猪脑微血管内皮细胞和星型胶质细胞共培养模拟体外血脑屏障模型

血脑屏障是隔离外周体循环和中枢神经系统的屏障结构.它可以选择性允许血液中的一些物质通过脑微血管内皮细胞进入大脑中.除了脑微血管内皮细胞之外,血脑屏障的组成主要还有星型胶质细胞、周细胞、神经元和细胞外基质.本试验通过原代培养猪脑微血管内皮细胞和星型胶质细胞,以Transwell细胞板为载体构建猪体外血脑屏障的共培养模型.经过4h渗漏试验和TEER电阻的测定试验显示所构建的猪体外血脑屏障的模型具备了血脑屏障基本的生物学特性,可以用于猪血脑屏障相关疾病尤其是人兽共患性疾病致病机制的研究.     

A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders.     

The blood-brain barrier and its role in inflammation

The unique microenvironment within the central nervous system (CNS) relies upon the integrity of the blood-brain barrier (BBB). This selectively permeable barrier comprises interendothelial tight junctions located at the capillaries and postcapillary venules. Cells and structures in the local environment are required to maintain normal BBB function. When inflammation is present, the BBB itself plays an integral role in the inflammatory response by either producing or expressing a variety of cytokines, adhesion molecules, metalloproteinases, serine proteases, products of arachidonic acid metabolism, and nitric oxide. Understanding the role of the BBB during inflammation is essential when creating and employing a therapeutic regime for animals with CNS disease. This review focusses on recent discoveries about the BBB and its role in inflammation, and applies this knowledge to our current understanding of inflammatory CNS disease in dogs and cats.     

Immunophenotypic and histological characterisation of 109 cases of feline lymphosarcoma

OBJECTIVE: To determine and analyse the immunophenotype and histological appearance of naturally occurring cases of lymphosarcoma in Australian cats. DESIGN: A prospective multi-institutional study of naturally occurring feline lymphosarcoma. METHODS: One hundred and eighteen cats were referred for diagnosis and/or management of suspected lymphosarcoma. Tissue samples for histopathological analysis and immunophenotyping were collected as biopsies or at necropsy from 109 cases. Histological classification of the neoplasms followed the Working Formulation Classification System. Four multi-species cross-reactive antibodies were used to classify tumours as having a B or T cell phenotype. RESULTS: Seventy-six (70%) cases were B cell tumours and 28 (26%) were T cell tumours. The remaining 5 (4%) specimens failed to stain with the four antibodies. Histologically, 11 (10%) cases were classified as low-grade, 72 (66%) were medium-grade and 26 (24%) were high-grade tumours. There were no significant associations between age and either histological grade or immunophenotype. Mediastinal and leukaemic cases were significantly more likely to be T cell tumours (P < 0.001 and P < 0.001, respectively). CONCLUSIONS: In contrast to previously documented studies in the cat, the majority of cases of lymphosarcoma were of B cell phenotype and intermediate histological grade. Based on our data, the histological phenotype of lymphosarcoma is unlikely to predict immunotype, nor are cases of certain immunotypes likely to be of specific histological subtype. Considered in relation to previous reports, the findings suggest that epidemiological factors operating in these cats to produce lymphosarcoma may be different to those operating in North American and UK cat populations.     

Nylon wool column fractionation and characterisation of feline lymphocyte subpopulations

Feline separated mononuclear cells (SMC) were obtained from peripheral blood by ficoll-diatrizoate gradient separation. SMC were further fractionated on nylon wool columns into nylon wool adherent cells (NWAC) and nylon wool effluent cells (NWEC). The three cell populations, SMC, NWAC and NWEC, were characterised using direct immunofluorescent staining for surface immunoglobulin (sIg) as a B cell marker and neuramidase treated guinea pig erythrocyte-rosette formation (E-rosettes) and mitogen-induced lymphocyte blastogenesis (LB) as possible T-cell markers. Feline SMC consisted of 30.1 +/- 4.0% sIg+ cells 36.6 + 5.4% E-rosette forming cells and 33.3% null cells i.e. cells which were sIg- and non E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming cells. NWAC were 51.0% +/- 10.8% sIg+, approximately 20% of cells were lost. The LB responsiveness of NWEC to concanavalin A (Con A) and phytonaemagglutinin-P (PHA-P) was enhanced compared to SMC. NWAC were non-responsive to Con A and PHA-P at all concentrations tested. It was concluded that nylon wool column fractionation of feline SMC was an efficient procedure for T cell enrichment and that the enriched cells retained the properties of E-rosette formation and blastogenesis by mitogens.     

Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and persistently infected cats.

The study determines the sequence evolution of feline calicivirus both in cell culture and in persistently infected cats and relates this to changes in virus neutralisation.     

Variable sensitivity of a feline embryo cell line and of three kitten kidney cell cultures to feline herpes- and caliciviruses

The number of plaques produced in a feline embryo (FEmb) cell line and in three independently derived kitten kidney (KK) cell cultures varied in a consistent and reproducible manner when each was inoculated with the same number of feline herpesvirus 1 (FHV1) plaque forming units (PFU); the three KK cells produced 2-9 times more plaques than FEmb cells. One of the three KK cells produced FHV1 plaques that were smaller in diameter than those FEmb cells. Each of the three KK cell cultures inoculated with the same number of FEmb cell PFU of a strain of feline calicivirus (FCV) produced different numbers of plaques; two of the three KK cell cultures produced 2-3 times more plaques than FEmb cells. The plaque diameter of FCV in the three KK cells was 30-50% smaller than the plaque diameter in FEmb cells.     

Induction of feline immunodeficiency virus from a chronically infected feline T-lymphocyte cell line

The infection of the feline T-lymphocyte cell line FeT-J with the feline immunodeficiency virus (FIV) Petaluma strain led to the establishment of nonvirus-producing cells. One clone (C15) obtained by limiting dilution was found to express FIV in response to chemical inducers of retroviruses. The chemical treatment of C15 cells led to not only FIV protein synthesis but also an augmentation of viral production. Examination of the C15 cell derivatives obtained by recloning revealed that 10-40% of treated cells constitutively expressed FIV antigens, whereas 100% with expressed FIV antigen in response to the inducer. Chemical induction resulted in more than a 100-fold increase in infectious viral production. The results suggest that a majority of FeT-J cells that are infected with FIV exist in a non-productive state. Establishing a cell line that can be non-productively infected by FIV may help determine the mechanisms of FIV latency.     

Phylogenetic characterisation of naturally occurring feline immunodeficiency virus in the United Kingdom

Feline immunodeficiency virus (FIV) is a significant pathogen of domestic and non-domestic felids worldwide. In domestic cats, FIV is classified into five distinct subtypes (A-E) with subtypes A and B distributed most widely. However, little is known about the degree of intrasubtype viral diversity and this may prove critical in determining whether monovalent vaccines are likely to protect against FIV strains within a single subtype. Here, we characterise novel env sequences from 47 FIV strains recovered from infected cats in the United Kingdom and its environs. Phylogenetic analyses revealed that all bar one sequence belonged to subtype A, the predominant subtype in Western Europe. A single sequence was identified as a likely subtype A/C recombinant, intriguing given that subtype C does not appear to exist in either the UK or North Western Europe and suggestive of a recombination event predating its introduction into the UK. Subtype A strains from the UK were not significantly differentiated from representative subtype A isolates found elsewhere suggesting multiple introductions of FIV into the country. Divergence among isolates was comparable to that observed for subtype A isolates worldwide, indicating that FIV in the UK covers the full spectrum of subtype A diversity seen globally. This study demonstrates that while subtype A is predominant in the UK, novel introductions may result in the emergence of novel subtypes or intersubtype recombinants, potentially circumventing vaccine strategies. However, the dominance of subtype A suggests that the development of a regional or subtype-specific protective vaccine for the UK could be achievable.     

Characteristics of a feline mammary carcinoma cell line

The establishment of an epithelial cell line, JM, from a feline mammary adenocarcinoma is described. In vitro, the morphological and cultural properties of these cells, their surface features such as lectin binding and their response to hormones were ascertained. In vivo, they are tumorigenic in athymic nude mice inducing tumour nodules composed of epithelia-like cells.     

Tissue culture of feline normal mammary gland

To study the growth and morphology of feline mammary epithelial cells in vitro, normal feline mammary tissues as well as cells collected from feline milk were cultured and examined using phase-contrast microscopy and immunohistochemistry. The method employed was a modification of Hiratsuka et al., and was found to be useful for the culture of dissociated feline mammary tissue. Small polygonal cells and large spindle-shaped cells that were positively stained with anti-keratin and anti-actin antibodies formed colonies with a pavement-like structure in culture of mid-pregnant mammary gland. Cultures of involuting mammary gland were composed mainly of small spindle-shaped cells, which were negative for keratin immunostaining and thought to be fibroblasts. Myofibroblasts, which participate in feline mammary carcinogenesis, and show keratin negative, actin positive, were not encountered in the present culture system of normal mammary gland. The cells from milk formed small colonies, but did not grow to reach confluent monolayer.     

Diagnosis and treatment of a feline oral mast cell tumor

A cat was diagnosed with an oral mast cell tumor following incisional biopsy. The location of the tumor, possible metastasis, financial restraint and patient disposition severely limited therapeutic options. The patient was treated with six doses of 1-(2-chloroethyl)3-cyclohexyl-1-nitrosurea (CCNU) and methylprednisolone acetate. Complete remission was obtained after the third dosing regimen. This is the first documented case of feline oral mast cell tumor and one of a small group of cats with various cancers to be responsive to CCNU treatment.     

Infection studies in kittens, using feline infectious peritonitis virus propagated in cell culture

The propagation of feline infectious peritonitis virus (NW1-FIPV strain) in cell culture is described. Tissue culture-propagated virus was used to inoculate specific-pathogen-free kittens intraperitoneally, intratracheally, or orally. Intraperitoneal inoculation caused seroconversion and effusive peritonitis in 100% of the kittens. Intratracheal inoculation produced disease in 60% of the kittens, and oral inoculation in only 20%. Seroconversions without production of disease occurred in 10% of the kittens inoculated by either the intratracheal or the oral route. The remainder of the kittens inoculated by the intratracheal (30%) and oral (70%) routes did not develop serum antibodies or disease.     

Studies of enteric coronaviruses in a feline cell line

Development is reported of a feline cell line which can support the growth of coronaviruses from canine (CCV), feline (FIPV) and porcine (TGEV) species. The cell culture has been serially transferred over 100 times and has retained its initial growth requirements, proliferative capacity and morphologic features. Each virus had specific growth characteristics in this cell culture although all produced a similar CPE and plaques under agar. Cross neutralization studies demonstrated a two-way relationship between TGEV and CCV and between TGEV and FIPV, whereas a one-way relationship was demonstrated between CCV and FIPV.     

单核细胞增多性李斯特菌对小鼠血脑屏障通透性的影响

为研究小鼠感染单核细胞增多性李斯特菌(LM)90SB2株后血脑屏障通透性的变化,本实验采用ELISA方法检测小鼠血清中髓鞘碱性蛋白(MBP)的水平和甲酰胺-紫外分光光度法检测脑组织中伊文思蓝(EB)的含量。结果显示:MBP于感染后4 h开始升高,10 h后维持较高水平,10 h、24 h、48 h、120 h和144 h各时间点与对照组比较均有显著性差异(p<0.05);EB在感染后24 h之内无明显增加,24 h后有较大幅度升高,并且与对照组比较存在显著性差异(p<0.05);但72 h后开始缓慢下降。表明小鼠感染LM后会导致血脑屏障通透性一定程度的升高。