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T Kealey

Publications and source records attributed to T Kealey.

At least 37 records · Page 2Linked to original sources
PubMed1997

The role of IGF-I in human skin and its appendages: morphogen as well as mitogen?

Previous studies have investigated the expression of insulin-like growth factor-I (IGF-I) and its receptor in cultured skin cells or in whole skin. In order to fully understand the role of IGF-I in the skin and its appendages, however, a comprehensive study that details the expression of IGF-I and the IGF-I receptor in sections of human skin is needed. Therefore, we now report an immunocytochemical and in situ hybridization localization study of the cell types expressing IGF-I and its receptor in human adult skin and its appendages. We have observed that (i) dermal fibroblasts produce IGF-I, (ii) the epidermal basal keratinocytes are IGF-I negative but IGF-I receptor positive, and (iii) the keratinocytes of the stratum granulosum produce IGF-I. These observations indicate either that the mitogenesis of the basal keratinocytes is regulated by IGF-I expressed both in the dermis and in the stratum granulosum, or that dermal fibroblasts are responsible for sequestering IGF-I to the basal keratinocytes and that the stratum granulosum-derived IGF-I may be an autocrine regulator of epidermal differentiation. The distribution of IGF-I and its receptor in the hair follicle indicates that IGF-I may be a morphogen, not a mitogen, at those sites, because their proliferating cells, but not their differentiating cells, are IGF-I receptor negative. Further, IGF-I receptor expression by the dermal papilla appears to be switched off during the transition from anagen to catagen, which implies a regulatory role for IGF-I during the hair growth cycle.

Adult↗
PubMed1996

Whole hair follicle culture.

In this article the authors have reviewed the historical background behind the organ culture of whole hair follicles. The methods developed by the authors and others for the isolation and whole organ maintenance of hair follicles from both human and other species are described. How whole organ models have been used to further understanding of the biology of the hair follicle and how they may be used in the future are discussed.

Animals↗
PubMed1996

Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-alpha in alopecia areata.

The immune system may be involved in the regulation of normal hair follicle growth as well as in the pathogenesis of some hair diseases. Immunomodulatory cytokines not only act as mediators of immunity and inflammation but also regulate cell proliferation and differentiation and, as such, may play an important part in regulating hair growth. We have investigated the effects of a number of interleukins (IL), colony stimulating factors and tumour necrosis factors (TNF) on hair follicle growth in vitro. Dose-response studies showed that IL-1 alpha, IL-1 beta and TNF-alpha were potent inhibitors of hair follicle growth. The histology of hair follicles maintained with inhibitory doses of IL-1 alpha, IL-1 beta and TNF-alpha showed similar changes in hair follicle morphology, resulting in the formation of dystrophic anagen hair follicles. These changes in histology were characterized by the condensation and distortion of the dermal papilla, marked vacuolation of the hair follicle matrix, abnormal keratinization of the follicle bulb and inner root sheath, disruption of follicular melanocytes and the presence of melanin granules within the dermal papilla. Moreover, these changes in hair follicle morphology are similar to those reported in alopecia areata and suggest that IL-1 alpha, IL-1 beta and TNF-alpha may play an important part in the pathophysiology of inflammatory hair disease.

Alopecia Areata↗
PubMed1996

In the absence of streptomycin, minoxidil potentiates the mitogenic effects of fetal calf serum, insulin-like growth factor 1, and platelet-derived growth factor on NIH 3T3 fibroblasts in a K+ channel-dependent fashion.

There is considerable evidence to suggest that the opening of K+ channels plays an important role in stimulating mitogenesis. K+ channel blockers have been shown to inhibit mitogenesis in vitro, mitogens increase cytosolic membrane K+ channel permeability, K+ channel openers stimulate hair growth in vivo, and the Ras/Raf signal transduction pathway induces K+ channel activity. Paradoxically, however, K+ channel openers such as minoxidil have been reported in vitro not to modulate, or even to inhibit, mitogenesis in a range of cell types. Only untherapeutic concentrations have stimulated mitogenesis. These experiments, however, appear to have been carried out in the presence of aminoglycoside antibiotics, which inhibit potassium channel activity. We now report that in the absence of aminoglycoside antibiotics, minoxidil at 10 microg/ml (0.05 mM) causes a significant stimulation of proliferation of NIH 3T3 fibroblasts maintained over a 10-d period in 5% fetal calf serum-supplemented medium. Further, we show that in the presence of 100 microg streptomycin per ml, minoxidil at 10 microg/ml produces an initial inhibition of proliferation, which apparently confirms, in NIH 3T3 fibroblasts, that the inhibition of mitogenesis by minoxidil in the presence of streptomycin is an artifact. The potentiation of NIH 3T3 cell growth by minoxidil can be attributed to the opening of potassium channels, because the potassium channel blocker tolbutamide (5 mM) or combinations of the blockers tolbutamide (1 mM)/tetraethylammonium (2 mM) or glibenclamide (1 microM)/apamin (10 nM) block the minoxidil-induced stimulation of growth. We also demonstrate that minoxidil is able to significantly potentiate the mitogenic effects of both platelet-derived growth factor and insulin-like growth factor 1 on NIH 3T3 fibroblasts in the presence of CPSR-2 (a cytokine free serum substitute). Thus we have shown that minoxidil potentiates the mitogenic effects of fetal calf serum in vitro on NIH 3T3 fibroblasts by opening potassium channels and is also able to potentiate the mitogenic effects of the growth factors platelet-derived growth factor and insulin-like growth factor 1.

3T3 Cells↗
PubMed1996

Modeling acne in vitro.

To help elucidate the factors responsible for the infundibular changes seen in acne, the human sebaceous pilosebaceous infundibulum was isolated by microdissection and maintained for 7 d in keratinocyte serum-free medium supplemented with 50 micrograms/ml bovine pituitary extract, 100 units/ml penicillin and streptomycin, 2.5 micrograms/ml amphotericin B and CaCl2(10H2O) to give a final Ca2+ concentration of 2 mM. Infundibular structure was maintained over 7 d in this medium; the pattern of cell division mimicked that in vivo. The rate of cell division was significantly higher than previously described for infundibula maintained in supplemented William's E medium, and moreover did not fall over 7 d. The addition of 1 ng/ml interleukin-1 alpha (IL-1 alpha) caused hypercornification of the infundibulum similar to that seen in comedones; this could be blocked by 1000 ng/ml interleukin-1 receptor antagonist (IL-1ra). In about 20% of subjects there was spontaneous hypercornification of the infundibulum that could be blocked by 1000 ng/ml IL-1ra, suggesting that the infundibulum is capable of synthesising IL-1 alpha. The addition of 5 ng/ml epidermal growth factor or 5 ng/ml transforming growth factor-alpha to the medium caused a disorganisation of the keratinocytes of the infundibulum that resulted in rupturing similar to that seen in the more severe, purulent grades of acne. The addition of 1 microM 13-cis retinoic acid caused a significant reduction in the rate of DNA synthesis and apparent parakeratosis. We are now, therefore, able to model histologically the major infundibular changes in acne.

Acne Vulgaris↗
PubMed1996

The improved organ maintenance of the human sebaceous gland: modeling in vitro the effects of epidermal growth factor, androgens, estrogens, 13-cis retinoic acid, and phenol red.

We have previously reported that human sebaceous glands can be maintained for up to 14 d as whole organs with full retention of the physiological rate and pattern of new cell formation, but we have also reported that the newly formed cells did not differentiate normally, causing a progressive loss of lipogenesis in vitro. We now show that this abnormal sebocyte differentiation was attributable to the presence of epidermal growth factor (EGF) and phenol red in our maintenance medium. In their absence, human sebaceous glands apparently retain in vivo rates of cell division and lipogenesis over 7 d of maintenance in addition to a retention of in situ morphology. This is reversible on the re-addition of 10 ng EGF/ml and 10 mg phenol red/ml. The addition of 600 pM 17 beta-estradiol results in a significant fall in the rate of lipogenesis over 7 d of maintenance, without affecting the rate of cell division. This effect is apparently due to abnormal differentiation of newly formed sebocytes. Neither 1 nM testosterone nor 1 nM dihydrotestosterone (DHT) has any effect on rates of cell division of lipogenesis over 7 d. In the presence of phenol red, however, 1 nM testosterone or 1 nM DHT cause a significant reduction in the rate of lipogenesis over 7 d of maintenance. One micromolar 13-cis retinoic acid caused a significant reduction in the rate of lipogenesis over 7 d in both the presence and absence of phenol red. These findings show that we can model the physiological effects of steroids, EGF, and 13-cis retinoic acid in vitro.

Adult↗
PubMed1994

Human hair growth in vitro: a model for the study of hair follicle biology.

The factors that regulate hair follicle growth are still poorly understood. In vitro models may be useful in elucidating some aspects of hair follicle biology. We have developed an in vitro human hair growth model that enables us to maintain isolated human hair follicles for up to 10 days, during which time they continue to grow at an in vivo rate producing a keratinised hair fibre. We have shown that epidermal growth factor (EGF) in our system mimics the in vivo depilatory action of EGF in sheep, and suggest that this occurs as a result of EGF stimulating outer root sheath (ORS) cell proliferation which results in the disruption of normal mechanisms of cell-cell interaction in the hair follicle. We identify transforming growth factor-beta (TGF-beta) as a possible negative regulator of hair follicle growth and show that physiological levels of insulin-like growth factor-I (IGF-I) can support the same rates of hair follicle growth as supraphysiological levels of insulin. Furthermore, in the absence of insulin hair follicles show premature entry into a catagen-like state. This is prevented by physiological levels of IGF-I. Finally we demonstrate that the hair follicle is an aerobic glycolytic, glutaminolytic tissue and discuss the possible implications of this metabolism.

Aerobiosis↗
PubMed1994

Effects of EGF on the morphology and patterns of DNA synthesis in isolated human hair follicles.

We have previously reported that human hair grows at a normal rate in vitro for up to 10 d. We have also reported that, on gross observation, epidermal growth factor appears to induce a catagen-like effect on cultured hair follicles, but we have not characterized the details of this. We now report that when isolated human hair follicles are maintained in the presence of epidermal growth factor, the rate of hair follicle elongation is significantly stimulated but hair fiber production is inhibited. Light microscopy showed that epidermal growth factor stimulated a thickening and vacuolation of the cells of the lower outer root sheath of the hair follicle and that the matrix cells of the hair follicle underwent an upward migration resulting in the formation of a 'club hair'-like structure that remained connected to the dermal papilla by a thin strand of epithelial cells. [Methyl-3H] thymidine autoradiography was carried out to investigate the patterns of DNA synthesis and showed that epidermal growth factor inhibited DNA synthesis in the hair follicle matrix cells but dramatically stimulated DNA synthesis in the outer root sheath. We conclude from these studies that epidermal growth factor may be inducing an artificial 'catagen-like' effect by stimulating outer root sheath proliferation, which uncouples the normal patterns of proliferation and migration that occur in the anagen hair follicle and that result in an anagen-to-catagen-like transition. Moreover, these results also suggest that, under certain conditions, outer root sheath cells in the hair follicle may be capable of downward migration.

Adult↗
PubMed1994

Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro.

Insulin stimulated hair follicle growth in a dose-dependent manner over the range of 0.01 to 100 micrograms/ml. Maximum rates of hair follicle growth were observed when follicles were maintained in medium containing 10 micrograms/ml insulin, which is supraphysiologic. Hair follicles maintained in the absence of insulin or at physiologic levels showed premature entry into a catagen-like state. Insulin-like growth factor (IGF)-I and -II had no significant effect on hair follicle growth when maintained in the presence of 10 micrograms/ml insulin. However, in the absence of insulin, both IGF-I (0.01-100 ng/ml) and IGF-II (0.01-100 ng/ml) stimulated hair follicle growth in a dose-dependent manner. IGF-I was more potent than either insulin or IGF-II, stimulating maximum rates of hair follicle growth at 10 ng/ml, whereas IGF-II gave maximum stimulation at 100 ng/ml. The rates of hair follicle growth stimulated by 10 ng/ml IGF-I were identical to those stimulated by 10 micrograms/ml insulin. IGF-II (100 ng/ml), however, was unable to stimulate hair follicle growth to the same extent as insulin. Both IGF-I (10 ng/ml) and IGF-II (100 ng/ml) were more potent than insulin at preventing hair follicles from entering into a catagen-like state. Growth hormone had no effect on hair follicle growth or morphology in the absence of insulin. These data suggest that in vitro IGF-I may be an important physiologic regulator of hair growth and possibly the hair growth cycle. Moreover, the removal of insulin from tissue culture medium may be a useful method of generating large numbers of catagen hair follicles for further in vitro studies.

Cells, Cultured↗
PubMed1994

The isolation and maintenance of the human pilosebaceous unit.

We have previously developed methods for the isolation and maintenance of human sebaceous glands and hair follicles. However, in long-term culture the maintenance of both is suboptimal. This may be due to a lack of stem cells, which are thought to be located in the bulge area of the hair follicle, and this region is not present in either model. Isolation of the entire pilosebaceous unit would retain this region, and may lead to improved maintenance of both structures. We describe a method for the isolation of viable, individual, pilosebaceous units by microdissection from human scalp face-lift skin. The viability of isolated pilosebaceous units has been determined by light microscopy, patterns of DNA synthesis by [methyl-3H] thymidine autoradiography, and lipogenesis by [U-14C] acetate uptake into lipids. When maintained for 7 days in supplemented Williams E medium, isolated pilosebaceous units showed a significant increase in length. This was due to the production of a keratinized hair fibre which grew at the in vivo rate of 0.3 mm/day. Light microscopy and [methyl-3H] thymidine autoradiography confirmed that after 7 days maintenance the hair follicle retained apparently normal morphology and patterns of DNA synthesis, However, the morphology of the sebaceous gland on maintenance was more variable, generally showing luminal keratinization. Moreover [methyl-3H] thymidine autoradiography of sebaceous glands showed a marked reduction on maintenance. The rates and patterns of lipogenesis by the whole pilosebaceous unit were, respectively, lower and different from those seen with isolated human sebaceous glands; this indicates that the bulk of pilosebaceous lipogenesis is derived from the hair follicle. Rates of recovery of [14C] from 2 mM-[U-14C] sodium acetate into thin-layer chromatography plates after 7 days maintenance decreased, although this was not statistically significant, indicating that rates of lipogenesis may fall on maintenance. Pilosebaceous units were maintained for 7 days on Gelfoam (an absorbable gelatin sponge) at the media-air interface. Initial results show a marked improvement in sebaceous gland morphology. It is possible, therefore, to obtain viable human pilosebaceous units by microdissection, and to maintain them in vitro for up to 7 days, with apparently full retention of hair follicle function, but only partial retention of sebaceous gland function.

Autoradiography↗
PubMed1994

The human hair follicle engages in glutaminolysis and aerobic glycolysis: implications for skin, splanchnic and neoplastic metabolism.

On maintenance in supplemented Williams E medium, human hair follicles grow at the normal rate, and retain their normal anagen morphology, for up to 10 days. This permits us to study their metabolism under near-physiological conditions. The ATP content of freshly isolated follicles was 124.4 +/- 10.6 pmol/follicle (mean +/- SEM; n = 50). The energy charge was 0.81 +/- 0.08 and the glycogen content 2.3 +/- 0.3 nmol/follicle. These did not alter significantly during any metabolic studies, which were performed for up to 6 h in supplemented Williams E medium. We found that the major fuel was glucose, which at physiological concentrations yields 5.47 +/- 0.77 nmol ATP/follicle/h, but 90% of the glucose was metabolised to lactate, and only 10% oxidised. Glutamine was also an important fuel, generating 2.16 +/- 0.33 nmol ATP/follicle/h, but this too was largely metabolised to lactate rather than oxidised. Lipid fuels such as palmitate or beta-hydroxybutyrate only yielded 0.72 +/- 0.15 and 0.72 +/- 0.14 nmol ATP/follicle/h, respectively, and their oxidation did not inhibit glucose utilisation. No glucose-fatty acid cycle operates in the hair follicle, therefore, but a glucose-glutamine cycle does, since the presence of glutamine will inhibit glucose utilisation.

Adenosine Triphosphate↗
PubMed1993

Alpha-adrenoceptor stimulation of porcine pulmonary arteries.

The effects of alpha 1- and alpha 2-adrenoceptors stimulants on vascular tone of 188 isolated rings of pulmonary arteries from 24 pigs have been studied. The rings were pretreated with indomethacin, to inhibit cyclo-oxygenase. Isometric tension was recorded and concentrations of cyclic 3'5'-guanosine monophosphate (cGMP) and cyclic 3'5'-adenosine monophosphate (cAMP) were measured. Rings with endothelium contracted with phenylephrine (10(-5) M) (n = 41) and the alpha 1-adrenoceptor agonist methoxamine (10(-3) M) (n = 24). cGMP did not change with methoxamine, but rose with phenylephrine, peaking at 30 to 45 s. This preceded the maximum rise in tension with phenylephrine which occurred later at 120 to 360 s. The alpha 2-adrenoceptor agonist, clonidine (10(-5) M) (n = 33) and the muscarinic receptor agonist acetylcholine (10(-5) M) (n = 30) relaxed precontracted pulmonary arterial rings, minimum tension occurring after 120 s, whilst cGMP rose after 30-45 s. After removal of endothelium (n = 24), the tension after phenylephrine (10(-5) M) was higher and the rise in cGMP was abolished. The cAMP levels did not change with phenylephrine (10(-5) M), acetylcholine (10(-5) M), clonidine (10(-5) M) nor methoxamine (10(-3) M). Activation of alpha 1-adrenoceptors on pulmonary arteries smooth muscle cause contraction, whilst activation of alpha 2-adrenoceptors on endothelial cells cause relaxation probably through a release of nitric oxide and a rise in cGMP.

Acetylcholine↗
PubMed1993

Metabolism of freshly isolated human hair follicles capable of hair elongation: a glutaminolytic, aerobic glycolytic tissue.

The metabolism of the human hair follicle was investigated in vitro under conditions that maintained glycogen and adenosine triphosphate (ATP) content and the growth rate of the follicle at values observed in vivo. We have shown that only 10% of the total glucose utilized was oxidized to CO2 and 40% of this was oxidized via the pentose phosphate shunt. Although fatty acids and ketone bodies were oxidized by the hair follicle, they are poor energetic substitutes for glucose. Nor will fatty acids or ketone bodies sustain hair growth in vitro. Glutamine, however, was shown, both biochemically and by comparing growth rates, to be an important fuel with 23% of uptake being oxidized, generating a possible 2.16 +/- 0.32 nmoles ATP/follicle/h (mean +/- SEM) (glucose metabolism generates 4.54 +/- 0.61 nmoles ATP/follicle/h). Sixty-four percent of the glutamine taken up was calculated to be metabolized to lactate, showing that the hair follicle engages in both glycolysis and glutaminolysis. The glucose-fatty acid cycle appears to be unimportant in the hair follicle but our data indicates that a glucose-glutamine cycle does operate.

3-Hydroxybutyric Acid↗
PubMed1993

Isolation and maintenance of the human pilosebaceous duct: 13-cis retinoic acid acts directly on the duct in vitro.

The human pilosebaceous duct was isolated and maintained for 7 days in defined medium, and defined medium supplemented with 1 microM 13-cis retinoic acid. Freshly isolated ducts retained their in vivo morphology, showing a stratified squamous keratinizing epithelium. On maintenance there was a loss of basic duct architecture, and a significant reduction in the rate of [methyl-3H] thymidine uptake. The addition of 1 microM 13-cis retinoic acid resulted in an improved duct architecture and caused a further significant reduction in the rate of [methyl-3H] thymidine uptake. [Methyl-3H] thymidine autoradiography showed that freshly isolated ducts maintained their in situ pattern of cell division. It was difficult to discern the region of cell division in ducts maintained for 7 days, but the degree of graining reflected the measured rates of [methyl-3H] thymidine uptake into PCA precipitable material. The pattern of keratin synthesis of the freshly isolated duct was similar to patterns previously described for the duct in situ. This study reports the successful isolation and maintenance of the human pilosebaceous duct, and demonstrates that 13-cis retinoic acid acts directly at the level of the duct.

Cell Division↗
PubMed1993

Prolonged maintenance of human hair follicles in vitro in a serum-free medium.

We have previously reported the in vitro growth of human hair follicles for up to 4 days in a partially defined medium containing serum. We now report the prolonged in vitro growth of isolated human hair follicles for at least 9 days. This was achieved after analysis of the contribution of certain components of the original medium and, by a process of elimination, deriving a completely defined medium supplemented only with antibiotics, L-glutamine, insulin and hydrocortisone. We have shown, by [methyl-3H] thymidine autoradiography, that the hair follicles grown in this medium maintain an in vivo pattern of DNA synthesis, and that the gross morphology and histology of these maintained hair follicles remains similar to that of freshly isolated hair follicles. We have also shown that the patterns of keratin synthesis, as determined by [35S] methionine labelling, do not alter with maintenance.

Autoradiography↗