Characterization of Haemophilus ducreyi-specific T-cell lines from lesions of experimentally infected human subjects.

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease that facilitates the transmission of human immunodeficiency virus. In the human model of infection, the histopathology of infected sites in part resembles a delayed-type hypersensitivity (DTH) response. In this study, T cells were isolated from skin biopsy specimens obtained from 24 subjects who were infected for 7 to 14 days. One clone and 12 lines that responded to H. ducreyi antigens were obtained from 12 of the subjects. Fluorescence-activated cell sorter analysis showed that the antigen-responsive lines and clone were predominantly CD3(+) and CD4(+). The lines and clone responded to H. ducreyi antigen in a dose-dependent manner and produced gamma interferon (IFN-gamma) alone or IFN-gamma and interleukin-10 (IL-10) but no IL-4 or IL-5 in response to H. ducreyi. Proliferation of T cells was dependent on the presence of autologous antigen-presenting cells. The lines showed little response to antigens prepared from other members of the Pasteurellaceae and responded to different fractions of H. ducreyi separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that T cells that recognize H. ducreyi antigens are recruited to sites experimentally infected with the organism. The lack of cross-reactivity to the Pasteurellaceae and the response of the lines to different antigen fractions suggest that subjects are sensitized to H. ducreyi during the course of infection.

Sensitivity of partially purified ice nucleation activity of Fusarium acuminatum SRSF 616.

Factors that affect bacterial ice nucleation, including growth medium, growth phase, nutrient deprivation, and cold-temperature exposure, were investigated in the ice nucleation active (INA) fungus Fusarium acuminatum SRSF 616. Ice nucleation activity remained relatively constant throughout the growth cycle, and the cell-free culture supernatant consistently displayed higher ice nucleation activity than the hyphal pellet. Although nutrient starvation and low-temperature exposure enhance bacterial ice nucleation activity, reducing the concentration of C, N, or P in synthetischer nährstoffarmer broth (SNB) did not increase fungal ice nucleation activity, nor did exposure to 4 degrees C or 15 degrees C. From the SNB supernatant, selected INA chromatography fractions were obtained that demonstrated increased sensitivity to proteinase K and heat compared with culture supernatant. We propose that partial purification of the fungal ice nuclei resulted in removal of low-molecular-weight stabilizing factors.

Use of the "spiral" flap for closure of small defects of the nasal ala.

BACKGROUND: Multiple wound closure options exist for cutaneous defects of the nasal ala. The best option depends on the depth, size, and location of the defect. OBJECTIVE: To demonstrate a modification of the traditional rotation flap for closure of small alar defects. METHODS: The design and execution of the "spiral" flap for closure of a representative defect are described. RESULTS: Immediate and delayed postoperative views demonstrate expected outcome. CONCLUSION: The spiral flap modification of the rotation flap is a simple and elegant closure option for small nasal alar defects.

Expression of the melanocortin 5 receptor on rat lymphocytes.

The expression of melanocortin-5 receptor (MC5-R) mRNA and protein was characterized from isolated rat lymphocytes. The presence of MC5-R mRNA in spleen and thymus tissues was demonstrated by RT-PCR. The RT-PCR product was sequenced to confirm the identification of MC5-R. Tissues from lachrymal glands, adipose, adrenals, thymus, pancreas, and isolated splenic lymphocytes were detergent solubilized. The crude proteins were resolved by SDS-PAGE, transblotted to a nitrocellulose membrane, and probed for MC5-R using anti-receptor rabbit antisera. Two different types of polyclonal rabbit antisera were raised against synthetic peptides representing epitopes found at the amino (alphaN-MC5-R) and the carboxyl termini (alphaC-MC5-R) on the MC5-R. A prominent band at 77,000 (p77) was detected in all tissues except the pancreas. Preimmune sera did not detect p77 by Western analysis and the addition of peptide antigen neutralized the detection of p77 by the specific antisera. The receptor protein was purified from spleen and thymic lymphocytes using protein A agarose that precipitated material complexed to alphaN-MC5-R. The purified MC5-R was detected by Western analysis using alphaC-MC5-R. Both anti-receptor antisera, alphaN-MC5-R and alphaC-MC5-R, detected the p77. The p77 was treated with protein endoglycosidase F to produce a smaller protein band between 34-38,000 (p35); the inferred size is 37,000 based on the cDNA sequence. The data suggest that Asn-linked carbohydrate groups account for much of the p77 mass of the MC5-R. The data also demonstrate the expression of MC5-R protein on rat lymphocytes, thus, supporting the hypothesis that MC5-R is the ACTH receptor on lymphocytes.

Skin cancer: recognition and management.

Skin cancer is the most common malignancy in humans and accounts for one third of newly diagnosed cancers--it will be diagnosed in approximately 1 in 5 Americans in their lifetime and > 1 million cases are diagnosed each year. Skin cancer can cause local tissue destruction, disfigurement, and even death if left untreated; therefore, timely recognition, treatment, and appropriate referral are critical to reducing morbidity. As the incidence of skin cancer rises each year, the primary care physician needs to be familiar with the clinical presentation, treatment options, and means of prevention of the most common skin cancers: basal cell carcinoma, squamous cell carcinoma, and malignant melanoma.

Molecular studies of hemichordate development: a key to understanding the evolution of bilateral animals and chordates.

Using the Hawaiian acorn worm, Ptychodera flava, we began molecular studies on the development of hemichordates, a phylum previously unstudied at this level. Here we review results garnered from the examination of a few specific genes selected to help understand the evolution of vertebrate structures. These studies suggest new ideas about the evolution of developmental mechanisms in the deuterostomes. In a seminal observation, we noted an unexpected zone of expression of the Brachyurygene in the early anterior embryonic ectoderm where the mouth will form. Typically, the Brachyury gene is closely linked to development of the notochord and is expressed around the blastopore and in the posterior mesoderm in most animals. This first expression of Brachyury at the blastopore may represent a regulatory program associated with organizing the original animal head and gut opening, as suggested by the expression of Brachyury during hypostome formation in hydra. We believe that the anterior expression of Brachyury in deuterostomes represents the cooption of the program for organizing the original animal gut opening to form the deuterostome mouth. Recent data from the trochophore larva of a polychaete show that an anterior zone of expression of Brachyury is produced in this protostome by splitting of the Brachyury field during the formation of a gut with a mouth and anus by the lateral fusion of the sides of the blastopore. The ability to initiate independently a secondary regulatory program to organize the new mouth leading to an anterior field of Brachyury expression may be a signal event in the evolution of the deuterostomes. We also noted that the P. flava homolog of T-brain/Eomes, a gene closely related by sequence and expression around the blastopore to Brachyury and associated with development of the vertebrate brain, also exhibits early posterior expression around the blastopore and a field of de novo anterior ectoderm expression during later embryogenesis. The tissue in the zone of de novo anterior ectoderm expression of Pf-Tbrain produces the apical organ, a larval neural structure that has been touted as an evolutionary precursor of the chordate dorsal brain. The gene regulatory mechanisms responsible for initiating the anterior zone of de novo expression of T-brain may represent a cooption to specify early neuroectoderm of the regulatory program evolved first to drive anterior Brachyury expression for deuterostome mouth formation. It will be interesting to examine the possibilities that an ability to initiate the de novo anterior expression of the program that includes T-brain may be a key event in the evolution of the developmental mechanisms leading to the chordate dorsal nervous system.

Mast cells and dendritic cells in basal cell carcinoma stroma.

BACKGROUND: The interaction of tumors with the surrounding stroma has become an important topic in tumor biology. Basal cell carcinoma (BCC) stroma has been characterized as hypervascular and rich in mast cells. The presence of dermal dendrocytes thought to have both antigen presenting and wound healing functions has recently been reported in BCC stroma. GP1b-alpha is a newly described vascular adhesion molecule with potential significance in tumor biology. OBJECTIVE: To further characterize the cellular phenotype of BCC stroma. METHODS: Eleven BCCs (8 nodular, 2 sclerosing, 1 adenoid-cystic) were examined using immunohistochemical techniques for the presence of antigens specific to vascular endothelium, mast cells, and dermal dendrocytes. RESULTS: The stroma of all BCCs demonstrated increased vascularity, increased numbers of mast cells, and increased numbers of dermal dendrocytes expressing both CD34 and GP1b-alpha adjacent to tumor nests. No differences in antigen expression were observed between histologic subtypes of BCC. CONCLUSION: The close proximity of stromal mast cells and dermal dendrocytes surrounding BCC nests suggests a biologically significant interaction. The pattern observed is similar to that observed in healing wounds.

Cutaneous metastasis of chordoma.

BACKGROUND: Chordomas are rare neoplasms that arise from the notochord remnant. They develop in the sacrococcygeal (50%) or cervical (15%) region and are generally regarded as a locally aggressive tumor with a slow progressive growth rate and a metastatic incidence ranging from 3 to 48%. Skin involvement by chordoma is rare, but can occur by direct extension, by local recurrence and by metastases. OBJECTIVE: To illustrate by a case report the clinical presentation and management of this disease. METHODS: We present a case of sacral chordoma with metastases over a 10-year period to the lungs, the soft tissue of the chest wall, the triceps tendon, and distant cutaneous metastases to the back and the nose. RESULTS: The cutaneous metastases were treated by excision. CONCLUSION: Chordoma is a slow growing tumor of the notochord remnant that may metastasize to the skin. Physicians and pathologists should be aware of this entity.

T-Brain expression in the apical organ of hemichordate tornaria larvae suggests its evolutionary link to the vertebrate forebrain.

T-box genes encode a novel family of sequence-specific activators that appear to play crucial roles in various processes of animal development. Although most of the T-box genes are involved in the mesoderm formation of chordate embryos, mammalian T-Brain is expressed in the developing central nervous system, and defines molecularly distinct domains within the cerebral cortex. Here we report the first invertebrate T-Brain homologue from the hemichordate acorn worm, Ptychodera flava, which we designate Pf-Tbrain. Developmental expression of Pf-Tbrain was examined by whole mount in situ hybridization to various stages of P. flava embryos. A weak, broad in situ hybridization signal of the Pf-Tbrain transcript is first detected during gastrulation in cells around the archenteron, but this signal disappears as gastrulation proceeds. At mid-gastrula an intense signal appears in several apical ectoderm cells of the gastrula. This signal becomes restricted to the apical region, where the eyespots or the light-sensory organ of the tornaria larva form. Expression of Pf-Tbrain in the apical sensory organ of the tornaria and vertebrate T-Brain in the forebrain suggests an evolutionary relationship between the non-chordate deuterostome larval apical sensory organ and the chordate forebrain.

Developmental expression of the hemichordate otx ortholog.

The phylogenetic location of hemichordates is unique because they seem to fill an evolutionary gap between echinoderms and chordates. We report here characterization of Pf-otx, a hemichordate ortholog of otx, with its embryonic and larval expression pattern. Pf-otx is initially expressed in the vegetal plate of the blastula. Expression remains evident in the archenteron through gastrulation and then disappears. A new expression domain appears near the mouth along the preoral and postoral ciliated bands in the early tornaria larva.

Molecular characterization of TgHBox4, a Drosophila Abd-B homolog found in the sea urchin Tripneustes gratilla.

We have isolated and sequenced a cDNA clone that, as judged by the sequence of the homeobox region, encodes a sea urchin homolog of the homeobox containing the gene Abdominal-B of Drosophila. The total length of the cDNA is 3634 nucleotides and includes an open reading frame, which encodes a protein that is 32,321 Da. The N-terminal region of the homeodomain includes consensus sequences found in some of TgHBox4's Abdominal-B relatives. A genomic clone representing the 5' part of the message was also isolated. This clone and a previously isolated clone were found to represent the full-length cDNA sequence. We have also raised antibodies against a bacterially expressed portion of the TgHBox4 protein and used them to determine the location of TgHBox4 proteins during development. The protein displays ubiquitous expression early in development but becomes more restricted, to posterior regions, late in embryogenesis. Thus, in contrast to its Abd-B homologs in bilateral metazoans, TgHBox4 is probably not involved in pattern formation but may have a posterior-defining role late in embryogenesis.

Characterization of gill-specific genes of the acorn worm Ptychodera flava.

Acorn worms are hemichordate deuterostomes that have remarkable gills thought to be homologous to pharyngeal gills in urochordates and cephalochordates, and pharyngeal pouches in vertebrates. In search of molecular keys to analyzing the origin and evolution of the anterior gut and neck region of the chordate body, the present study isolated cDNA clones for six gill-specific genes, designated PfG1 to PfG6, from Ptychodera flava using differential screening of a cDNA library of RNA from gills. Northern blotting confirmed that these genes were all expressed only in the gills. In situ hybridization showed that the expression of these genes is limited to the endodermally derived columnar epithelium of the pharynx. PfG1 encodes a 42-kDa polypeptide containing sequence similar to D-domains, protein domains characteristic of extracellular proteins. Expression of PfG1 is localized in a delimited pattern along the columnar epithelium of the inner gill apparatus. Expression in the epibranchial ridge appears as two stripes running longitudinally in the epithelium just lateral of the midline. A stripe of expression also appears in a slightly posterior portion on the curve of each band of columnar epithelium on the pharyngeal surface of the secondary gill bars. The five other gill-specific genes, PfG2 to PfG6, encode a family of C-type lectin polypeptides that appear to be secreted proteins. PfG2 to PfG6 are also expressed in the columnar epithelium of the epibranchial ridge as two parallel stripes, but at the lateral margin of the ridge. One of the genes, PfG6, is additionally expressed in the innermost curve of the epithelium on the pharyngeal surface of each secondary gill bar. The localization of expression of PfPax1/9, a gill-specific transcription factor gene, was examined and shown to also be primarily in the endodermal columnar epithelium on the pharyngeal faces of the gill bars. On the secondary gill bars, where PfG1 and PfG6 are also expressed in the columnar epithelium, PfPax1/9 is expressed in the anterior and posterior portions but signal is not evident in the epithelium on the central, innermost curve of the gill bar. The anterior domain of PfPax1/9 expression is more extensive but overlaps the anterior domain of PfG1 expression, whereas its posterior domain of expression is more posterior and complementary to that of PfG6.

Characterization of a hemichordate fork head/HNF-3 gene expression.

Based on anatomical and developmental similarities, hemichordates are thought to be most closely related to chordates. However, so far very few developmental genes have been characterized from hemichordates. To gain molecular insight into the developmental mechanisms involved in the origin and evolution of chordates, we investigated the expression of a fork head/HNF-3 (PfHNF3) gene in the acorn worm embryo. Chordate fork head genes are implicated in the formation of endoderm, notochord and floor plate. We found that a PfHNF3 transcript was first detected at the early blastula stage; the signal of in situ hybridization was found in the vegetal plate cells, invaginating endoderm and then in the archenteron. By the late gastrula and into the early tornaria larva stages, an intense signal remained in the anterior region of the archenteron, while the expression in the other regions of archenteron decreased. The intense signal was retained in the pharynx of the tornaria larva. A comparison of the pattern of PfHNF3 with that of HNF-3 genes of sea urchin, ascidian, amphioxus and vertebrate suggests a possible acquisition of new functions of the gene during deuterostome evolution.

Treatment of superficial basal cell carcinoma and squamous cell carcinoma in situ with a high-energy pulsed carbon dioxide laser.

BACKGROUND: High-energy pulsed carbon dioxide (CO2) lasers have been used extensively to resurface wrinkled and photodamaged skin with a low risk of scarring. Results of histological studies demonstrate precise ablation depths in treated skin with minimal thermal damage to underlying tissue. Our objective was to determine if a pulsed CO2 laser could effectively ablate superficial malignant cutaneous neoplasms (superficial multifocal basal cell carcinoma [BCC] and squamous cell carcinoma [SCC] in situ). OBSERVATIONS: Thirty superficial neoplasms (17 BCCs and 13 SCCs) and their surrounding 3-mm margins were treated with either 2 or 3 passes of a pulsed CO2 laser (500 mJ, 2-4 W) using a 3-mm collimated handpiece. The treated areas were subsequently excised and evaluated histologically by serial sectioning at 5-micron intervals for residual tumor at the deep and lateral margins. Average patient age was greater for those with SCCs than for those with BCCs (76.5 vs 56.7 years; P = .001). The average tumor thickness of SCC in situ was significantly greater than that of superficial BCC (0.57 vs 0.34 mm; P = .01). All (9 of 9 patients) BCCs were completely ablated with 3 passes, and residual tumor in the deep margins was seen in 5 of 8 patients treated with 2 passes of the pulsed CO2 laser (P = .005). Incomplete vaporization of the SCC depth was seen in 3 of 7 patients treated with 3 passes and in 2 of 6 patients treated with 2 passes. Those SCCs incompletely treated were significantly thicker than those completely ablated (0.65 vs 0.41 mm; P = .01). Positive lateral margins were seen in 1 BCC and 3 SCC specimens. CONCLUSIONS: Pulsed CO2 laser treatment can be effective in ablating superficial BCC. Treatment of the neoplasm and a minimum of 4-mm margins with 3 passes (500 mJ, 2-4 W) is recommended for complete vaporization using this laser system. Because 3 passes did not completely ablate all SCC in situ, use of this modality alone is not recommended for treatment of thick or keratotic lesions. No direct comparison of efficacy can be made with other destructive modalities that have not been evaluated with comparably sensitive histological techniques. Further study is needed to establish any cosmetic advantage of pulsed CO2 lasers over other destructive modalities for treatment of superficial malignant neoplasms and long-term cure rates.

Novel pattern of Brachyury gene expression in hemichordate embryos.

Together with echinoderms and chordates, hemichordates constitute the third major group of the deuterostomes, which share a number of common developmental features. The Brachyury gene is responsible for the formation of notochord, the most defining feature of chordates. Therefore, isolation and characterization of the hemichordate homolog of Brachyury is key to understand the origin and evolution of chordates. Here we show that the hemichordate Brachyury gene (PfBra) is expressed in two regions of the gastrula and young tornaria larva, the archenteron invagination region and the stomodeum invagination region.

The Spawning and Early Development of the Hawaiian Acorn Worm (Hemichordate), Ptychodera flava.

The spawning and early embryogenesis of the hemichordate, Ptychodera flava, in Hawaii are described in detail and illustrated with photographs of living material. Natural spawning in the evenings of early December was induced by a shift of seawater temperature from about 22 degrees C to about 26 degrees C. The fertilized egg divides equally and slowly at first, reaching 8 cells at about 5 hr after insemination at room temperature (20-24 degrees C). Divisions then appear to become slightly unequal and by 9 hr the embryo has divided into about 100 cells. The blastocoel forms during cleavage as an irregular space that, when viewed from the side, tends to appear oblate and ultimately appear crescent-shaped as the vegetal plate thickens into the blastocoel. The archenteron forms at about 18 hr as a cleft beginning at the vegetal pole and extending into the vegetal plate. As development proceeds, the embryo expands and by 24 hr forms a typical deuterostome gastrula with an outer sphere of ectoderm and a inner tube of endoderm connected at the blastopore. An out-pocketing of the gut appears at the tip of the archenteron over the next 4 hr to form the protocoel which will become the proboscis coelom. Approaching 40 hr the gut becomes asymmetric and over the next few hr contacts the ectoderm to form a mouth. Hatching occurs during this time at about 45 hr of development. Morphogenesis continues to produce an early tornaria larva by about 60 hr.

Repair of defects of the nasal ala.

BACKGROUND: Alar defects present a reconstructive challenge. OBJECTIVE: To define closure options for alar defects of variable thickness and location. METHODS: The repair options for closure of alar defects are reviewed and discussed with regard to depth of defect and complexity of reconstruction. CONCLUSION: Surgeons repairing defects of the nose should develop a variety of reconstructive approaches for the ala including but not limited to those presented here.