Distinct patterns of microvascular endothelial cell morphology are determined by extracellular matrix composition.

Endothelial interactions with the extracellular matrix (ECM) play important roles in angiogenesis but whether specific ECM signals can determine specific cellular morphologies is unclear. The authors compared in vitro ECM-induced morphological responses of the phenotypically distinct human placental microvascular endothelial cells (HPMECs) with large vessel endothelial cells (HUVECs). HPMECs showed distinct patterns of reorganization in response to collagen-I or collagen-IV (monolayer disruption, sprouting, migration) and Matrigel or laminin-A (intussusception, cord formation, tubulogenesis), and an intermediate response to fibrin; whereas HUVECs responded similarly to collagen-1 and Matrigel (elongation, lattice formation, vacuolation) and showed little response to fibrin. Although the extent of collagen and Matrigel responses of HPMECs were increased by serum, acidic or basic fibroblast growth factor (aFGF, bFGF), or vascular endothelial growth factor (VEGF), and varied with matrix protein concentration, the basic patterns were matrix specific, and were independent of fibronectin. The collagen responses correlated with disruption of adherens and tight junctions and the formation of filopodial protrusions. Matrigel responses were associated with up-regulated junctional localization of VE-cadherin, and tubulogenesis developed mainly through paracellular remodeling rather than intracellular vacuolation. Overall, these findings suggest that distinct ECM interactions stimulate specific morphological responses. These signals may regulate morphological behaviour in the angiogenesis cycle, switching endothelial cells between migratory and vasculogenic phenotypes.

Does low penetration of human skin by the normal mode ruby laser account for poor permanent depilatory success rates?

Studies reported to date have shown a good depilatory response from patients treated with the normal mode ruby laser (NMRL) over 12 weeks, but a low response over a time period greater than this. Previous publications have suggested that this could be accounted for by the apparently poor skin penetration of laser light and so this study attempted to assess whether this was indeed the case. Skin samples of varying thicknesses were taken from six Caucasian patients and their depths measured. Each was laid individually on an energy meter before having pulses from an NMRL compatible with clinical doses (4.75 J/cm2, 9.24 J/cm2 and 13.41 J/cm2) fired on the epidermis. Several samples had the laser fired repetitively on the surface to assess whether this caused any change in laser/skin fluence depth profiles. Repetitive firing of the NMRL on the epidermis of skin samples did not alter the energy recorded by the meter beneath. The fluence/depth profiles were constructed showing the majority of energy was lost within the first 1 mm of the skin surface (50%) which then further reduced over distance but at a much slower rate. The maximum depth of penetration was 14.8 mm (SD +/- 0.478) which appeared to be a function of wavelength and not fluence. The results suggest that laser penetration of skin should be adequate for generating enough heat at the hair bulge and bulb, potentially causing permanent damage. The implications of this study are that it is probably the presence of the correct chromophore in large enough amounts which is required for successful permanent depilation to occur.

Successful reduction in skin damage resulting from exposure to the normal-mode ruby laser in an animal model.

Normal-mode ruby laser (NMRL) irradiation of skin has now become an acceptable method of producing depilation. However, side effects, which include superficial burning and changes in skin pigmentation, still occur and, although temporary, can be distressing to the patient. This paper reports a method by which the skin can be protected (or preconditioned) from damage during NMRL treatment by pre-heating to a lower, non-damaging level prior to irradiation. Using the black-haired mouse (C57B1/10) as an animal model, an appropriate 'preconditioning' temperature was established by exposing the mouse skin to a range of temperatures, taking biopsies and staining the skin immunohistochemically for heat shock protein 70 (HSP 70) expression within the keratinocyte cells. Increased HSP 70 expression is stimulated by exposure to environmental stressors such as heat, so it was hypothesised that its increased expression conveyed increased cellular protection. The appropriate temperature (45 degrees C for 15 min) allowed for the superficial skin cells to be protected (as assessed by maximal HSP 70 staining) but undamaged (as assessed by haematoxylin and eosin staining), leaving the target hair-producing cells unprotected. Eight mice (16 flanks) were then exposed to this preconditioning temperature (eight of the flanks being growing-hair sites and eight resting-hair sites) and 5 h later exposed to a laser fluence known to cause mild skin damage and depilation (6J/cm2). This exposure was to both the preconditioned and the adjacent non-preconditioned sites. A statistically significant reduction in skin damage (P <0.001), as measured by the time taken to heal and noted both clinically and histologically, was seen in the preconditioned sites in resting-hair regions but not in growing-hair regions. Depilation, established over an 8 week period, was successful in growing-hair regions within both preconditioned and non-preconditioned sites, but complete hair regrowth had occurred in preconditioned and non-preconditioned sites within resting-hair regions by 5 weeks. The latter finding was consistent with work already reported suggesting that NMRL-assisted depilation in this animal model is not successful for hairs in the telogen phase. Successful preconditioning of mouse skin prior to laser exposure appears to reduce NMRL-induced skin side effects. In addition, the technique does not appear to adversely affect successful depilation.

The temperatures reached and the damage caused to hair follicles by the normal-mode ruby laser when used for depilation.

Although it is proposed that heat is the cause of follicular damage leading to depilation, this has never been proved. This study aims to determine the mode by which depilation is effected and, if heat is the mechanism, what temperatures are reached within treated follicles and if sufficient damage is produced therein. Two excised specimens of hair-bearing skin from 5 patients undergoing facelifts were dissected to reveal the hair bulbs/shafts on the deep surface. They were placed on a jig, and one pulse from a normal-mode ruby laser (NMRL) of 15 J per square centimeter was fired on the epidermal surface. A thermal imaging camera recorded dermal temperature changes on the deep surface in real time. Specimens were then examined histologically for the site and extent of cellular damage by immunohistochemical staining for a protein marker of cell damage (p53). The NMRL targeted hair follicles specifically. The most common follicular temperature increase ranged from 5 to 10 degrees C. In specimens from 1 patient the increase was more than 30 degrees C (p < 0.001). Heat dissipation into interfollicular tissue in all specimens occurred 2 seconds after exposure. Evidence of laser-induced damage to follicle-lining cells was found only in those follicles with damaged hair shafts. The changes were found to a greater depth (to the bulb) and greater extent (beyond the bulge) in those follicles reaching higher temperatures. These findings suggest that the NMRL should produce permanent depilation. The variability between follicles and between patients explains, perhaps, the uneven outcome regarding depilation using the NMRL. Success appears to depend on peak follicular temperatures achieved during laser exposure, which may result from the follicular characteristics of the individual patient.

A review of the ruby laser with reference to hair depilation.

There is a clinical need in the fields of reconstructive and cosmetic plastic surgery for a safe, simple, and effective method of hair depilation. Depilatory clinics have been established throughout the country, commonly using the ruby laser, to treat a cohort of the population, estimated to be between 6% and 10%, recognized as being hirsute. Clinical trials performed to date have not established a protocol that suits the previously mentioned criteria and have been, usually, small in number and short in follow-up. With the increased use that this form of laser treatment will inevitably undergo, it is the belief of the authors that the only way of ascertaining whether the treatment is safe, simple, and effective is first to establish how the ruby laser works. This review relates the knowledge that is currently available regarding the function of the ruby laser to a number of the clinical studies that have been undertaken, including three that have used other types of laser. Using this information, future areas in which research is required can be defined, ultimately to improve the clinical efficacy of ruby laser-assisted hair removal while lessening the current side effects (namely, superficial burning, and hypo- and hyperpigmentation).

TMJ morphology and function in a patient with Klippel-Trenaunay syndrome. Case report.

Klippel-Trenaunay syndrome is a rare congenital mesodermal disturbance of uncertain etiology in variable expression. The classic manifestation is the triad of congenital mesodermal abnormalities. Clinically a diversity of phenotypes with subjacent malformations may be encountered. The deviations of the mesioblastic germ layer affecting angioblastic, lymphoblastic and osteoblastic structures, may give rise to malformations either alone or in an unlimited diversity of associations. While this syndrome may be diagnosed by chance in the course of ultrasonic scanning during pregnancy, it is normally diagnosed during infancy or early childhood. Evaluation and carefully coordinated medical treatment are important in minimizing morbidity and relieving multiple complaints. The aim of our investigation was to evaluate and correlate the clinical, functional, radiographic and MRI findings in a patient suffering from this syndrome. We present a 13-year-old male patient suffering from extreme facial asymmetry in association with hypertrophy of the complete right side of the body. In spite of pronounced functional and morphologic asymmetry, no signs of degenerative joint disease were identified by radiography or MRI. Since temporomandibular joint dysfunction and facial asymmetry can result in irreversible degenerative joint disease, close follow-up monitoring is indispensable if joint damage is to be prevented.

The effect of 5-fluorouracil on Dupuytren fibroblast proliferation and differentiation.

INTRODUCTION: Dupuytren's disease is a proliferative disease with contractile properties, prone to recur after surgery. Intra-operatively applied 5-fluorouracil has been used to avoid scar problems in the eye after glaucoma filtration surgery and was therefore investigated as a means to inhibit proliferation and myofibroblast differentiation in Dupuytren fibroblasts in vitro. METHOD: Primary cell lines were obtained by explants from Dupuytren's tissue (n = 6), non-diseased palmar fascia from patients with Dupuytren's disease (n = 3) and carpal ligament from patients undergoing carpal tunnel release (n = 3). The effect of 5-fluorouracil on proliferation was assessed by cell counting. Myofibroblast differentiation, an intergral part of Dupuytren's contracture, was investigated by staining for alpha smooth muscle actin, a marker for contractile cells, using immunohisto-chemical methods. RESULTS: A single exposure to 5-fluorouracil caused a sustained inhibition of proliferation in Dupuytren's and non-diseased fascia cultures, whilst the effect on carpal ligament cultures was transient. Untreated Dupuytren's fibroblasts exhibited the highest myofibroblast differentiation, whilst differentiation in non-diseased fascia cultures was shown to be proportional to cell density and virtually non-existent in carpal ligament cultures. After 5-fluorouracil exposure, the differentiation was significantly reduced in Dupuytren's fibroblasts cultures, reduced at high cell densities in non-diseased fascia and unchanged in carpal ligament cell cultures. DISCUSSION: 5-fluorouracil inhibits both proliferation and myofibroblast differentiation in Dupuytren's cell cultures and may have a potential use as an adjuvant treatment to Dupuytren surgery in order to reduce the rate of recurrence and contracture.

The effect of ruby laser light on cellular proliferation of epidermal cells.

In ruby laser-assisted hair removal, microscopic damage is often seen in the basal epidermal cells, where melanosomes are concentrated. It is not known whether this treatment leads to cellular hyperproliferation. It was the aim of this study to investigate this. Ten white patients were treated with the Chromos 694-nm Depilation Ruby Laser, and biopsies taken before and after treatments to assess the presence of cell hyperproliferation, which normally accompanies epidermal damage, with immunohistochemical staining of keratin 16 and Ki67. No evidence of cell hyperproliferation was seen in all specimens examined after ruby laser irradiation. The authors conclude that despite the possible microscopic damages seen in the basal epidermis after laser hair removal, there is no evidence of cellular hyperproliferation. This is in contrast to ultraviolet-irradiated cell damage, in which increased basal cell turnover is seen.

Ruby laser irradiation (694 nm) of human skin biopsies: assessment by electron spin resonance spectroscopy of free radical production and oxidative stress during laser depilation.

Human skin biopsies (hair-bearing scalp skin and non-hair-bearing breast skin) were treated with t-butylhydroperoxide, irradiated with UV light (UVR) or irradiated with 694 nm ruby laser red light. Free-radical production and oxidative stress were assessed with electron spin resonance spectroscopy (ESR) using the ascorbate radical as a marker. In comparison with both UVR and t-butyl-hydroperoxide (which readily induce the ascorbate radical in hair-bearing and hairless skin), 694 nm red light does not result in the formation of the ascorbate radical in detectable concentrations. Spin-trapping experiments with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that while free radicals could be detected after treatment of skin with t-butylhydroperoxide, radicals could not be trapped after laser treatment. Treatment of lasered skin (containing DMPO) with t-butylhydroperoxide produced radical adducts as well as the ascorbate radical, demonstrating that the laser neither depletes endogenous ascorbate nor the preadministered spin trap. It is concluded that 694 nm red light does not induce oxidative stress in human skin in levels comparable either to t-butyl hydroperoxide or UV light.

Hair removal using the ruby laser: clinical efficacy in Fitzpatrick skin types I-V and histological changes in epidermal melanocytes.

The ruby laser is effective in removing unwanted body hair. The occurrence of cutaneous side-effects such as blistering, hypopigmentation and hyperpigmentation, however, remains problematic. These side-effects are more commonly seen in patients with dark coloured skin, which partly explains the relative scarcity of information on the efficacy of ruby laser hair removal in such patients. The mechanisms of the occurrence of these side-effects are also not known. It was the aim of this study to evaluate the efficacy of ruby laser-assisted hair removal in patients with Fitzpatrick skin type V in a retrospective clinical study and to evaluate the mechanism of post-treatment pigmentary change in a prospective clinical study. The percentage reduction in hair density in patients with skin type V was assessed after a variable period following treatment with the Chromos 694 Depilation Ruby Laser, and was compared with the results of those with skin types I-IV. To study the pigmentary change and melanocyte numbers after laser irradiation, ex-vivo scalp skin and serial patient biopsies were taken and stained with S-100, dopa oxidase and Masson-Fontana methods. Laser treatment reduced melanocyte numbers as measured by DOPA stain but not by S100. Laser treatment resulted in the clearance of pigment from the epidermis on histology. Ruby laser was shown to be effective in removing unwanted hair from patients with dark coloured skin, but with a higher incidence of cutaneous side-effects. The occurrence of hypopigmentation after laser irradiation was thought to be due to the suppression of melanogenesis in the epidermis rather than to destruction of the melanocytes.

The presence of melanin in genomic DNA isolated from pigmented cell lines interferes with successful polymerase chain reaction: a solution.

We have found that polymerase chain reaction (PCR) amplification of genetic material is unsuccessful when using template genomic DNA derived from certain normal melanocyte and melanoma cell lines. We demonstrated that this phenomenon only occurs with pigmented cell lines and appears to be due to the association of genomic DNA with remnants of the pigment melanin. We therefore describe a simple and rapid technique that rids genomic DNA samples of melanin, resulting in a genomic DNA template that allows successful PCR.

Ruby laser-assisted hair removal success in relation to anatomic factors and melanin content of hair follicles.

Ruby laser-assisted hair removal is thought to work via selective photothermolysis, which relies on light reaching the deeper layers of skin, and the absorption of light by the target chromophore, melanin. It is therefore possible that efficacy of treatment is affected by anatomic factors that determine the amount of light reaching the hair bulbs (i.e., skin color, depth of intracutaneous hair, epidermal thickness and dermal density) and the melanin content of hair. To examine this hypothesis, a prospective study was performed. Forty-eight volunteers were treated with the Chromos 694 Depilation Ruby Laser at a single standard fluence of 11 J/cm2. Treatment efficacy was determined by measuring hair density at 3 and 7 months after treatment. Epidermal depth and dermal density were measured from 2-mm biopsies taken before treatment, and the intracutaneous hair length was determined from plucked hair. Skin color was assessed using a spectrophotometer, and melanin content of dissolved hair was assessed using spectrophotometry. Efficacy of treatment for each patient was compared with the patient's age, intracutaneous hair length, epidermal depth, dermal density, skin color, and total melanin content and relative eumelanin content of hair. No correlation was found between the efficacy of treatment and age and the various anatomic factors. Patients with higher eumelanin content in their hair had better long-term results (Spearman rank test, p = 0.00219). The results suggested that the efficacy of treatment did not depend solely on the amount of laser light penetrating the skin but correlated well with the eumelanin content of hair. The clinical implication of this finding is discussed.

The effect of ruby laser light on ex vivo hair follicles: clinical implications.

Several clinical studies on the efficacy of ruby laser-assisted hair removal have reported that regrowth of hair after treatment is common. One of the reasons for the regrowth of hair is the incomplete destruction of germinative hair cells due to the insufficient penetration of the ruby laser in the skin. It was the aim of this study to estimate the extent of damage to the hair follicles after one ruby laser treatment and to determine whether the ruby laser destroyed the bulbs and the bulge regions of hair follicles. The extent of laser damage in hair shafts was determined by serial examination of six specimens of ex vivo scalp skin lasered with the Chromos 694 Depilation Ruby Laser at 14 J per square centimeter and 20 J per square centimeter. Another nine specimens of ex vivo scalp skin were similarly lasered, and monoclonal antibody LP2K was used to identify the bulge regions of the hair follicles using the immunoperoxidase technique. Damage to the bulge region was assessed from consecutive specimens, which were stained with hematoxylin-eosin stain. The mean depth of laser damage sustained by hair follicles was 1.34 mm (14 J per square centimeter) and 1.49 mm (20 J per square centimeter) underneath the skin surface. Most of the laser damage involved the bulge regions but fell short of the hair bulbs. The laser damage did not seem to extend far enough down the hair shafts to result in permanent hair destruction. The clinical implications of this finding are discussed.

Ruby laser-assisted hair removal: a preliminary report of the correlation between efficacy of treatment and melanin content of hair and the growth phases of hair at a specific site.

An unpredictable response, even in patients with dark hair, often undermines successful ruby laser hair removal. A prospective clinical study was carried out to evaluate the roles of melanin content and growth phases of hair in treatment efficacy. Thirty-six volunteers with white skin and dark hair were recruited for the study, and were all treated using the Chromos 694 Depilation Ruby Laser. The overall efficacy of treatment was assessed at the end of 3 months. The efficacy of laser treatment is not due solely to the proportion of hair in the growing or static phase of the hair cycle. There is a lack of correlation between the melanin content and the overall efficacy of laser hair removal in those treated once, but patients with darker hair responded better after repeated treatments. The proportion of hair in the growth phase and the melanin content of hair do not contribute solely to the efficacy of ruby laser hair removal.

Ruby laser-assisted hair removal: an ultrastructural evaluation of cutaneous damage.

Ruby laser-assisted hair removal is thought to act via selective photothermolysis of melanin in the hair follicles. Although initial clinical trials of permanent hair removal using ruby lasers are promising, the exact mechanisms of hair destruction and the potential damage to other structures of skin are not known. The aim of this study was to evaluate the cutaneous ultrastructural changes following ruby laser hair removal. Nineteen healthy Caucasian patients with dark (brown/black) hair were treated with the ruby laser and biopsies taken after 0, 2, 3, 5, 7, 14 and 21 days. Specimens were examined by light and electron microscopy. Laser-treated specimens showed widespread coagulation and charring of subcutaneous hair shafts. These obviously damaged follicles were randomly dispersed amongst intact follicles within the same treatment sites. Microscopic changes were also seen in the basal epidermis where melanin was concentrated, irrespective of any obvious macroscopic damage. A low level of inflammatory response seen up to 2 weeks after treatment always followed laser treatment. Suprabasal epidermal necrosis was only seen in patients with blister formation after treatment. Ruby laser irradiation results in selective damage to the hair follicles, with microscopic changes to the basal epidermis. The damage is probably compounded by the inflammatory response to the damaged hair. The normal appearance and distribution of collagen in the dermal layer supported the clinical evidence that laser-assisted hair removal, if performed correctly, does not lead to scar formation.

Ruby laser-assisted hair removal reduces the coarseness of regrowing hairs: fallacy or fact?

There have been anecdotal reports that hairs that regrow after ruby laser-assisted hair removal are finer in appearance. If true, this phenomenon adds to the improved aesthetic effect of laser treatment of unwanted hair. It is the aim of this study to determine whether this phenomenon indeed occurs, and if so, assess its permanence and its mode of action. In this prospective clinical study, 71 patients with 94 treatment sites were treated with the Chromos 694 Depilation Ruby Laser. Hair diameter was measured pre-treatment, and at 3 and 7 months post-treatment. In addition, ex vivo scalp skin was used to assess if the ruby laser selectively damaged coarser hairs. Laser-treated and matched untreated skin samples were histologically assessed and the diameters of hair shafts (normal or obviously damaged) were measured. Results of this study were analysed using Kruskal-Wallis one-way analysis. There was no statistically significant difference between the hair diameter of non-lasered specimens and the hair diameter of the normal hair in lasered specimens. However, a statistically significant difference was seen between the hair diameter of non-lasered specimens and diameters of damaged hair in lasered specimens (P < 0.05). There was a statistically significant difference (P < 0.05) between pre-treatment and 3 month hair diameters, but no statistically significant difference was found between pre-treatment and 7 month hair diameters. In conclusion, ruby laser-assisted hair removal results in a temporary reduction in hair diameter of regrowing hair. This is not due to the selective targeting of larger hair follicles.

Interactions of CD80 and CD86 with CD28 and CTLA4.

CD80 and CD86 are cell surface glycoproteins expressed on a variety of professional APCs. They have attracted much attention due to their function as potent costimulators of T lymphocyte function through their interaction with CD28 and possibly CTLA4. Because inhibitors of this interaction may have therapeutic relevance in human autoimmune disease, we investigated the properties of linear peptides derived from conserved regions of CTLA4 and CD80 known to be essential for binding. None of these peptides were sufficient to bind ligand, nor did they act as potent competitive inhibitors. Conformationally constrained versions of the CTLA4 motif were also inactive. These results suggested that other parts of the proteins are important in determining binding, so a series of modified CD80 and CD86 molecules were constructed in an attempt to identify other binding determinants. Insertion of two residues between the two Ig domains of CD80 resulted in decreased affinity for CTLA4, but a similar mutation in CD86 was without effect. We also identified another asymmetry between CD80 and CD86 in that the V domain of CD86 but not that of CD80 is sufficient for CTLA4 binding. The CD86-V domain appears to have CTLA4 binding properties equivalent to that of intact CD86. These data illustrate a fundamental difference between these costimulatory molecules and suggest a mechanism by which they may be differentially recognized by receptors on the T cell surface.

Establishment and maintenance of normal human keratinocyte cultures.

Keratinocytes are the major cellular component of the epidermis, which is the stratified squamous epithelia forming the outer-most layer of skin. The keratinocytes lie on a basement membrane and are organized into distinct cell layers which differ morphologically and biochemically These regions from the basement membrane outward are the basal, spinous, granular, and cornified layers. Cellular proliferation takes place mainly in the basal layer. On division, keratinocytes give rise to either replacement progenitor cells and/or cells that are committed to undergo the process of terminal differentiation These latter cells leave the basal layer and gradually migrate upward, simultaneously progressing along the differentiation pathway as they go. Finally they reach the outer surface of the epidermis in the form of fully mature functional cells, the corneocytes. The function of these mature cells is the protection of the underlying viable tissues from the external milieu.