Purinergic receptor expression in the regeneration epidermis in a rat model of normal and delayed wound healing

This study investigated changes in the protein expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalized. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps, and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day four after wounding. The re-epithelializing wound edges were analyzed immunohistochemically for P2X(5), P2X(7), P2Y(1) and P2Y(2) receptors, and immunostaining of keratinocytes was quantified using optical densitometry. In normal rat epidermis, P2Y(1) and P2Y(2) receptors were found in the basal layer where keratinocytes proliferate; P2X(5) receptors were associated with proliferating and differentiating epidermal keratinocytes in basal and suprabasal layers; P2X(7) receptors were associated with terminally differentiated keratinocytes in the stratum corneum. In the regenerating epidermis of denervated wounds, P2Y(1) receptor protein expression was significantly increased in keratinocytes (P<0.001) but P2Y(1) receptors (P<0.001) compared with untreated denervated wounds. In innervated wounds, NGF treatment enhanced expression in keratinocytes. P2X(5) (P>0.001) and P2Y(1) receptor protein (P<0.001) expression in keratinocytes. P2X(7) receptors were absent in all experimental wound healing preparations. P2X(5), P2X(7), P2Y(1) and P2Y(2) receptor protein expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.     

结缔组织生长因子在口腔黏膜下纤维性变组织中的角质形成细胞中的表达

目的探讨结缔组织生长因子(CTGF)在口腔黏膜下纤维性变(OSF)发病机理中的作用.方法应用原位杂交法对34例OSF、10例正常口腔黏膜组织角质形成细胞(KC)中CTGF mRNA进行检测.结果24例(70.6%)OSF组织KC中有CTGF mRNA表达,早期OSF组织KC中CTGF mRNA阳性表达明显高于中晚期OSF(P＜0.05),10例正常口腔黏膜组织KC中CTGF mRNA表达呈阴性.结论OSF组织KC可合成分泌CTGF,在OSF发病机理中CTGF可能起重要作用,CTGF可能作为一中间介质参与了OSF的发病过程.     

Defective expression of a desmosome-related keratinocyte membrane antigen in squamous cell carcinoma

Intercellular junctions of keratinocytes are essential for coordination of the epithelial tissue function. Neoplastic transformation is often accompanied by changes in the glycolipid and glycoprotein composition of the cell membranes, although no evident structural modifications can be observed. We have investigated one aspect of the keratinocyte cell-cell contact in squamous cell carcinoma using KM48 monoclonal antibody as a differentiation-related marker for desmosomes. Quantitative analysis of the immunogold labelling revealed that the tumor cells lose the KM48 antigen during their neoplastic transformation. The process could also be observed, although to a lesser degree, in clinically and ultrastructurally normal marginal epidermis. The desmosome frequency in the tumor tissue also decreased, but not as radically as did the KM48 expression. Decreased expression of the desmosome-related membrane antigen suggests qualitative and perhaps functional modifications of the junction in squamous cell carcinoma.     

A genetic mechanism for cecal atresia: the role of the Fgf10 signaling pathway

BACKGROUND: Intestinal atresia represents a significant surgically correctable cause of intestinal obstruction in neonates. Intestinal development proceeds as a tube-like structure with differentiation along its axis. As the intestine differentiates, the cecum develops at the transition from small to large intestine. Fgf10 is known to serve a key role in budding morphogenesis; however, little is known about its role in the development of this transitional structure. Here we evaluate the effect of Fgf10/Fgfr2b invalidation on the developing cecum. MATERIALS AND METHODS: Wild-type C57Bl/6, Fgf10(-/-), and Fgfr2b(-/-) embryos harvested from timed pregnant mothers were analyzed for cecal phenotype, Fgf10 expression, and differentiation of smooth muscle actin. RESULTS: Wt cecal development is first evident at E11.5. FGF10 is discreetly expressed in the area of the developing cecum at early stages of development. One hundred percent of Fgf10(-/-) and Fgfr2b(-/-) mutant embryos demonstrate cecal atresia with absence of epithelial and muscular layers. The development of neighboring anatomical structures such as the ileocecal valve is not affected by Fgf10/Fgfr2b invalidation. CONCLUSIONS: FGF10 expression is localized to the cecum early in the normal development of the cecum. Fgf10(-/-) and Fgfr2b(-/-) mutant embryos demonstrate cecal atresia with complete penetrance. Epithelial and muscular layers of the cecum are not present in the atretic cecum. The Fgf10(-/-) and Fgfr2b(-/-) mutants represent a genetically reproducible animal model of autosomal recessive intestinal atresia.     

Mutation frequencies in murine keratinocytes as a function of carcinogenic status

A link between genetic abnormalities and carcinogenesis is well established. It follows that a correlation exists between mutation frequency and malignant progression. We have determined the spontaneous and DNA damage-induced mutation frequencies for a series of cell lines derived from SENCAR mouse keratinocytes at various stages of malignant progression. Nontumorigenic mouse keratinocytes (3PC), papillomas (MT1/2), squamous-cell carcinomas (CH72), and spindle-cell carcinomas (CH72T4) were transfected with damaged or undamaged shuttle vectors containing a supF mutation reporter gene. The plasmid mutation frequencies were determined by blue/white screening. The spontaneous plasmid mutation frequency of the squamous-cell carcinoma line was slightly higher than the mutation frequencies of the other cell lines tested. The DNA damage induced by triplex-directed psoralen crosslinks increased the mutation frequencies sixfold to eighteenfold in all cell lines tested, with no significant differences among the cell lines. Sequence analyses revealed that the spindle-cell carcinoma line had a different spontaneous mutation spectrum from the other cell lines. DNA damage-induced mutations were predominantly point mutations at the triplex-duplex junction in all of the cell lines tested, as expected. These data suggested that a strong mutator phenotype was not required for progression to an advanced malignant phenotype in our model system.     

Ultraviolet B induces hyperproliferation and modification of epidermal differentiation in normal human skin grafted on to nude mice

BACKGROUND: For ethical and technical reasons, the in vivo biological effects of ultraviolet (UV) radiation on skin are difficult to study in human volunteers. The use of human skin grafted on to nude mice may circumvent this difficulty. OBJECTIVES: To investigate the effects of a single moderate UVB exposure on human skin grafted on to nude mice. METHODS: Modifications of epidermal differentiation markers and patterns of keratin expression were assessed from 24 h to 14 days after a physiological UVB irradiation characterized by the induction of sunburn cells. RESULTS: During the first 48 h postexposure, involucrin, loricrin, transglutaminase type I, filaggrin and keratin K2e expression were altered together with the formation of abnormal horny layers. Constitutive keratin K14 was increased while keratin K10 expression was delayed. Newly synthesized keratins K6, K16, K17 and K19 were induced in parallel with an increase in the epidermal proliferation rate. A progressive normalization of both keratinocyte proliferation and differentiation took place during the following days, reaching completion within 2 weeks. CONCLUSIONS: Exposure of human skin to a UVB dose corresponding to a mild sunburn reaction induces epidermal hyperproliferation and alterations of several constitutive differentiation markers, as well as a drastic modification in the pattern of epidermal keratins. Although these modifications were shown to be progressively reversed in a single exposure model, the data also suggest that subsequent UV exposures occurring during the recovery period may lead to potentially deleterious long-term consequences, such as photoageing and photocarcinogenesis. Grafted human skin appeared to be an attractive and promising model for investigating the biological consequences of UVB radiation in vivo.     

Biologic therapy of inflammatory bowel disease

Advancing knowledge regarding the biology of chronic inflammation has led to the development of specific biologic therapies that mechanistically target individual inflammatory pathways. Many biologic therapies are being evaluated for the treatment of the chronic inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Biologic compounds proven to be effective for Crohn's disease include monoclonal antibodies to tumor necrosis factor (infliximab and CDP571) and to the leukocyte adhesion molecule alpha4 integrin (natalizumab). Other biologic compounds for which there is insufficient evidence to judge efficacy for inflammatory bowel disease include: p55 tumor necrosis factor binding protein (onercept); interferon alpha; interferon beta-1a; anti-interferon gamma antibody; anti-interleukin 12 antibody; p65 anti-sense oligonucleotide (blocks NF-kappaB); granulocyte colony stimulating factor, and granulocyte macrophage colony stimulating factor; anti-interleukin 2 receptor antibody; epidermal growth factor; keratinocyte growth factor 2 (repifermin); human growth hormone; anti-CD4 antibody; and anti-alpha4beta7 antibody. Biologic therapies that have been proven ineffective for inflammatory bowel disease include: interleukin 10; interleukin 11; anti-sense intercellular adhesion molecule-1; and the tumor necrosis factor receptor fusion protein etanercept. Based on the early successes of infliximab, CDP571 and natalizumab, it seems certain that biologic therapy will play an important role in the future treatment of inflammatory bowel disease.     

Characterization of the human Ly-6 antigens,the newly annotated member Ly-6K included,as molecular markers for head-and-neck squamous cell carcinoma

The E48 antigen is a successfully explored molecular marker for the diagnosis and therapy of HNSCC. The applicability of E48 as an HNSCC-associated antigen, however, is restricted due to its heterogeneous expression in 30% of tumors; and identification of additional target antigens is therefore desired. E48 belongs to the Ly-6 antigen family, comprising a group of highly homologous, low m.w., GPI-anchored surface proteins, of which some show tissue-restricted expression patterns. To identify novel human HNSCC-associated Ly-6 members with squamous cell-associated expression patterns, we performed comprehensive gene-screening consisting of BLAST searches within GenBank databases, followed by expression analysis. Using this approach, the Ly-6K gene could be annotated as a novel member of the human Ly-6 family. Expression of the human Ly-6 genes E48, Ly-6K, PSCA, GML, RIG-E, G6C and Ly-6H was prescreened by qualitative RT-PCR and subsequently analyzed by quantitative RT-PCR in normal keratinocytes, HNSCC cell lines, normal mucosa, HNSCC tumors as well as normal peripheral blood and bone marrow cells. PSCA was highly expressed in normal mucosa, but 100-fold decreased expression was seen in HNSCC. For Ly-6H, GML and G6C, no or very low expression was observed in keratinocytes and HNSCC. Expression of RIG-E was high in normal and malignant squamous cells and in peripheral blood and bone marrow cells, thus limiting its applicability as an HNSCC-associated marker. In contrast, besides the E48 gene, the Ly-6K gene also appeared to be selectively expressed in HNSCC and normal squamous cells. Moreover, expression of Ly-6K was shown in HNSCC cell lines, in which no E48 expression could be detected. These data justify further evaluation of Ly-6K as potential target antigen for the diagnosis and therapy of HNSCC.