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Characterization of a novel recombinant hyaluronan binding protein for tissue hyaluronan detection.

Tumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HA-binding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods.

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Dermatofibroma and dermatofibrosarcoma protuberans: an immunohistochemical study reveals distinctive antigenic profiles.

Recent studies of mesenchymal cells of the dermis using antibodies to factor XIIIa (FXIIIa) and CD34 have demonstrated immunophenotypic heterogeneity amongst the normal resident spindle/dendritic cells of the dermis. These immunohistochemical markers also have been reported to be useful in the distinction between two dermal mesenchymal tumors of uncertain histogenetic origin - the dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP). DFs are FXIIIa positive, CD34 negative while DFSPs are FXIIIa negative and CD34 positive. Expression of CD34 may also have histogenetic implications for these cutaneous neoplasms. In order to further study these tumors we studied 13 DFs and 12 DFSPs immunohistochemically using a microwave antigen retrieval technique in formalin fixed, paraffin embedded tissue with antibodies to FXIIIa, CD34, CD45, factor VIII related antigen (FVIII-RA), the Ki-67 antigen (MIB-1 antibody) and the lectin Ulex europaeus. Of the DFs, all 13 were FXIIIa positive; 12/13 were CD34 negative and 1 was strongly CD34 positive. All DFSPs were FXIIIa negative and CD34 positive. One DFSP also contained an area of fibrosarcoma which was negative for both markers. All tumors were negative with anti-FVIII-RA Ulex europaeus, and anti-CD45. MIB-1 staining demonstrated nuclear staining of the tumor cells in both DFs and DFSPs. Image analysis of MIB-1 stained sections revealed a significant difference in mean percent positive nuclear area between DFs (1.16% +/- 0.405) and DFSPs (2.265% +/- 0.963). In summary, FXIIIa reliably distinguished between DFs and DFSPs; however, CD34 immunoreactivity can be seen in DFs. No evidence for vascular or hematopoietic origin of these tumors was found using microwave antigen retrieval and anti-FVIII-RA, Ulex europaeus, or CD45 staining. With microwave enhancement trypsin was not necessary for FXIIIa staining; however, it did not significantly enhance detection of FVIII-RA, CD45, or Ulex antigens. DF and DFSP tumor cells are in the cell cycle as demonstrated by MIB-1 staining and there are significant differences in percent positive nuclear area between these neoplasms, being higher in DFSP compared to DF.

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Distributions of melanoma growth stimulatory activity of growth-regulated gene and the interleukin-8 receptor B in human wound repair.

The alpha-chemokines have been implicated as regulators of proliferation and differentiation of normal keratinocytes and as mediators of keratinocyte maturation and migration in inflammatory processes that involve the skin. Using the cutaneous wound repair model, we examined the sites and temporal sequence of the appearance of melanoma growth stimulatory activity or growth-regulated gene (MGSA/GRO;ligand) and the type B interleukin (IL)-8 receptor (IL-8RB) to which MGSA/GRO binds. Human burn tissues (n = 44) representing days 2 to 12 after injury were obtained during surgical debridement, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunolocalizations were performed with polyclonal antisera for both ligand and receptor, as well as a monoclonal antibody for the IL-8 RB. Western blot analysis confirmed the presence of the IL-8 RB in immunoprecipitates of epidermal keratinocyte lysates. In normal skin, MGSA/GRO protein was restricted to sites populated by differentiated keratinocytes (suprabasal compartments, inner root sheath cells, and dermal sweat ducts). MGSA/GRO protein was barely detectable within epithelial margins and islands of burn wounds where the migrating/proliferating keratinocyte populations reside, but staining intensities increased as cells matured into the outer layers. Weak diffuse staining was detected in areas of neutrophilic infiltration (granulation tissue and overlying exudates). By contrast, in normal skin the IL-8 RB was detected in specific locations within epidermal and dermal compartments of healing wounds. In the dermis, polyvalent antibodies detected receptor immunoreactivity most prominently in dermal sweat ducts and endothelium of capillaries, whereas this immunoreactivity was inconspicuous in sections stained with the monoclonal antibody. Receptor immunostaining was noted in migrating/proliferating keratinocytes in epithelial margins and islands but was in the outer layers or in hypertrophic epidermis adjacent to wounds. This same pattern was observed in epidermal appendages such as hair follicles and eccrine sweat ducts. In granulation tissues, IL-8 RB was noted in numerous fibroblasts and in subpopulations of macrophages and smooth muscle. The presence of both MGSA/GRO and its receptor in human burn wounds implicate this cytokine as an autocrine or paracrine mediator of epidermal regeneration in both the inflammatory and proliferative phases of cutaneous wound repair.

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Low frequency of codon 61 Ha-ras mutations and lack of keratin 13 expression in 7,12-dimethylbenz[a]-anthracene-induced hamster skin tumors.

Alterations in the pattern of keratin expression are a common feature of skin-tumor development. In this study, we investigated whether the loss of epidermal keratin 1 (K1) and its replacement by mucosal keratin 13 (K13) is unique to mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), since it has been reported that human epidermal tumors do not exhibit aberrant expression of K13. With that purpose, we analyzed the keratin profiles of 16 DMBA-induced hamster skin tumors using monospecific antibodies against K1 and K13. Although all the tumors expressed K1, they also showed an overall tendency towards loss of this keratin; furthermore, none of the tumors expressed K13. Previous studies have suggested that the induction of K13 in mouse skin is related to the mutation of the Ha-ras gene by the initiating agent DMBA, a mutation consistently found in murine DMBA/TPA-induced tumors and rarely found in human skin tumors. Therefore, we also evaluated the tumors for the presence of codon-61 mutations by direct sequencing of DNA extracted from paraffin-embedded tissue sections. Only three tumors showed an A-->T transversion in the second nucleotide of Ha-ras codon 61. However, presence of the mutation did not correlate with K1 staining. Although hamster skin tumors were induced by the same initiator as were mouse skin tumors, hamster skin tumors did not show the same keratin profile. Moreover, their immunohistochemical expression of K1 and K13 and their codon 61 sequences resembled that of their human counterparts. These results suggest that the aberrant expression of K13 may be unique to murine skin. Furthermore, although codon 61 Ha-ras mutation appears to be related to keratin alterations in the mouse model, this mutation is not sufficient to produce the same biochemical changes in other species.

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Leishmania (Viannia) braziliensis: comparative pathology of golden hamsters infected with isolates from cutaneous and mucosal lesions of patients residing in Tres Bracos, Bahia, Brazil.

The histopathology of primary forepaw and metastatic lymph node, spleen, and liver lesions produced in golden hamsters infected with cutaneous leishmaniasis (CL) strains (LTB 111 and LTB558) and mucocutaneous leishmaniasis (MCL) strains (LTB12 and LTB201) of Leishmania (Viannia) braziliensis isolated from patients residing in Tres Bracos, Bahia, Brazil is described. No pathological features providing clear differentiation of the CL and MCL strains were found. Although amastigotes were plentiful early in the development of primary forepaw lesions, they were either absent or could not be identified with certainty in sections of late stage lesions. Similarly, amastigotes were not found in histologic lesions at metastatic sites; however, leishmanial DNA was detected in both early and late stage forepaw lesions and metastatic lesions using Leishmania kinetoplast DNA and the gene coding for gp63 as hybridization probes. The DNA recovered from metastatic lesions was extracted from formalin-fixed paraffin-embedded tissues that had been stored at room temperature for prolonged periods.

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A comparative light microscopic analysis of the sensory innervation of the mystacial pad. I. Innervation of vibrissal follicle-sinus complexes.

This comparative study was conducted to provide a detailed, comprehensive description of the innervation to the follicle-sinus complex (F-SC) of mystacial vibrissae and to determine if interspecies variability in the innervation of the F-SCs may be related to differences in the structure or existence of barrels in the primary somatosensory (SI) cortex. Two silver techniques (Winkelmann on 100 micron-thick-frozen sections and Sevier-Munger on 8 micron-thick paraffin sections) were applied to comparable mystacial skin samples from adult hamsters, mice, rats, gerbils, rabbits, guinea pigs and cats. The basic structure and innervation of the F-SCs is the same in all species. Six distinct populations of sensory receptors are identified at consistent locations: Merkel endings in the epidermal rete ridge collar at the mouth of the follicle; circularly disposed presumptive lanceolate, Ruffini, and free nerve endings (FNE) in the inner conical body; longitudinal lanceolate endings in a dense palisade in the mesenchymal sheath at the level of the ring sinus; Merkel endings in the external root sheath at the level of the ring sinus; scattered corpuscular and FNEs (possibly lanceolate or Ruffini endings) in the cavernous sinus; and a few FNEs in the dermal papilla. In each F-SC, the first two locations are supplied by several superficial vibrissal nerves that arise from several small nerves that also innervate the skin between the vibrissae. These superficial nerves may innervate more than one F-SC. The next three locations are supplied by a single large deep vibrissal nerve that is derived directly from a row fascicle of the infraorbital nerve. Each deep nerve innervates a single F-SC. The source of the papilla innervation was not found. The ring sinus locations are consistently the most heavily innervated in all species. The number of axons in comparable deep vibrissal nerves is similar among the rodents, higher in the cat, and lower in the rabbit. Innervation of the inner conical body varies considerably, being dense in species that vigorously whisk their vibrissae (hamster, mouse, rat, and gerbil) and sparse or absent in species that minimally or never whisk (guinea pig, rabbit, and cat). Innervation to the cavernous sinus is sparse particularly in hamsters and gerbils. The innervation to the rete ridge is uniquely absent in the rabbit.(ABSTRACT TRUNCATED AT 400 WORDS)

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