from 22 unoperated control fetuses (99-145 days gestation; term, 145 +/- 2 days), from seven pairs of twins infused with either saline or cortisol for five days preceding delivery at 127-133 days, and from four 139-143-day-old fetuses adrenalectomized at 120-123 days. Developmental increases (2-8-fold) occurred in protease concentrations in the fetal abomasum and in amylase and chymotrypsin contents in the fetal pancreas. These increases paralleled the normal prepartum rise in fetal plasma cortisol. In addition, the enzyme values were significantly higher in cortisol-infused than in saline-infused fetuses (with the exception of pancreatic amylase) and were significantly lower in adrenalectomized fetuses than in control fetuses at term. The pH of abomasal fluid remained neutral (pH 6.8-8.0) during late gestation and was not affected by cortisol treatment or adrenalectomy. The results suggest that cortisol stimulates the development of the exocrine abomasum and pancreas in fetal sheep and may, thereby, increase the digestive capacity in neonatal lambs. Compared with the pig, another long-gestation species, the sheep has an early development of gastric pepsinogen but a late development of gastric acidity and pancreatic protease activities.

Abomasum↗
PubMed1995

Fetal and maternal plasma lipids in chronically catheterized mares in late gestation: effects of different nutritional states.

The effects of different nutritional states on plasma lipid concentrations have been examined in pregnant mares and their fetuses. Maternal and fetal arterial catheters were inserted into 12 pony mares between 244-303 days' gestation (term 320-360 days) and observations made from 5 days following the insertion of catheters. After recovery from surgery maternal and fetal arterial samples were withdrawn from 7 mares with normal feeding patterns (Group IA), from four of these mares at the end of a 30 h fast and 3 h later following refeeding (Group IB) and six mares who failed to re-establish normal feeding patterns (Group II). The fatty acid concentrations and composition of the plasma free fatty acid (FFA), triacylglycerol and phospholipid fractions were analysed. Maternal FFA, triacylglycerol and phospholipid concentrations were significantly raised in the fasted (Group IB) and under-fed (Group II) mares. Fetal concentrations of FFA and phospholipid increased significantly in the group of under-fed (Group II) mares but not in the fasted (Group IB) mares. In the fetal plasma the proportions of polyunsaturated fatty acids derived from essential fatty acids in the FFA and phospholipid fractions were much higher than those in the mare. In the fasted (IB) and under-fed (II) groups the relative amounts of the polyunsaturated fatty acids in each fraction remained unchanged (P > 0.05). These results show a short fast or prolonged undernutrition result in raised maternal plasma lipid concentrations which in turn can effect the total amount of lipid in the fetal circulation. However any increases in polyunsaturated fatty acids in the fetus (e.g. in Group II) are unlikely to come from the maternal circulation; likely sources of these fatty acids are the placenta or fetal tissues.

Animals↗
PubMed1995

Renal function in the chronically cannulated fetal llama: comparison with studies in the ovine fetus.

Samples of maternal and fetal plasma, fetal urine, and amniotic fluid were collected from 8 chronically cannulated pregnant llamas, in the last third of gestation. The samples were obtained for up to 18 days post-surgery. Osmolality, sodium (Na), potassium (K), chloride (Cl), and urea were measured on 40 samples collected on days 1, 2, 3, 4-5, 6-7, 8-9, and 10-19. The osmolalities of maternal and fetal plasma, fetal urine and amniotic fluid, averaged over these 7 time periods, were, respectively, 312 +/- 2, 311 +/- 1, 484 +/- 14, and 317 +/- 1 mosmol kg-1. Values are given as mean +/- s.e. The major differences from fetal fluid values in the ovine fetus (from previously published values) were the higher osmolality and urea concentration of llama fetal urine. Urine flow rate measured in 6 fetuses, 4.5-6.5 kg body weight, was 5.8 +/- 0.4 mliter h-1; urea clearance rate was 55.5 +/- 11.8 mliter h-1. Glomerular filtration rate (GFR), measured with 51Cr-EDTA in 5 fetuses on 1-4 occasions, was 111.4 +/- 23.3 mliter h-1. Fractional reabsorptions (FR) of Na, K and Cl were 97.9 +/- 1, 75.9 +/- 13.5 and 97.7 +/- 0.4% respectively. The GFR (25 mliter kg-1 h-1) and urine flow rate (1 mL kg-1 h-1) were less than half and about one-tenth the respective values in ovine fetuses. As Na reabsorption is the major oxygen-consuming activity of the kidney, the llama fetal kidney requires only half the oxygen needed by the ovine fetal kidney to reabsorb the filtered sodium load. The reason for the formation of hypertonic, rather than hypotonic, urine in the fetal llama may be due to both greater morphological maturity of the kidney and the excretion of as yet unidentified osmotically active organic substances.

Amniotic Fluid↗
PubMed1995

Immunohistochemical localisation of steroidogenic enzymes and phenylethanolamine-N-methyl-transferase (PNMT) in the adrenal gland of the fetal and newborn foal.

An increase in fetal adrenal cortisol output signals the onset of parturition in many animal species but, in the fetal horse, plasma concentrations of cortisol remain low for much of late pregnancy, with a rise occurring only very close to the time of birth (term 320-360 days). Immunohistochemistry was used to determine the localisation and changes in distribution of key steroidogenic enzymes for cortisol production; P450scc, P450C17 and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in adrenal tissue from fetal and newborn horses and these findings were correlated with the appearance of immunoreactive (IR)-phenylethanolamine-N-methyl-transferase (PNMT), a cortisol-dependent enzyme. Five micron sections of adrenal tissue from fetuses at Day 100-156 (n = 5), Day 244-295 (n = 8), greater than Day 300 (n = 4) and from newborn foals (n = 6), were stained using specific antibodies and the avidin-biotin-peroxidase technique. All 3 steroidogenic enzymes were present by Day 150, but in less than 20% of the cortical cells. By late gestation the steroidogenic enzymes were present in approximately 30% of the cells, but the distribution varied. P450SCC and P450C17 predominated in cortical cells proximal to the medulla; 3 beta HSD was present throughout the cortex, but more in the zona fasciculata. In foals after birth, IR-3 beta HSD and IR-P450SCC had increased substantially throughout the adrenal cortex, and IR-P450C17 was present in most cells of the presumptive zonae fasciculata and reticularis. IR-PMNT was localised to nuclei of scattered medullary cells at the medullary-cortical interface by Day 150.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Localisation of 15-hydroxy prostaglandin dehydrogenase (PGDH) and steroidogenic enzymes in the equine placenta.

15-hydroxy prostaglandin dehydrogenase (PGDH) is the critical enzyme that determines metabolism of primary prostaglandins. Its expression is determined in part by steroid hormones, particularly progesterone, formed from delta(5) steroids through 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity. To assess whether the regulation of PGDH might occur in a paracrine, autocrine or intracrine fashion, we used immunohistochemistry (IHC) to determine the localisation of key steroidogenic enzymes in the equine placenta and compared these patterns to the distribution of immunoreactive (IR-) PGDH. Placental tissue was obtained from pony or Thoroughbred mares at about Days 150, 250-280 and >300 of pregnancy (term 320 to 360 days; n=5-8 each group). IR-PGDH, 3beta-HSD, cholesterol side chain cleavage enzyme (P450(scc)) and 17-hydroxylase/lyase (P450(C17)) were localised using specific antibodies and the avidin-biotin peroxidase technique and visualised using diaminobenzidine as substrate. IR-P450(scc) was present in trophoblast cells, but not in maternal tissues of the microcotyledons. In contrast, at Days 150 and 280, IR-PGDH was present in maternal epithelial and interstitial cells in the microcotyledons, but was not detected in trophoblast epithelium, chorioallantois or endometrial glands. After Day 300, IR-PGDH was present in the maternal epithelium and interstitial cells of the placenta and it was also present in trophoblast cells in some specimens.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Vaginal birth versus elective caesarean section: effects on gastric function in the neonate.

Plasma gastrin concentration increases in late gestation and reaches a peak at birth or shortly after birth in many species ('neonatal hypergastrinaemia'). We investigated the hypothesis that gastrin and gastric acid secretion in the neonate is influenced by the final rise in plasma cortisol associated with spontaneous (vaginal) birth. Caesarean-delivered (CD, n = 28) or vaginally delivered (VD, n = 24) premature or full-term piglets (97-115 days gestation) were killed immediately after birth (using pentobarbitone). Compared with newborn CD pigs, the newborn VD pigs had significantly higher (P < 0.05) concentrations of cortisol (669 +/- 60 versus 223 +/- 2 nM) and gastrin (57 +/- 4 versus 33 +/- 1 pM) in plasma, and significantly lower gastric fluid pH (3.3 +/- 0.2 versus 4.7 +/- 0.3), amidated (bioactive) gastrin in the antrum (550 +/- 77 versus 1220 +/- 29 pmol g-1) and glycine-extended (precursor) gastrin in the antrum (81 +/- 10 versus 143 +/- 5 pmol g-1). There were significant linear correlations between log plasma cortisol values and plasma gastrin (r = 0.40, P < 0.05) or gastric fluid pH (r = -0.51, P < 0.05) in newborn pigs. The effects of cortisol in the immediate postnatal period were investigated in forty-one CD pigs born at 111-112 days gestation and treated with saline, metyrapone (an inhibitor of cortisol synthesis) or adrenocorticotrophic hormone (ACTH) from 0 to 7 days after birth. At 7 days, plasma gastrin in ACTH-treated pigs (elevated plasma cortisol) was significantly lower than in saline-treated pigs, but not different from that in metyrapone-treated pigs (low plasma cortisol). No treatment effects were observed postnatally for antral gastrin and fundic cobalamin-binding protein concentrations. These results suggest that in the intrapartum period the high circulating cortisol levels stimulate the normal rise in gastrin and acid secretion associated with spontaneous vaginal delivery in the piglet, whereas postnatally, cortisol is unlikely to play an important role in the subsequent development of gastrin and acid secretion.

Adrenal Glands↗