TGF-β and TNF-α stimulate MMP-9 production in murine epidermal keratinocytes

Matrix metalloproteinases 2 and 9 (MMP‐2 and ‐9) are zinc‐dependent metalloenzymes and have gelatin‐degrading activity. Both MMP are known to be secreted by many types of cells and play important roles in several biological changes including tissue remodeling and wound healing. In the present study, a primary culture of murine epidermal keratinocytes was prepared and effects of transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) on expression of MMP‐2 and MMP‐9 by the keratinocytes was examined. Gelatin zymography revealed that murine epidermal keratinocytes secreted proenzyme forms of MMP‐2 and MMP‐9, but the active forms of both MMP were hardly detectable, indicating that in vitro autoactivation of these proenzymes did not occur. Both TGF‐β and TNF‐α stimulated MMP‐9 production in a dose‐dependent manner, but the MMP‐2 level was not changed. Interferon‐γ hardly affected production of MMP‐2 or MMP‐9. Ribonuclease protection assay demonstrated that TNF‐α increased the level of MMP‐9 mRNA 6‐fold compared to the control, whereas TGF‐β slightly up‐regulated it. These results suggest that expression of MMP‐9 could be regulated by several cytokines in murine epidermal keratinocytes.     

Pattern recognition receptors in companion and farm animals - the key to unlocking the door to animal disease?

The innate immune system is essential for host defence and is responsible for early detection of potentially pathogenic microorganisms. Upon recognition of microbes by innate immune cells, such as macrophages and dendritic cells, diverse signalling pathways are activated that combine to define inflammatory responses that direct sterilisation of the threat and/or orchestrate development of the adaptive immune response. Innate immune signalling must be carefully controlled and regulation comes in part from interactions between activating and inhibiting signalling receptors. In recent years, an increasing number of pattern recognition receptors (PRRs), including C-type lectin receptors and Toll-like receptors (TLRs), has been described that participate in innate recognition of microbes, especially through the so called pathogen-associated molecular patterns (PAMPs). Recent studies demonstrate strong interactions between signalling through these receptors. Whereas useful models to study these receptors in great detail in the murine and human system are now emerging, relatively little is known regarding these receptors in companion and farm animals. In this review, current knowledge regarding these receptors in species of veterinary relevance is summarised.     

Orf virus immuno-modulation and the host immune response

Orf virus encodes a range of immuno-modulatory genes that interfere with host anti-virus immune and inflammatory effector mechanisms. The function of these reflects the pathogenesis of orf. The orf virus interferon resistance protein (OVIFNR) and virus IL-10 (vIL-10) inhibit interferon production and activity. In addition the vIL-10 suppresses inflammatory cytokine production by activated macrophages and keratinocytes. The virus GM-CSF inhibitory factor (GIF) is a novel virus protein that binds to and inhibits the biological activity of GM-CSF and IL-2. Together, these immuno-modulators target key effector mechanisms of host anti-virus immunity to allow time for virus replication in epidermal cells.     

Upregulation of TNF-α Production by IFN-γ and LPS in Cultured Canine Keratinocytes: Application to Monosaccharides Effects

Activated keratinocytes play a key role in the cutaneous immune system by their interactions with other cell types through the production of cytokines with both autocrine and paracrine activity. But there is little knowledge about epidermal cytokines in the dog. In this study, cultured canine keratinocytes were stimulated by human recombinant interferon γ (IFN-γ) and lipopolysaccharide (LPS) and cell supernatants were tested for tumour necrosis factor α (TNF-α) concentration using a cell viability assay on a murine cell line. We show that IFN-γ in combination with LPS significantly increases TNF-α secretion by canine keratinocytes. The best stimulations were obtained using confluent cultures and the association of IFN-γ (400 ng/ml) and LPS (40 μg/ml). The experimental protocol we describe represents a new method for studying keratinocyte activation and its modulation in the dog. We provide an example of application of our method: the study of the effects of different monosaccharides on canine keratinocyte activation.     

Leukocyte endothelial cell interactions in pig to human organ xenograft rejection

In cases where antibody- and complement-mediated hyperacute rejection (HAR) of vascularized organ xenografts has been prevented, acute vascular rejection (AVR) and acute T cell-mediated rejection (ACR) cause graft destruction. Infiltration of leukocytes (innate and graft-primed T cells) into the graft execute the latter two rejection modalities. The leukocyte extravasation process, which is a prerequisite for graft infiltration, is governed by adhesion molecules, including the selectin, integrin and immunoglobulin protein families, and the chemokine protein family. The compatibility between porcine endothelial cell and human leukocyte adhesion molecules was investigated in dynamic adhesion and static transendothelial migration assays. The effect of human anti-pig antibodies on human leukocyte adhesion to, and transendothelial migration across, porcine endothelium was assessed under dynamic and static conditions, respectively. In contrast to previously published results, no difference in the ability of neutrophils to adhere to pig and human endothelium was observed. Furthermore, no evident quantitative or qualitative differences in the capacity of human and porcine endothelium to support transendothelial migration of human leukocytes (T, B and natural killer (NK) cells, monocytes, and neutrophils) could be detected. The presence of human anti-pig antibodies (Abs) modulated the migration of leukocytes across porcine endothelium, as well as neutrophil adhesion to porcine endothelium under conditions of flow. Antibodies specific for pig endothelial adhesion molecules can potentially be used as species (graft)-specific immunosuppressive reagents in order to prevent cellular organ xenograft rejection.     

Advances on the Experimental Infectious Models of Avian Pathologenic Escherichia coli

Avian pathogenic Escherichia coli(APEC) is an important pathogen that causes localized and systemic infection in avian species of all ages.It is also an important reservoir or source of virulence genes of human extraintestinal pathogenic Escherichia coli (ExPEC).In order to understand deeply the infection progresses,pathogenesis,host immune responses and genetic resistance mechanism of APEC,and evaluate the efficacy of drugs and vaccines,several experimental infection models were established to evaluate the virulence of APEC through different approaches.According to the different systems involved,it can be divided into respiratory system,vascular system,musculoskeletal system,dermatological system,reproductive system,gastrointestinal system and chicken embryo system.In addition,there are infection experiments in mice and rats,tissue culture cells and explants infection experiments in vitro.The author highlights the establishment,pathogenesis,host responses and application of the different APEC experimental infection models.     

Primary neoplasms of bones in mice: retrospective study and review of literature

To compare and summarize the mechanisms, frequencies of occurrence, and classification schemes of spontaneous, experimental, and genetically engineered mouse skeletal neoplasms, the literature was reviewed, and archived case material at The Jackson Laboratory was examined. The frequency of occurrence of spontaneous bone neoplasms was less than 1% for most strains, with the exceptions of osteomas in CF-1 (5.5% and 10% in two studies) and OF-1 outbred strains (35%), and osteosarcomas in NOD/ShiLtJ (11.5%) and NOD-derived (7.1%) mice. The frequency was 100% for osteochondromas induced by conditional inactivation of exostoses (multiple) 1 (Ext1) in chondrocytes, osteosarcomas induced by tibial intramedullary inoculation of Moloney murine sarcoma virus, and osteosarcomas induced by conditional inactivation of Trp53-with or without inactivation of Rb1-in osteoblast precursors. Spontaneous osteogenic neoplasms were more frequent than spontaneous cartilaginous and vascular types. Malignant neoplasms were more frequent than benign ones. The age of occurrence for spontaneous neoplasms ranged from 37 to 720 days (M = 316.35) for benign neoplasms and 35 to 990 (M = 299.28) days for malignant. In genetically engineered mice, the average age of occurrence ranged from 28 to 70 days for benign and from 35 to 690 days for malignant. Histologically, nonosteogenic neoplasms were similar across strains and mutant stocks; osteogenic neoplasms exhibited greater diversity. This comparison and summarization of mouse bone neoplasms provides valuable information for the selection of strains to create, compare, and validate models of bone neoplasms.     

禽致病性大肠杆菌实验感染模型研究进展

禽致病性大肠杆菌（avian pathogenic Escherichia coli,APEC）是导致不同日龄禽类局部和全身感染的重要病原菌,也是人肠外致病性大肠杆菌（extraintestinal pathogenic Escherichia coli,ExPEC）毒力基因的重要储存宿主或来源。为深入理解APEC的感染过程、致病机理、宿主免疫应答及遗传抵抗机制、评估药物和疫苗防治效果,研究者通过不同途径建立了多种评估APEC毒力的实验感染模型。根据所涉及的系统不同可分为呼吸系统、脉管系统、肌肉系统、皮肤系统、生殖系统、消化系统及鸡胚系统等。此外,尚有小鼠与大鼠感染试验及组织培养细胞和外植块感染试验等。作者重点介绍了不同APEC实验感染模型的建立、致病机制、宿主应答及应用。     

Cell adhesion molecules, leukocyte trafficking, and strategies to reduce leukocyte infiltration

Leukocyte-endothelial cell interactions are mediated by various cell adhesion molecules. These interactions are important for leukocyte extravasation and trafficking in all domestic animal species. An initial slowing of leukocytes on the vascular endothelium is mediated by selectins. This event is followed by (1) activation of beta2 integrins after leukocyte exposure to cytokines and pro-inflammatory mediators, (2) adherence of leukocyte beta2 integrins to vascular endothelial ligands (eg, intercellular adhesion molecule-1 [ICAM-1]), (3) extravasation of leukocytes into tissues through tight junctions of endothelial cells mediated by platelet and endothelial cell adhesion molecule-1 (PECAM-1), and (4) perivascular migration through the extracellular matrix via beta1 integrins. Inhibiting excessive leukocyte egress and subsequent free radical-mediated damage caused by leukocyte components may attenuate or eliminate tissue damage. Several methods have been used to modify leukocyte infiltration in various animal models. These methods include nonspecific inhibition of pro-inflammatory mediators and adhesion molecules by nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, inhibition of cytokines and cytokine receptors, and inhibition of specific types of cell adhesion molecules, with inhibitors such as peptides and antibodies to beta2 integrins, and inhibitors of selectins, ICAMs, and vascular cell adhesion molecule-1 (VCAM-1). By understanding the cellular and molecular events in leukocyte-endothelial cell interactions, therapeutic strategies are being developed in several animal models and diseases in domestic animal species. Such therapies may have clinical benefit in the future to overcome tissue damage induced by excessive leukocyte infiltration.     

FC‐27 A spontaneous dog model for in vivo gene therapy of junctional epidermolysis bullosa

Junctional epidermolysis bullosa (JEB) is a mechanobullous skin disease associated with mutations in the basement membrane components of the dermo‐epidermal junction. This condition is a suitable prototype for proving the feasibility of a gene therapy approach to genodermatoses. As successful genetic treatment requires immunocompetent animal models to study the possible host immune response to the transgene, we have characterized a breed of short‐haired pointers suffering from a mild form of JEB. These animals exhibit skin blistering and erosions following minor trauma. Immunohistochemical analysis of the skin biopsies and immunoprecipitation of spent medium from cultured JEB keratinocytes showed reduced expression and secretion of the α3 chain of laminin 5 (α3, β3, γ2), the major adhesion ligand of basal keratinocytes. The search for genetic mutations detected a homozygous insertional mutation (4818 + 207 ins6.5 kb) in intron 35 of the lama3 gene. Consequently, keratinocytes from dogs with JEB secrete reduced amounts of wild‐type laminin 5, and adhesive properties of the keratinocytes are compromised. Retroviral transfer of wild‐type dog α3 cDNA into JEB keratinocytes enhanced secretion of laminin 5 in the extracellular matrix and restored the adhesion, differentiation and proliferative capacity of the transduced cells. Transplantable fibrin‐based skin equivalents made with transduced JEB keratinocytes and grafted onto SCID mice generated normal cohesive and stratified epithelia and showed stable localized deposition of laminin 5 at the dermo‐epidermal junction. Our results form the basis for preclinical assays of gene therapy on a unique immunocompetent animal model for an inherited skin disease. Funding: DEBRA (UK) Foundation, Association Francaise Contre les Myopathies.     

Coordinated host defense through an integration of the neural,immune and haemopoietic systems

Interactions between the neural and immune systems exist through humoral factors operating via the hypothalamic-pituitary-adrenal axis and cytokines acting over a relatively long distance. Anatomical evidence also suggests direct, hard-wired pathways of interaction and control through innervation of lymphoid organs and peripheral sites involved in host defense, including the thymus, spleen, lymph nodes, and skin. Recent evidence has demonstrated: 1) neural control of the bone marrow haemopoietic system, 2) interactions between peripheral nerve endings in the skin and epidermal Langerhans cells, and 3) peripheralization of leukocytes in the initial stages of stress. This leads us to propose that the nervous system is involved in host monitoring and coordination of host defense systems. If the brain is to have appropriate control of host defense mechanisms it must have: (a) afferent inputs monitoring host defense status, (b) efferent control pathways that modulate primary reactions to infection and damage, (c) efferent activation pathways to the myeloid defense system while the specific, lymphoid immune system is activated, and (d) inhibition of the proliferative lymphocytic response if the infection has been dealt with. We are investigating whether such a model, which allows for control and coordination of both the initial myeloid defense system and of the acquired immune response, is observed in mammals.     

Endothelial–platelet interactions in influenza-induced pneumonia: A potential therapeutic target

Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro-thrombotic milieu and infiltration of immune effector cells. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza-mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza-induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.     

Comparison of growth and differentiation of normal and neoplastic canine keratinocyte cultures

Neoplastic canine keratinocytes derived from a spontaneous oral squamous cell carcinoma were maintained in culture for more than 45 passages. The presence of desmosomes and keratin filaments was demonstrated by electron microscopy and immunohistochemistry. The keratinocytes were grown in two different culture conditions to induce variations in the stage of differentiation, i.e., in submerged cultures and at the air-liquid interface. For comparison, normal canine keratinocytes were grown under the same conditions. Anisocytosis was present in neoplastic cultures grown submerged in medium. Grown at the air-liquid interface, neoplastic keratinocytes differentiated into a well-organized, multilayered stratified squamous epithelium analogous to normal keratinocytes. Rare areas of irregular growth and formation of whorls were detected. Expression of lectin binding sites and specific cell surface antigens of neoplastic and normal keratinocytes demonstrated marked similarities between the two cell lines. Neoplastic cells lacked certain surface antigens that are present on normal cells. Squamous cell carcinoma cells grew faster than normal canine keratinocytes as demonstrated by growth curve evaluation. Neoplastic keratinocytes responded to growth stimulation by epidermal growth factor and cholera toxin as do normal keratinocytes. Neoplastic cells grown in medium lacking these factors proliferated faster than growth factor stimulated normal keratinocytes.     

Phenotypic analysis for a cell line of canine epidermal keratinocytes

Epidermal keratinocytes have the potential to produce inflammatory mediators that are considered to play an important role in skin diseases such as atopic dermatitis (AD). Thus, cell lines of canine epidermal keratinocytes are useful for studying the biological reactivity of keratinocytes in vitro. However, there has been no report on properly analyzing the phenotype of canine keratinocyte cell lines. In this work, we performed phenotypic analysis of CPEK, which was derived from the epidermis of an adult dog in order to examine the phenotypic similarity with epidermal keratinocytes. The present findings indicated that CPEK cells expressed markers for epidermal keratinocytes including cytokeratin 14, alpha6 integrin and PCNA. Our findings demonstrated that CPEK could be a useful cell line for investigating the central role of epidermal keratinocytes in the pathogenesis of AD in vitro.     

Powder and particle-mediated approaches for delivery of DNA and protein vaccines into the epidermis

The epidermis of the skin is both a sensitive immune organ and a practical target site for vaccine administration. However, administration of vaccines into the epidermis is difficult to achieve using conventional vaccine delivery methods employing a needle and syringe. A needle-free vaccine delivery system has been developed that efficiently delivers powdered or particulate DNA and protein vaccines into the epidermal tissue. The delivery system can be used to directly transfect antigen presenting cells (APCs) by formulating DNA or protein vaccines onto gold particles (particle-mediated immunization). Antigen can be directly presented to the immune system by the transfected APCs. Antigen can also be expressed and secreted by transfected keratinocytes and picked up by resident APCs through the exogenous antigen presentation pathway. Alternatively, protein antigens can be formulated into a powder and delivered into the extracellular environment where they are picked up by APCs (epidermal powder immunization). Using any of these formulations, epidermal immunization offers the advantage of efficiently delivering vaccines into the APC-rich epidermis. Recent studies demonstrate that epidermal vaccine delivery induces humoral, cellular, and protective immune responses against infectious diseases in both laboratory animals and man.     

病原微生物荚膜多糖的生物学功能

荚膜多糖(capsular polysaccharide,CPS)是一种广泛存在于细菌、支原体、部分真菌等菌体表面的碳水化合物.同时,荚膜多糖有助于菌体抵抗干燥和低温等不利环境,并通过在菌体表面形成物理屏障阻碍宿主补体的杀伤与吞噬作用.在长期多种应激-压力环境下,病原菌已进化出多种免疫逃避机制并促进宿主感染;在非病原微...     

Cytologic evaluation of otic exudates

The multifactorial nature of otitis externa requires accurate etiologic information to ensure therapeutic success. The collection and preparation of cytologic samples of otic exudates are simple to perform, and information of immediate diagnostic and therapeutic value can be attained. Evidence of epidermal hyperplasia and increased glandular secretory activity suggests a noninfectious cause. Large numbers of microorganisms and infiltrating leukocytes confirm the presence of infection. The presence of ear mites, particularly O. cynotis, is always significant. Abnormal epithelial cells that appear singly or in sheets and clusters support the diagnosis of neoplasia; the extent of cell differentiation determines malignancy. Inflammatory cells and acantholytic keratinocytes from vesicular otic lesions suggest autoimmune skin disease.     

Identification of heterologous monoclonal antibodies that cross-react with cervine leukocyte subpopulations

The degree to which cross-reactivity between monoclonal antibodies developed against cells of the human, mouse, bovine and ovine immune systems, and cells of the cervine immune system occurs was investigated. It was found that within the ruminants a considerable degree of cross-reactivity does exist while there is virtually none between the cervine and murine or human systems. The highest incidence of cross-reactivity was found between ovine monoclonals and cervine leukocytes (46% cross-reactive) with 25% of bovine monoclonal antibodies cross-reacting with deer leukocytes. Ovine monoclonals were found to be the most useful in identifying a wide range of cervine leukocyte subpopulations. Bioassays showed that ovine anti-class I and II monoclonals detected molecules on cervine leukocytes that are functionally similar to MHC antigens. The possibility that cross-reactive monoclonals detect similar subpopulations in both the homologous and heterologous species is discussed.     

Critical role of the vascular endothelial cell in health and disease: a review article

Objective: To review the human and veterinary literature on the role of the vascular endothelial cell in health, as well as during hypoxic and inflammatory disease states. Data sources: Data from human and veterinary literature were reviewed through a Pubmed search and a manual search of the references listed in articles covering some aspect of vascular endothelial cell function. Human data synthesis: The development of techniques that allow the maintenance and growth of endothelial cells in culture has produced an explosion of new research in the area of endothelial cell physiology. This plethora of data has revealed the critical role that vascular endothelial cells play in both health and disease states. Interspecies variations can occur with respect to the vascular endothelial cell physiology and its response to pathologic conditions. Veterinary data synthesis: There is a paucity of information regarding the role of the vascular endothelial cell in health or disease of small animals. Many human studies use species cared for by veterinarians, providing information that may be applied to small animals and that may be used to construct future studies. Conclusion: An organ system itself, the vascular endothelium is an essential component of all organs in the body. The endothelial cell lining functions to maintain selective permeability between the blood and the tissue it supplies, regulate vascular tone, sustain blood fluidity through regulation of coagulation, and modulate interaction of leukocytes with the interstitium and inflammatory reactions. During disease states, the endothelial cell functions locally to limit the boundaries of the disease process. If these functions are not controlled, they can become a part of the pathogenic process, contributing to blood stasis and thrombosis, potentiation of local inflammation and interstitial edema formation, subsequent tissue hypoxia, and multiple organ dysfunction. Pharmacological investigations targeting the modulation of endothelial function during disease states have not yet advanced treatment protocols. Since all critically ill animals are at risk for some degree of endothelial cell dysfunction, treatment regimens should focus on promoting capillary blood flow and tissue oxygen delivery.