Development of a peptide inhibitor of hyaluronan-mediated leukocyte trafficking.

Hyaluronan (HA), a high molecular weight glycosaminoglycan, is expressed abundantly in the extracellular matrix and on cell surfaces. Although HA is known to bind many adhesion molecules, little information has been available with respect to its direct physiological role. In this study, we developed a novel 12-mer (GAHWQFNALTVR) peptide inhibitor of HA, termed "Pep-1," by using phage display technology. Pep-1 showed specific binding to soluble, immobilized, and cell-associated forms of HA, and it inhibited leukocyte adhesion to HA substrates almost completely. Systemic, local, or topical administration of Pep-1 inhibited the expression of contact hypersensitivity responses in mice by blocking skin-directed homing of inflammatory leukocytes. Pep-1 also inhibited the sensitization phase by blocking hapten-triggered migration of Langerhans cells from the epidermis. These observations document that HA plays an essential role in "two-way" trafficking of leukocytes to and from an inflamed tissue, and thus provide technical and conceptual bases for testing the potential efficacy of HA inhibitors (e.g., Pep-1) for inflammatory disorders.

Survey of Japanese layer farms for Salmonella enteritidis with vaccination- and infection-specific antigens for egg yolk antibodies.

Japanese layer farms were surveyed for Salmonella Enteritidis vaccination and infection with specific antigens for egg yolk antibodies with the use of vaccination-specific antigen Salmonella Enteritidis FliC-specific 9-kDa polypeptide (SEP9) and infection-specific antigen deflagellated Salmonella Enteritidis whole cell (DEWC). The specific antibodies in eggs from 201 commercial layer farms throughout Japan were surveyed. The percentages of farm flocks with a mean enzyme-linked immunosorbent assay (ELISA) titer of over 0.1 were 56.2% (113 of 201) in DEWC-ELISA and 22.3% (45 of 201) in SEP9-ELISA. Flocks indicating high titers in SEP9-ELISA always showed high titers in DEWC-ELISA. Because both specific antibody titers of the vaccinated flocks monitored long term remained high throughout life, flocks with high titers of both ELISAs in this survey must be vaccinated. On the other hand, 34.3% (69 of 201) of flocks had high titers of DEWC-specific antibody alone. Because Salmonella Enteritidis infection induces the DEWC-specific antibody but not the SEP9-specific antibody, detecting only high ELISA titers of DEWC-specific antibody can be an effective monitoring tool for Salmonella Enteritidis exposure rather than vaccination. These results suggest that vaccination programs in Japanese layer farms would be insufficient to control Salmonella Enteritidis infection, and egg screening to detect specific antibodies would be valuable in obtaining the necessary information to control Salmonella Enteritidis infection.

Enhanced contact hypersensitivity in human monocyte chemoattractant protein-1 transgenic mouse.

Monocyte chemoattractant protein (MCP)-1 is a chemotactic cytokine for monocytes, memoryT cells and dendritic cells (DC). However, the precise role of MCP-1 in a variety of immunological responses remains unclear. In the present study, we analyzed contact hypersensitivity (CHS) using human MCP-1 transgenic mice (hMCP-1Tgm) that constitutively produce high levels of hMCP-1 in the sera. Following 2,4-dinitrofluorobenzene (DNFB) sensitization, enhancement of CHS was demonstrated in Tgm as compared with that in non-Tgm. Anti-hMCP-1 antibodies significantly inhibited the CHS in Tgm. A prominent accumulation of B7-1+I-Ad+ Langerhans' cells (LC) bearing haptens was detected in draining lymph nodes (DLN) of Tgm 24 h after DNFB or fluorescein isothiocyanate (FITC) sensitization. Similar results were obtained with BALB/c mice administrated recombinant (r) hMCP-1. Langerhans' cells (LC) in the epidermal sheets of Tgm increased in size and expressed high levels of I-Ad and B7-1 12 h after FITC application compared with those of non-Tgm. After 18 h, the number of LC in the epidermis was reduced in Tgm. It was also shown that the B7-1 expression on LC of BALB/c mice was augmented after culture with rhMCP-1. These findings demonstrate that MCP-1 not only accelerates LC migration from epidermis into the DLN after sensitization with haptens but also up-regulates the I-Ad and B7-1 expressions, which results in the enhanced T cell activation and CHS.

Study of lipid in the ear canal in canine otitis externa with Malassezia pachydermatis.

An epidemiological investigation of 120 canine otitis externa cases in 1,370 dogs was done on the incidence rate, ear pinna shapes, breeds and their relationships. Eighty-five cases (12.6%) in 672 dogs with pendulous ears and 35 cases (5.0%) in 698 dogs with erect ears had otitis externa, and the difference between them was significant (P<0.05). Ninety-five auditory cerumen specimens were cultured for Malassezia pachydermatis (M. pachydermatis) and analyzed for concentrations of major fatty acids. Although rates of cases positive for M. pachydermatis in both ear pinna shapes were almost the same, i.e. 55.2% in the pendulous group and 53.6% in the erect group, the average total fatty acid level of the pendulous ear group was significantly (P<0.05) higher than that in the erect ear group after dismissing extraordinary levels in the Siberian husky. Isolated M. pachydermatis strains were examined for the effects of fatty acid supplementation on their growth. The majority of the strains utilized fatty acids and grew faster in fatty acid supplemented broth. These results suggest that M. pachydermatis, the predominant causative agent of canine otitis externa, prefers the auditory canal of dogs with lipid-rich earwax and grows fast, but growth strongly depends upon the canine breed.

[Analysis of contact hypersensitivity response in human monocyte chemoattractant protein (MCP)-1 transgenic mice].

Epidermal Langerhans cells (LC) belong to the dendritic cell lineage and represent the major antigen-presenting cells (APC) within the skin. The molecular mechanisms responsible for LC migration to the skin have not fully been defined. MCP-1 is a cytokine of C-C chemokine subfamily that is chemotactic in vitro for monocytes, memory T cells and dendritic cells. In the present study, to examine the roles of LC and MCP-1 in hapten-induced contact hypersensitivity response (CHR), we utilized human MCP-1 transgenic mice (hMCP-1 Tgm) that produce constitutively high levels of hMCP-1 in the sera. Following DNFB sensitization, enhancement of CHR was demonstrated in Tgm as compared with CHR in non-Tgm at the entire period examined. Anti-hMCP-1 antibody significantly inhibited the DNFB-included CHR in Tgm. For analysis of the mechanisms underlying the enhanced CHR, migration and activation of LC were examined in Tgm and non-Tgm. The number of LC in the Tgm skin was within a normal range. However, the Tgm showed an accumulation of NLDC-145+ cells in the draining lymph nodes (DLN) 24 h after DNFB sensitization. When Tgm and non-Tgm were treated with FITC, the number of FITC+NLDC-145+ cells was larger in Tgm DLN than that in non-Tgm DLN 24 h after FITC application. Moreover, administration of recombinant hMCP-1 to BALB/c mice led to an increase of FITC+NLDC-145+ cells in the DLN. In addition, 12 h after application of FITC, LC in the epidermal sheets of Tgm increased in size and expressed high levels of I-Ad as compared with those of non-Tgm. Expression of B7-1 in 24h-cultured LC of BA LB/c mice was augmented by addition of recombinant hMCP-1 in a dose-dependent manner. These findings suggest that MCP-1 accelerates LC migration from epidermis into the DLN and up-regulates I-Ad and B7-1 expressions on the LC, which eventually enhances CHR.

Açäo de etomidato sobre os potenciais evocados auditivos do tronco cerebral de ratos / Effect of etomidate on the brainstem auditory evoked potentials of rats

A utilizaçäo dos potenciais evocados auditivos durante cirurgias exige o emprego de anestésicos que näo interfiram sobre as suas características como latência, amplitude e configuraçäo. Alguns compostos, como os anestésicos voláteis, modificam as respostas evocadas a um estímulo sonoro, tanto do córtex como do tronco cerebral. Os agentes venosos possuem efeito diverso, em geral interferindo apenas com as respostas corticais. Na pesquisa foram comparados os efeitos do etomidato (5 mg.kg-1) e do pentobarbital sódico (40 mg.kg-1) sobre os potenciais evocados auditivos do tronco cerebral de 27 ratos. Esses animais foram submetidos a duas sessöes sucessivas com intervalo de uma semana, para registro dos potenciais evocados auditivos do tronco cerebral, sob efeito de etomidato e pentobarbital sódico. Foram usados pulsos retangulares de 0,8 ms, por um gerador de dois canais, estímulos de 100 dB-NHL e seqüência de repetiçäo de 10-1s. As respostas foram registradas e analisadas a latência, a amplitude e a configuraçäo das ondas. Observou-se que o etomidato apresenta um efeito näo distingüível do pentobarbital quanto à latência e configuraçäo do traçado. Houve um aumento estatisticamente significante da amplitude das ondas II e V pelo etomidato em relaçäo ao pentobarbital. Esses resultados mostram que o etomidato näo deprime a conduçäo sensitiva do nível do tronco encefálico