Ratio of size of recipient and donor areas in treatment of vitiligo by autologous cultured melanocyte transplantation.

BACKGROUND: Autologous melanocytes can be expanded in vitro, allowing the treatment of large lesions of vitiligo in one session. Theoretically, this procedure could provide a higher donor/recipient size ratio (DR ratio) compared with that in noncultured cell transplantation (with a DR ratio < 1 : 10). However, the exact DR ratio obtained from this procedure has not been reported. OBJECTIVES: To study whether transplantation of cultured pure melanocytes at a high DR ratio is as efficient as that at a low DR ratio. METHODS: One hundred and two patients with vitiligo were treated by transplantation of cultured pure melanocytes and were divided into two groups: a low DR ratio group, including patients with DR ratio ≤ 1 : 10 (mean 1 : 8, 35 cases) and a high DR ratio group with DR ratio > 1 : 10 (mean 1 : 27, 67 cases). The extent of repigmentation between these two groups was compared. RESULTS: There was no significant difference in repigmentation between the low DR ratio group (mean ± SD 77·4 ± 22·5%) and the high DR ratio group (77·6 ± 24·8%). Multiple regression analysis showed that even after adjustment for age, sex, type of vitiligo and transplanted cell density, there was no significant correlation between the extent of repigmentation and the DR ratio, indicating that patients treated with high DR ratio obtained a satisfactory result and showed no difference from the low DR ratio group. CONCLUSIONS: Various surgical procedures for the treatment of vitiligo which involve melanocyte transplantation or skin grafts have different inherent DR ratios. Transplantation of cultured pure melanocytes is an expensive and complicated procedure; however, it provides the highest DR ratio (> 1 : 10 and up to 1 : 60). Surgeons can select one of these methods for the treatment of vitiligo based on their experience and skill, on the size of lesions, and the availability of laboratory support.

Treatment of vitiligo in children and adolescents by autologous cultured pure melanocytes transplantation with comparison of efficacy to results in adults.

BACKGROUND: Transplantation of autologous cultured pure melanocytes is a well-established procedure for the treatment of refractory and stabilized vitiligo. However, there was no report specifically comparing the efficacy with the regard to defined age groups (children-adolescence-adult). OBJECTIVE: We analysed the efficacy of this procedure in the treatment of vitiligo in children and adolescents and compare it with the results in adults treated during the same period and using identical procedures. METHODS: Melanocytes were isolated from the roof of suction blister, cultured and expanded with Hu16 medium in vitro, and transplanted to laser-denuded receipt area. A total of 12 children (8-12 years), 20 adolescents (13-17 years) and 70 adults with vitiligo were treated using this procedure. RESULTS: The patients obtained satisfactory results (repigmentation of 50% or more) results in children, adolescents and adults were 83.3%, 95.0% and 84.0% respectively. The mean extent of repigmentation in children, adolescents and adults was 80.7%, 78.9% and 76.6% respectively. There was no statistical difference in repigmentation among these three groups. After adjusting for all factors (gender, type of vitiligo, period of stability, location of the lesion or transplanted cell density) individually or totally using multiple regression analysis, age still did not correlate to the extent of repigmentation. CONCLUSIONS: The satisfactory results obtained in the treatment of vitiligo in children and adolescents by transplantation of cultured autologous pure melanocytes are comparable with the results in adults. Therefore, this procedure can be considered in refractory and stable vitiligo in children and adolescents, especially in patients with large vitiliginous lesions.

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.

Repair of a critical size defect in the rat mandible using allogenic type I collagen.

Mandibular fractures, resulting from either trauma or reconstructive surgery, can be challenging craniofacial problems. The morbidity of failed fracture healing is significant and may require bone grafting. Donor site morbidity and finite amounts of autogenous bone are major drawbacks of autogenous bone grafting. Similarly, the use of allografts and xenografts may be associated with an increased risk of rejection, infection, and nonunion. To circumvent the limitations of bone grafting, research efforts have focused on formulating a suitable bone substitute. The purpose of our study was to evaluate the efficacy of type I collagen implants in repairing critical sized mandibular defects in rats. Twelve male Sprague-Dawley rats (200-300g) were divided equally into control and experimental groups. Full thickness, round, four millimeter in diameter defects were created in the ramus of the right mandible of all rats using an electrical burr at low speed. The defects were irrigated of all bone chips, and either filled with a precisely fitted disk of allogenic collagen type I gel (experimental animals) or left empty (control animals). Animals were killed 6 weeks after surgery and healing of the bone defects was assessed in a blinded fashion using radiologic and histologic analysis. Radiologic analysis of the control group revealed a clear circular right mandibular defect in all animals, whereas the collagen disk implant group revealed an indistinct to nonexistent right mandibular defect in all animals. Densitometric analysis revealed a significant difference between these groups (* P = 0.01). Similarly, gross analysis of control mandibles revealed a 4mm round, soft-tissue filled defect, while implanted defects demonstrated gross bone spanning the defect. Finally, histologic analysis of all control mandibles revealed clearly demarcated bony edges at the defect border with connective tissue spanning the defect. In contrast, histological analysis of all implanted mandibles revealed indistinct bony edges at the defect border with a thin layer of osteoblasts and viable bone spanning the defects. We have demonstrated the ability of type I collagen to promote healing of a membranous bony defect that would not otherwise heal at 6 weeks. The suitability of type I collagen as a carrier matrix provides ample opportunity for tissue-engineered approaches to further facilitate bony defect healing. Promoting bone formation through tissue engineering matrices offers great promise for skeletal healing and reconstruction.

Congenital intraocular teratoma associated with eyelid coloboma.

PURPOSE: To describe the first case of intraocular teratoma associated with eyelid coloboma and the second reported case of intraocular teratoma. DESIGN: Interventional case report. METHODS: A left intraocular tumor was surgically resected from a 2-day-old female with an associated lower eyelid coloboma. RESULTS: Pathologic evaluation revealed a completely intraocular tumor comprising derivatives of all three germ cell layers giving a diagnosis of intraocular teratoma. The eyelid coloboma was repaired, and a scleral-wrapped hydoxyapatite-integrated orbital implant was placed. CONCLUSION: To our knowledge, this is the second reported instance of teratoma originating within the globe and the only reported case of teratoma associated with eyelid coloboma. Although exceedingly rare, intraocular teratoma should be added to the differential diagnosis of congenital intraocular tumors.

A functional study on prostanoid receptors involved in cultured human iridal melanocyte stimulation.

The effects of various prostanoids on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (bFGF) (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium or various deleted media with or without various prostanoids at different concentrations. After 6 days, the numbers of cells and dendrites were counted and melanin content was measured and compared with controls. Prostaglandin E(2), an EP(2)receptor agonist (AH 13205) and AGN 192093 (thromboxane mimetic) stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. A mixed EP(1)and EP(3)receptor agonist (sulprostone), a EP(4)receptor agonist (ONO-AE1-329), IP receptor agonists (cicaprost or iloprost) and a TP receptor agonist (U-46619) showed no effect. Prostaglandin D(2)showed stimulating effects. However, these stimulating effects could not be blocked by the addition of a DP receptor antagonist (BW A868C). Furthermore, a DP receptor agonist (BW 245C) showed no effects, indicating that the effect of prostaglandin D(2)may involve receptors other than the DP receptor subtype. The present study indicates that: (1) among various EP receptor agonists, only an EP(2)receptor agonist has stimulating effects on iridal melanocytes; (2) DP, IP and TP receptor agonists do not have stimulating effects; and (3) the mechanisms of action of prostaglandin D(2)and AGN 192093 need further study.

Evaluation of acellular dermal graft (AlloDerm) sheet for soft tissue augmentation: a 1-year follow-up of clinical observations and histological findings.

OBJECTIVES: To evaluate and compare the long-term clinical persistence and histological appearance of subdermally implanted acellular dermal graft (AlloDerm) sheets and intradermal type I bovine collagen cross-linked with glutaraldehyde (Zyplast). PATIENTS: Ten adult patients (5 men and 5 women; average age, 46 years; age range, 37-59 years) not allergic to bovine collagen. METHODS: AlloDerm sheets were implanted surgically in a subdermal plane in one postauricular crease, and Zyplast was injected intradermally on the opposite side. AlloDerm and Zyplast implants were digitally photographed and their apparent volumes calculated at 1, 3, 6, 9, and 12 months after implantation. A specimen was removed at 3 and 12 months and examined histologically for collagen persistence, host tissue invasion, and inflammatory reaction. RESULTS: The apparent implant volume of the AlloDerm sheets decreased during the first 6 months and then stabilized over the next 6 months. By contrast, Zyplast was progressively absorbed, with complete loss of clinical effect by 6 months. Histological analysis of implanted AlloDerm sheets demonstrated progressive repopulation of the graft with minimal inflammation. CONCLUSIONS: AlloDerm sheets seem to provide stable soft tissue augmentation after an early period of resorption and are clearly superior to Zyplast injections for long-term, large-volume, soft tissue correction. Recommendations for clinical use include routine overcorrection, with subsequent augmentation delayed by at least 6 months.

Ultrasound biomicroscopic diagnosis of cyclitic membranes.

PURPOSE: To evaluate the utility of ultrasound biomicroscopy in imaging cyclitic membranes. METHODS: Patients with hypotony and suspected or known cyclitic membrane underwent ultrasound biomicroscopic examination. Histopathology of cyclitic membrane was correlated with ultrasound biomicroscopy in three cases. RESULTS: Six eyes of six patients were enrolled. Mean patient age was 62.2 +/- 18.4 (SD) years. The mean intraocular pressure in the affected eye was 4.3 +/- 3.4 mmHg. Three eyes were pseudophakic and three eyes were aphakic. All eyes had undergone two or more previous intraocular surgeries. Ultrasound biomicroscopy imaged the cyclitic membrane in all six eyes. Histopathology revealed fibroproliferative cyclitic membranes with associated inflammatory cells. CONCLUSION: Ultrasound biomicroscopy is useful in detecting the presence of those cyclitic membranes that may not be identified on clinical examination.

Iris and ciliary body melanomas: ultrasound biomicroscopy with histopathologic correlation.

OBJECTIVE: To correlate ultrasound biomicroscopic images of iris and ciliary body melanomas with their histopathologic features. METHODS: Ultrasound biomicroscopy was performed in 3 cases of iris melanoma and in 3 cases of ciliary body melanoma. Cross-sectional ultrasound biomicroscopic images were compared with findings from clinical examination and light microscopy to evaluate associations between their histopathologic, surface, and internal ultrasound characteristics. Unique images of intrastomal and obscured posterior tumor margins were visualized by ultrasound biomicroscopy. RESULTS: Results of this study revealed that ultrasound biomicroscopy offers an accurate method to evaluate tumor shape, reflectivity, and local invasion. Neoplastic tissue had only medium echogenicity. Enlarged vessels were correlated to echolucent spaces in the iris stroma. Anterior tumor margins were found within the iris stroma, within the anterior chamber angle, and on the endothelial surface of the cornea. Posterior tumor extension was noted to encroach onto the lens, into the sclera, and serous peripheral retinal detachments were associated with ciliary body tumors. CONCLUSION: Ultrasound biomicroscopic images correlated well with histopathologic features of anterior uveal melanomas including shape, reflectivity, and local extension. Arch Ophthalmol. 2000;118:1515-1521

Influence of autonomic neurotransmitters on human uveal melanocytes in vitro.

The influence of autonomic neurotransmitters on the growth and melanogenesis of cultured uveal melanocytes was studied. Uveal melanocytes were cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (complete medium). The cells were plated into multiple well plates, and various concentrations of adrenergic and cholinergic agents were added to the media (complete medium or various deleted media). After 6 days, the cells were detached for cell counting and melanin measurement and compared to controls. Epinephrine, isoproterenol, salbutamol and metaproterenol (adrenergic agonists that can activate beta(2)-adrenoceptors) substantially stimulated growth and melanogenesis of cultured uveal melanocytes in cAMP-deleted medium. Methoxamine, clonidine, prenalterol and D-7114 (adrenergic agonists that do not activate beta(2)-adrenoceptors) showed no effect under similar experimental conditions. Muscarine (a cholinergic agonist) inhibited the growth and melanogenesis of uveal melanocytes in complete medium. It indicates that adrenergic agents (beta(2)-adrenoceptor agonists) stimulate growth and melanogenesis in uveal melanocytes, while cholinergic agonist has an inhibitory effect. This effect appears to involve the cAMP second messenger system. These studies suggest that homeostasis of the uveal melanocytes may be maintained, in part, by regulating the autonomic nervous system in vivo.

Autologous collagen dispersion (Autologen) as a dermal filler: clinical observations and histologic findings.

OBJECTIVE: To assess the histologic behavior and clinical efficacy of autologous collagen dispersion (Autologen) in augmenting human dermis. SUBJECTS: Adult patients of the Facial Plastic Surgery Clinic at The New York Eye and Ear Infirmary who were undergoing facial aesthetic surgery with skin excision. METHODS: Five patients were injected intradermally with Autologen in one postauricular area and bovine cross-linked collagen (Zyplast) on the contralateral side. Patients were examined clinically for signs of infection, skin necrosis, or implant rejection/allergy 2, 4, and 12 weeks postinjection. Impressions and photographs of all implant sites were taken at all follow-up visits. Biopsy specimens of each implant were taken 4 and 12 weeks after injection and examined histologically for signs of integration, rejection, and resorption. RESULTS: All implants were well tolerated. No identifiable differences were noted in the clinical persistence of Zyplast vs Autologen. Histologically, there was more variability in the degree of fibroblast infiltration of Autologen vs Zyplast deposits. CONCLUSIONS: Our trial suggests that autologous collagen dispersion may represent a viable alternative to bovine collagen. Clinical persistence and histologic behavior of Autologen appear to be at least as favorable as those of Zyplast, and Autologen obviates the need for allergy testing and eliminates the possibility of disease transmission. Arch Facial Plast Surg. 2000;2:48-52

Modulation of tissue ingrowth into porous high-density polyethylene implants with basic fibroblast growth factor and autologous blood clot.

OBJECTIVE: To investigate the effect of direct application of biologic materials normally present in wounds (basic fibroblast growth factor [bFGF] and autologous blood clot [ABC]) to accelerate the bony and soft tissue ingrowth into porous high-density polyethylene implants. METHODS: We conducted a prospective, blinded animal histological study. Disks made of porous high-density polyethylene impregnated with bFGF or ABC were implanted into adult Sprague-Dawley rats in both subcutaneous and subperiosteal locations. Animals were killed and implants were harvested at 2, 4, and 10 weeks postimplantation and examined histologically for fibroblast invasion, collagen deposition, and inflammatory reaction.The results were compared with control (untreated) implants. RESULTS: As a group, the histological results showed significantly more fibroblasts within the ABC-treated implants than control implants or bFGF-treated implants. This difference in the number of fibroblasts between ABC-treated implants and bFGF-treated and control implants was also statistically significant 2 weeks after implantation. CONCLUSIONS: At the concentration of bFGF of 1 microg/10 microL, no acceleration of tissue ingrowth into porous high-density polyethylene implants was noted. However, when porous high-density polyethylene implants were treated with ABC, the implants were invaded to a greater degree by soft tissue, particularly in the early postoperative period (first 2 weeks). Bioactive substances associated with the coagulation and platelet cascades present in the ABC may be responsible for this accelerated incorporation of the porous implant and may have clinical implications. Arch Facial Plast Surg. 2000;2:27-33

Topical mitomycin chemotherapy for conjunctival malignant melanoma and primary acquired melanosis with atypia: clinical experience with histopathologic observations.

OBJECTIVES: To clinically evaluate topical mitomycin chemotherapy in patients with diffuse, multifocal, or recurrent primary acquired melanosis with atypia and/or conjunctival malignant melanoma and to histopathologically study ocular tissue samples obtained before and after treatment. METHODS: Chemotherapy with topical mitomycin, 0.04% 4 times daily, was administered for 28 days as the primary and only treatment in 7 patients (after biopsy) and for 7 days as adjuvant therapy to excision and cryotherapy in 5 patients. Mean follow-up was 38 months. Five patients developed subconjunctival recurrences, for which 2 underwent orbital exenteration and 3 were treated conservatively. Histopathologic specimens of conjunctival, adnexal, and ocular tissues obtained before and after chemotherapy were evaluated. RESULTS: Regression of tumor was observed in 11 patients with primary or adjuvant topical mitomycin chemotherapy. One patient with nodular melanoma was resistant to mitomycin chemotherapy. Histopathologic findings included regionally variable conjunctival epithelial atrophy and thinning. Dyskeratosis and focal keratinization in conjunctival epithelium were noted. Epithelial nuclei were occasionally pyknotic in areas of atrophic epithelium. Subepithelial inflammation was present and was most intense in areas with severe atrophy and/or keratosis. Two patients with primary treatment and 2 with adjuvant treatment developed subconjunctival recurrence. In patients with recurrent malignant melanoma, the deeper layers of the lamina propria were involved, with sparing of the epithelium and superficial lamina propria. Transient keratoconjunctivitis was observed in all patients during treatment. In evaluation of the exenteration specimens, corneal, scleral, episcleral, retinal, and anterior structures were within normal limits. CONCLUSIONS: Topical mitomycin chemotherapy was found to induce regression of conjunctival melanoma and primary acquired melanosis with atypia. When mitomycin chemotherapy was used as an adjuvant to excision and cryotherapy, 2 (40%) of 5 patients experienced tumor recurrence at a mean of 4.3 years' follow-up. Our histopathologic findings demonstrated a long-term mitomycin chemotherapy-related effect on the conjunctiva. The degree of chronic atrophy and inflammation was not clinically significant. The pattern of effect and location of recurrent disease suggest that this regimen of topical mitomycin chemotherapy was most effective for superficial tumors. No complications that would preclude use of our dose regimen were noted. Although subconjunctival or orbital recurrences were noted, topical mitomycin chemotherapy warrants further investigation as an alternative treatment for primary acquired melanosis with atypia and conjunctival malignant melanoma. Arch Ophthalmol. 2000;118:885-891

Improving degenerate oligonucleotide primed PCR-comparative genomic hybridization for analysis of DNA copy number changes in tumors.

Combining degenerate oligonucleotide-primed PCR (DOP-PCR) with comparative genomic hybridization (CGH) has made it possible to analyze genomic changes in single cells. Although DOP-PCR-CGH methodology has been reported, the reproducibility of the method has been uncertain. We have developed a reproducible DOP-PCR-CGH protocol by systematically evaluating different labeling methods (including nick translation, PCR incorporation, and random-primed labeling) and different hybridization mixtures (including amplified test DNA vs. amplified reference DNA, termed homo-hybridization; and amplified test DNA vs. unamplified reference DNA or vice versa, termed hetero-hybridization). We have analyzed DNA samples obtained from 16 tissue sources including fresh/frozen normal and tumor samples, formalin fixed and paraffin embedded tumor tissue, and tumor cell lines by using differently labeled probes and hybridization combinations, and we calculated the corresponding rate (CR) of DOP-PCR-CGH with standard CGH. We found that homo-hybridization produced reproducible results with high CRs as compared to standard CGH (91-100% CR, mean 97%); In contrast, hetero-hybridization failed to generate reproducible hybridization with low CRs (57-97% CR, mean 80%; chi(2) = 1245.8, P<0.0001), high background, uneven hybridization, and false deletions or amplifications. In addition, our improved DOP-PCR protocol raised the amplification efficiency at least five times as compared to previously reported protocols, allowing for the detection of genomic imbalances in as little as 12.5 pg of starting DNA. In conclusion, the DOP-PCR-CHG homo-hybridization method, especially when combined with labeling by nick translation, is reliable and reproducible. The method can be used in screening for genomic imbalances using minute amounts of tumor DNA, thereby facilitating CGH application. Genes Chromosomes Cancer 28:395-403, 2000.

Endoscopic foreheadplasty: a histologic comparison of periosteal refixation after endoscopic versus bicoronal lift.

Endoscopic brow lift techniques using temporary fixation rely on rapid readherence of the periosteum to calvarial bone. Little is known about the histologic events that occur during the early postoperative period after these procedures. An animal study was designed to compare and contrast periosteal fixation to bone and unelevated periosteum, with endoscopic and bicoronal brow lift techniques. One method of temporary fixation is the use of absorbable (polylactic/polyglycolic acid copolymer) LactoSorb screws; a histologic analysis of implanted LactoSorb screws was also performed. Sixteen rabbits underwent brow lifts; eight underwent endoscopic brow lift and fixation with LactoSorb screws without skin excision, and another eight underwent traditional bicoronal brow lift with skin excision and closure under tension. Animals were killed 1, 2, 6, and 12 weeks after the procedures were performed to evaluate the interaction of periosteum and bone and the normal, unelevated periosteum/calvarium interface at a site distant from the operative area. Histologic specimens were examined for the degree of apposition of periosteum to bone and for any fibrous or bony reaction at this interface. Histologic analysis showed various degrees of periosteal fibrosis and fixation to calvarial bone. After an initial phase of minimal periosteal adherence and moderate inflammation, the periosteum became progressively more adherent to bone in both groups, with no significant differences between treatment groups in rates of fixation. Fixation required at least 6 weeks. LactoSorb screws were surrounded by an area of mild inflammation and were progressively hydrolyzed and digested. Periosteal fixation increases over time for bicoronal and endoscopic brow lifts with minimal differences between the two techniques. With this animal model, periosteal adherence to calvarium requires at least 6 weeks with complete adherence by 12 weeks. In addition, the use of absorbable fixation screws seems to be both effective and well tolerated. The histologic changes associated with periosteal healing observed in this study suggest that permanent or semipermanent fixation may improve the accuracy and early postoperative maintenance of forehead advancement.