Cloning and expression of class I major histocompatibility complex genes of the rat.

Little is known about the organization of class I genes in the rat although there is prima facie evidence that it is distinct from that of the mouse. We report the cloning of 61 nonclassical rat class I genes into cosmid clusters with a total mapped length of 1,264 kb. It is certain that the total number of class I genes in the rat must exceed this number. From restriction maps it is possible to identify substantial regions of duplication. By transfection of cosmids into mouse L cells, it has been possible to demonstrate at least seven different nonclassical rat class I genes that are expressible on the cell surface. Crossreaction of a single mouse monoclonal antibody with all of these class I molecules is consistent with sequence homogenization within the rat nonclassical system. Attempts to find rat homologues of the mouse Tla genes by crosshybridization of rat cosmids with a range of different TLa-specific probes were unsuccessful, suggesting that this large group of divergent class I genes is absent or nearly so from the rat. The large number of class I genes in the rat appears to have arisen by expansion of genes more closely related to the classical sequence.

Ultrastructure of collagen thermally denatured by microsecond domain pulsed carbon dioxide laser.

BACKGROUND: Clinical improvement in photodamaged skin after carbon dioxide (CO2) laser resurfacing is thought to result in part from thermal collagen shrinkage. The presence of such collagen has not been unequivocally demonstrated. To identify and characterize the morphological features of collagen after CO2 laser exposure, we irradiated ex vivo human facial skin and bovine calcaneus tendon with microsecond domain pulsed CO2 laser energy and examined specimens for histopathological and ultrastructural changes in collagen. OBSERVATIONS: In dermis and tendon, 3 zones of collagen structure were apparent on electron microscopy. The first, most superficial zone demonstrated loss of collagen structure. The second zone consisted of admixed normal collagen fibers and thickened collagen fibers. Zone 3 consisted of normal-appearing collagen fibers. CONCLUSIONS: Ultrastructural examination of irradiated collagen revealed distinct morphological zones of denatured collagen fibers. Partially denatured fibers had an increased diameter consistent with lineal shrinkage. Zonal distinction was undetectable by light microscopy. Ultrastructurally, the zones of denatured collagen located above the normal fibers correlated with the zone of altered material seen on light microscopy. These findings suggest that collagen fiber shrinkage does occur after pulsed CO2 laser irradiation and that this phenomenon contributed, at least in part, to the immediate tissue contraction observed clinically.

Pulsed carbon dioxide laser resurfacing of photo-aged facial skin.

BACKGROUND AND DESIGN: public demand for procedures to rejuvenate photoaged skin have stimulated the use of high-energy short-pulsed carbon dioxide lasers as a precise and predictable treatment modality. The purpose of this study was to determine the degree of clinical improvement achieved in treating perioral and periorbital wrinkles with a high-energy, microsecond-domain pulsed CO2 laser. Photodamaged skin in the perioral (n=73) and periorbital (n=38) regions was treated with multiple passes of confluent single pulses of CO2 laser energy (10 600 nm, 3-mm collimated beam, <1- millisecond pulse, 450 mJ per pulse, 2 to 5 W), with the tissue being cleansed and débrided with normal saline between passes. A nine-point clinical scoring system was devised for evaluation of the degree of wrinkling and photodamage present. Preoperative and postoperative photographs were independently scored by four ¿blinded¿ reviewers. The patients were observed postoperatively for 1 to 12 months for the course of healing, and adverse events were recorded. RESULTS: All three classes (mild, moderate, and severe) of photoaging of the skin responded equally well, showing an average wrinkling score reduction of 2.25 for the periorbital region and 2.34 for the perioral region, the most superficial wrinkles and photodamage being eliminated and the more severe being markedly improved. An unexpected finding was tightening of loose and folded skin. Side effects included transient erythemia and postinflammatory hyperpigmentation, and one instance of an isolated hypertrophic scar. CONCLUSIONS: Resurfacing of photoaged skin by means of a high-energy, microsecond-domain pulsed CO2 laser with a specific clinical treatment protocol results in predictable improvement in perioral and periorbital wrinkling and photodamage with minimal risks. Heat-induced collagen shrinkage appears to contribute to these results by tightening loose skin and folds.

Tattoo darkening and nonresponse after laser treatment: a possible role for titanium dioxide.

OBJECTIVE: To examine relationships between chemical composition, biopsy findings, and clinical outcome in laser-treated tattoos. DESIGN: Observational nonblinded retrospective study. SETTINGS: University-based dermatology clinic and private practice. PARTICIPANTS: Twenty patients who underwent biopsy of laser-treated tattoos. MAIN OUTCOME MEASURES: Biopsy specimens were analyzed after laser treatment, and the depths of changed particles were recorded. Ultrastructure of the changed particles was examined by electron microscopy. Presence of inorganic chemicals was determined by x-ray diffraction. Correlation between x-ray diffraction, microscopy, and clinical response was attempted. RESULTS: Of the 20 tattoos, 7 lightened, 9 failed to change, and 4 darkened after laser treatment. There was a significant association between presence of titanium dioxide and poor response to laser therapy. Microscopic studies showed variable changes in the ink particles, but there was a trend toward residual deep green pigment in the resistant tattoos. Also, round dark stippling was observed superficially in the darkened specimens. CONCLUSIONS: Titanium is overrepresented in tattoos that respond poorly to laser treatment. Further studies are necessary to show whether this metal is the primary cause of this poor response.

Electrosurgical facial resurfacing: a prospective multicenter study of efficacy and safety.

BACKGROUND: A novel electrosurgical technology that uses a bipolar electrode-tipped stylet to deliver relatively low-radiofrequency energy through an electrically conductive medium has been developed. OBJECTIVE: To evaluate the efficacy and safety of the radiofrequency resurfacing system for the treatment of facial wrinkles. DESIGN: Multicenter, prospective, noncomparative study with longitudinal follow-up. SETTING: Four US academic dermatologic surgery clinics. PATIENTS: Ninety-five patients with mild to severe photodamage (Fitzpatrick classes I-III) involving periorbital (75 treatment sites) and perioral (50 sites) facial skin. INTERVENTION: Radiofrequency resurfacing with the use of 2 to 3 passes at 125 or 139 V. MAIN OUTCOME MEASURES: Wrinkle and cosmetic improvements evaluated by patients, investigators, and, by means of photographs, an independent panel of 5 evaluators. RESULTS: All evaluators determined a positive mean improvement in wrinkles for both periorbital and perioral anatomic sites, with greater improvement for patients with more severe wrinkles at baseline. An increased number of passes and higher voltage settings had a positive impact on wrinkle improvement. Transient postinflammatory hyperpigmentation occurred in 26% of periorbital and 4% of perioral sites. Hypertrophic scars occurred in 3.8% of treatment sites, with all but 1 scar resolving by 6 months. For the most part, healing was rapid, pain was minimal, and erythema largely resolved within 2 months. Other untoward effects were relatively few and short-lived. CONCLUSIONS: At the study settings used, radiofrequency resurfacing is an effective modality in the treatment of periorbital and perioral wrinkles in patients with Fitzpatrick class I, II, and III photodamage. There is less severe postoperative morbidity than seen with carbon dioxide or coagulating erbium:YAG lasers. The potential risks are similar to those seen with other resurfacing modalities.

Protoporphyrin IX fluorescence induced in basal cell carcinoma by oral delta-aminolevulinic acid.

Limited depth of penetration significantly limits photodynamic therapy of nodular basal cell carcinoma (BCC) using topical delta (5)-aminolevulinic acid (ALA). To demonstrate safety and efficacy of orally administered ALA in inducing endogenous protoporphyrin IX (PpIX) production in BCC, 13 patients with BCC ingested ALA in a dose-escalation protocol. All dose ranges (10, 20 or 40 mg/kg single doses) resulted in formation of PpIX in human skin and BCC, measurable by in vivo fluorescence spectrophotometry. The PpIX fluorescence peaked in tumors before normal adjacent skin from 1 to 3 h after ALA ingestion. Gross fluorescence imaging of ex vivo specimens revealed greater PpIX fluorescence in tumor than normal skin only at the 40 mg/kg dose. Fluorescence microscopy confirmed this finding by showing distinct, full-thickness PpIX fluorescence in all subtypes of BCC only after ALA given at 40 mg/kg. Side effects were dose dependent and self limited. Photosensitivity lasting less than 24 h and nausea coinciding with peak skin PpIX fluorescence occurred at 20 and 40 mg/kg doses. After 40 mg/kg ALA, serum hepatic enzyme levels rose to a maximum within 24 h, then resolved over 1-3 weeks. Transient bilirubinuria occurred in two patients.

Lack of selectivity of protoporphyrin IX fluorescence for basal cell carcinoma after topical application of 5-aminolevulinic acid: implications for photodynamic treatment.

Clinical trials of topical ALA in photodynamic therapy (PDT) of basal cell carcinoma (BCC) show significant recurrence rates. Exogenous 5-aminolevulinic acid (ALA) is converted by intracellular enzymes to photoactive protoporphyrin IX (PpIX) in human tissues. PpIX generates cytotoxic singlet oxygen when irradiated with visible light in the 400-640 nm range. To evaluate variability and heterogeneity in PpIX production by tumors in such trials, and to assess the usefulness of PpIX for marking skin tumors, we measured PpIX fluorescence distribution in BCC after topical application of 20% ALA cream. ALA cream was applied under occlusion for periods ranging from 3 to 18 h (average 6.9 h, SD 4 h) to 16 BCCs. ALA conversion to PpIX in the BCCs was assessed by in vivo photography, ex vivo video fluorescence imaging, and fluorescence microscopy. External macroscopic PpIX fluorescence, as assessed by in vivo and ex vivo imaging, correlated with the clinical presence of BCC. Examination by a digital imaging fluorescence microscope revealed inter- and intratumor fluorescence variability and heterogeneity. PpIX fluorescence corresponding to full tomor thickness was found in six superficial and four nodular tumors, and partial-thickness fluorescence was observed in five nodular tumors, but no PpIX fluorescence was observed in some areas of superficial, nodular and infiltrating tumors. In a significant number of nodular and infiltrating BCCs, topical ALA appeared to provide little or no PpIX in deep tumor lobules. In addition, no selectivity for tumor tissue versus normal epidermis was seen. The grossly brighter external PpIX fluorescence over tumors may be due, therefore, to enhanced penetration through tumor-reactive stratum corneum and to the tumor thickness.(ABSTRACT TRUNCATED AT 250 WORDS)

New methods in cutaneous resurfacing.

The ongoing effort to create an optimal method of skin rejuvenation has led to several new treatment options. Microdermabrasion and various nonablative laser resurfacing systems produce minimal skin injury, whereas current RF resurfacing devices may create both ablative or nonablative effects. The less invasive methods do limit the length of an uncomfortable healing time; however, studies appear to indicate that the results are less impressive than those produced by more destructive techniques and may prove only temporary over the time frame of a year. Efficacy and durability of clinical improvement seem to be inversely related to the wounding depth, the length of the healing period, or both. The search continues for the perfect method of skin rejuvenation, which minimizes cost, healing time, adverse effects, and discomfort and maximizes efficacy, response durability, and reproducibility. With advancements in technology, rejuvenation methods slowly approach these elusive goals.

Multi-electrode radio frequency resurfacing of ex vivo human skin.

BACKGROUND: A novel radio frequency multi-electrode surgical (MES) device has produced minimal residual thermal collagen damage (RTCD) and rapid healing in treating articular cartilage. OBJECTIVE: To assess the immediate tissue effects of the MES device on human skin. METHODS: Specimens of ex vivo human skin were subjected to 1 to 3 passes of the device at 3 voltage settings, then processed for routine and polarized light microscopy. The degree of tissue ablation, amount of RTCD, and level of follicular injury were assessed. RESULTS: Epidermal ablation did not occur; epidermal removal correlated with manual debridement after the first passes. Dermal ablation was not readily discernable even after 2 or 3 passes. The thickness of RTCD tended to increase with increased pass number. The mean thickness of this zone of damaged collagen measured 24-53 micro after 1, 63-83 micro after 2, and 80-97 micro after 3 passes, respectively. For a given number of passes, the thickness of the zone of RTCD did not consistently change with increasing voltage applied (86, 108, and 139 V). The small number of samples did not enable meaningful statistical analysis of these observations. In samples with pilosebaceous units, the electrosurgical keratinocyte injury extended down the follicular epithelium, but never beyond the level of the infundibulum. CONCLUSIONS: These findings suggest the MES device produces immediate tissue effects favorable for effective cutaneous resurfacing and rapid postoperative wound healing.

"Hot" KTP-laser treatment of facial angiofibromata.

BACKGROUND AND OBJECTIVE: Treatment of facial angiofibromata (AF) relied largely upon cutaneous resurfacing. While effective, resurfacing affects large areas with attendant risks of dyspigmentation, infection, and scarring. We investigated the pulsed KTP (532 nm) laser energy for its high absorption by melanin and hemoglobin as a photothermal destructive method for treating AF. STUDY DESIGN/MATERIALS AND METHODS: In five patients (Fitzpatrick phototypes II-;VI), AF were treated with the KTP laser (10 ms, 20 J/cm(2), 2 mm beam) using stacked pulses (2-3.3 Hz) or passes. No cooling device was employed. Each pulse evoked puffs of steam and caused progressive flattening of AF. Normal intervening skin was strictly avoided. Patients underwent one to five sessions in which as many as 100 lesions were treated. RESULTS: Individual lesions responded with complete flattening in one or two treatments. While this effect has persisted for 18-;24 months, slow recrudescence is expected. Transient hypopigmentation and hyperpigmentation were localized to treated skin. No scarring, infection, or other adverse events were observed. Patient satisfaction with this method was high due to rapid healing time ( < 10 days), minimal pain, ease of wound care, and efficacy. CONCLUSIONS: "Hot" KTP laser is an effective and safe method of treatment for facial AF. Limiting treatment only to lesional skin allowed rapid healing and very limited adverse effects despite the increased non-specific thermal damage caused by high fluence, long pulse duration, and an absence of superficial tissue cooling.

Merkel cell carcinoma. Histopathology, immunohistochemistry, and cytogenetic analysis.

BACKGROUND: Merkel cell carcinoma (MCC) is a cutaneous neoplasm, histopathologically difficult to differentiate from other small blue cell neoplasms. Immunohistochemical and ultrastructural analyses are usually helpful in differentiating these neoplasms. Recently, cytogenetic analysis has emerged as a potential tool in the diagnosis of solid neoplasms, including MCC. OBJECTIVE: To describe the immunohistochemical and cytogenetic features of a case of primary MCC and to review the cytogenetics literature on MCC. METHODS: Formalin-fixed tissue was processed routinely and labeled with a battery of antibodies. Metaphase cells from fresh tissue were prepared by Giemsa banding. RESULTS: Histopathologically, there were irregular aggregates of pyknotic cells with little cytoplasm. Immunohistochemically, the neoplastic cells stained positive for neurofilament, cytokeratin, neuron-specific enolase, and epithelial membrane antigen. Leucocyte common antigen, S-100, 013, and chromogranin were negative. Karyotyping of neoplastic cells showed loss of chromosome Y (-Y). CONCLUSIONS: Coexpression of cytokeratin and neurofilament is characteristic of MCC and allows it to be differentiated from similar neoplasms. The significance of Y chromosome loss is unclear. Further cytogenetic analyses are warranted to identify genetic mutations significant to the pathogenesis of MCC.

Rhinophyma's fibrous variant. Histopathology and immunohistochemistry.

Rhinophyma may present with either of two distinct histopathologic appearances. The most common shows histopathologic features of rosacea. The second pattern shows telangiectasia, diffuse dermal fibrosis with abundant mucin, and a virtual absence of pilosebaceous structures. These histopathologic features of the lesser-known fibrous variant of rhinophyma mimic those of fibrous papule of the nose. We report an unusual case that histopathologically resembled the second variant. Immunohistochemically, the dermal fibroblasts in this case showed intense staining with Factor XIIIa in a pattern similar to that demonstrated in fibrous papules. S-100 protein failed to stain these dermal fibroblasts. The presence of Factor XIIIa+ dermal fibroblasts may portend the evolution of fibrosis in rosacea and other inflammatory dermatoses.

Surgical pearl: phlebectomy hook Norplant extraction.

PIP: Removal of Norplant contraceptive implants may be rendered difficult by the size of the capsules and variations in insertion technique. In the standard removal technique, a single transverse incision is made close to and below the distal ends of the capsules, and a mosquito forceps is used for extraction. Described, in this article, is a simple technique for the isolation and removal of Norplant with a Ramelot phlebectomy hook. After anesthesia with 1% lidocaine with 1:100,000 of epinephrine, a 5 mm horizontal stab incision with a Number 11 blade is made at the base of the implants. Using digital palpation for stabilization, each capsule is captured with the Ramelot hook and delivered through the incision. After the implant is grasped with a mosquito forceps, gentle traction and an oscillating motion dislodge the implant from the surrounding fibrous tissue. Incision sites are closed with plain gut sutures. Complications associated with this procedure include broken capsules, cutaneous infection, and contusion.^ieng

Treatment of multiple eruptive hair cysts with erbium:YAG laser.

BACKGROUND: Eruptive vellus hair cysts (EVHC) frequently resist a variety of treatment modalities. While pulsed carbon dioxide (CO2) laser has been used effectively for facial EVHC, this laser presents significant risks for hypertrophic scarring when used on truncal sites. Due to absorption of 2940 nm energy by both tissue water and protein, the erbium:yttrium-aluminum-garnet (Er:YAG) laser ablates more cleanly and creates less residual thermal injury in the wound bed. This laser might prove efficacious and safe in treating nonfacial EVHC. OBJECTIVE: To assess treatment efficacy and wound healing after Er:YAG laser ablation of EVHC. METHODS: Two patients with 32 truncal EVHC were treated with pulsed Er:YAG laser using a drilling technique followed by second intention healing. RESULTS: Laser treatment sites healed without permanent dyspigmentation or hypertrophic scarring. No lesion recurrence was observed. CONCLUSION: Er:YAG laser ablation is an effective method for treating EVHC at anatomic sites prone to hypertrophic scar formation.

Ber-EP4-positive phenotype differentiates actinic keratosis from superficial basal cell carcinoma.

BACKGROUND: Well-defined histopathologic criteria exist to distinguish actinic keratosis (AK) from superficial basal cell carcinoma (BCC). A similar morphology of downwardly budding dysplastic keratinocytes may occur in both entities, creating potential for errors in diagnosis. A marker that could reliably distinguish these two lesions would overcome this difficulty in diagnosis. OBJECTIVE: To investigate whether Ber-EP4 staining is useful in distinguishing AK from superficial BCC, and to determine whether AK exhibits a cellular phenotype that is more consistent with BCC or squamous cell carcinoma (SCC). METHODS: We subjected tissue sections from superficial BCC, AK, and squamous intraepithelial neoplasia (SIN) demonstrating epidermal budding to immunohistochemical staining with monoclonal antibody Ber-EP4. RESULTS: Abnormal keratinocytes in all specimens of superficial BCC (5 of 5) were Ber-EP4 positive; all AK (10 of 10) and SIN (8 of 8) were Ber-EP4 negative. CONCLUSION: Ber-EP4 staining reliably distinguishes AK from superficial BCC. The lack of Ber-EP4 staining of AK supports the currently accepted pathogenetic dogma that SIN and SCC arise from AK, but BCC does not.