常用实验动物不同组织部位肥大细胞异质性的组织学特点比较

目的探讨并比较六种常用实验动物不同部位肥大细胞异质性的形态学特点。方法应用麻醉后处死的方法,取小鼠、大鼠、豚鼠、兔、犬和猴的皮肤、肺脏、乙状结肠和脾脏组织,经4%中性缓冲甲醛溶液或Bouin’s液固定后,制作常规组织切片,分别作HE、甲苯胺蓝、阿尔新蓝-藏红和P物质免疫组织化学染色,镜下观察并应用彩色病理图像分析软件进行形态学分析;另取皮肤组织固定于磷酸缓冲戊二醛溶液,制作常规超薄切片,透射电镜下观察肥大细胞超微结构。结果六种动物不同组织中肥大细胞各有其分布特点,且在形态大小、异染性及染色特性等方面表现各异,肥大细胞密度和形态学参数差异有显著性（P〈0.05）;豚鼠、犬和猴皮肤肥大细胞颗粒具有特殊亚微结构;小鼠皮肤组织内可见P物质免疫阳性肥大细胞和神经纤维,犬脾脏内可见P物质免疫阳性肥大细胞。结论在六种实验动物的同一组织中以及同一种动物不同组织中,肥大细胞具有明显的异质性,此异质性对采用实验动物开展与肥大细胞功能相关的动物实验研究具有重要参考价值。     

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.     

The Syndrome of Sea Cucumber (Apostichopus japonicus) Infected by Virus and Bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added,healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.     

The Syndrome of Sea Cucumber （Apostichopusjaponicus） Infected by Virus and Bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.     

The syndrome of sea cucumber (Apostichopus japonicus) infected by virus and bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75–200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%–100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%–80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal. Fundation items: Supported by commonweal program of State Oceanic Administration of China (200705007), Science and Technology Project of Liaoning Province (2004203001) and Science and Technology Project of Dlian (2004 BINS030).     

Immunohistochemical distribution of epimorphin in human and mouse tissues

A novel protein epimorphin has been identified as a mesenchymal signal factor. We reported previously ubiquitous expression of epimorphin in normal skin and a significant increased expression in diseased human skin. The present immunofluorescence study was conducted to determine systematically the distribution of epimorphin in adult human organs with an anti-epimorphin monoclonal antibody. Epimorphin was found to be widely distributed in all human organs examined. It was present in the connective tissue adjacent to or around various epithelial tissues, muscles and vessels. In particular, strong staining was present on the endomysium of muscles, the adventitia of blood vessels, along the sinusoidal lining of hepatocytes and connective tissue around epithelial cells, exocrine and endocrine glands. The results suggest that epimorphin may play a key role in maintaining normal tissue structure and interaction between mesenchymal tissue and epithelial tissue in vivo. ©; 1998 Chapman & Hall     

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.     

Characteristics on staining of leukocytes in experimental animals]

The staining characteristics of the peripheral blood cells from mouse, rat, guinea pig, rabbit, dog, marmoset and monkey were studied. In marmoset, it is easy to distinguish neutrophils from eosinophils by using the phosphate-buffered solution of pH 5 or 6. It was found in the special staining methods that neutrophil granules showed intense peroxidase and Sudan black B reactions in marmoset in comparison with those in the other species of experimental animals. Neutrophil granules rabbit was, however, intensely stained with esterase and acid phosphatase.     

Children with Avoidant or Disorganized Attachment Relate Differently to a Dog and to Humans During a Socially Stressful Situation

ABSTRACT

Human attachment representations are shaped in interaction with the primary caregiver and are generally transferred to further bonding/ social partners later in life. According to previous evidence, primary attachment representations acquired with humans do not seem to be transferred to companion animals. This was held as a major factor why such animals would effectively provide social support also to persons with insecure attachment. The aim of this study in 19 male children, 7–11 years of age and with insecure-avoidant or disorganized attachment, was to investigate differences in their social behavior and in physiological responses when socially supported by an unfamiliar therapy dog in a socially stressful situation. The Trier Social Stress Test for Children (TSST-C) was conducted to elicit stress in the children when in the presence of the dog and a female human investigator. We found that boys with disorganized attachment (n=11) communicated more intensely than avoidantly attached boys (n=8) with both the dog and humans present. Boys with a disorganized attachment had more physical contact with the dog during the TSST-C and talked more to the dog during and after the TSST-C than did boys with an insecure-avoidant attachment. While the prevailing wisdom holds that attachment representations acquired with the primary human caregiver would not transfer to companion animals, our data indicate otherwise. At least components of attachment-related interaction styles, such as degree of contact seeking in verbal and tactile interactions are also displayed in interaction with animal partners.     

Glycosaminoglycans in the touch corpuscle of the dog

Synopsis Alcian Blue staining of the touch corpuscle of dog skin indicates that glycosaminoglycans are present as globular masses in some of the basal cells and are also seen in high concentration in the connective tissue core. The function of these basal cells is not clear but they may be responsible for the production of the large amount of glycosaminoglycan present in the underlying dermis.     

Cellular proliferation potential during aging and caloric restriction in rhesus monkeys (Macaca mulatta)

Caloric restriction (CR) is the most successful method of extending both median and maximal lifespans in rodents and other short-lived species. It is not yet clear whether this method of life extension will be successful in longer-lived species, possibly including humans; however, trials in rhesus monkeys are underway. We have examined the cellular proliferative potential of cells from CR and AL (ad libitum fed) monkey skin cells using two different bioassays: colony size analysis (CSA) of dermal fibroblasts isolated and cloned directly from the skin and beta-galactosidase staining at pH 6.0 (BG-6.0) of epidermal cells in frozen sections of skin. Decreases in both proliferative markers occurred with age, but no differences were observed between CR and AL animals. Skin biopsies were obtained from AL and CR rhesus monkeys from two different aging colonies, one at the National Institute on Aging (NIA) and one at the University of Maryland-Baltimore (UMB). These biopsies were used as a source of tissue sections and cells for two biomarkers of aging assays. The CR monkeys had been maintained for 9–12 years on approximately 70% of the caloric intake of control AL animals. In the CSA studies, the fraction of small clones increased significantly and the fraction of large clones decreased significantly with increasing age in AL monkeys. The frequency of epidermal BG-6.0 staining cells increased with age in older (>22 years) AL monkeys, but most predominately in those of the UMB colony, which were somewhat heavier than the NIH AL controls. Old monkeys on CR tended to have fewer BG-6.0-positive cells relative to old AL-derived epidermis, but this effect was not significant. These results indicate that cellular proliferative potential declined with age in Macaca mulatta, but was not significantly altered by CR under these conditions. Although these experiments are consistent with an absence of effect of CR on monkey skin cell proliferative potential, we have found in previous experiments with mice that a longer duration of CR (as a fraction of total lifespan) was needed to demonstrate CR-related improvement in clone size in mice. Further studies on the now mid-aged monkeys will be needed as their age exceeds 20 years to conclusively rule out an effect of CR on proliferative potential of skin cells from these primates. J. Cell. Physiol. 180:123–130, 1999. © 1999 Wiley-Liss, Inc.     

犬皮肤成纤维细胞的分离、培养及鉴定

目的探索和建立适用于犬皮肤成纤维细胞的体外分离、培养及鉴定的技术方法。方法采用组织贴块培养法和胰蛋白酶、胶原酶Ⅰ联合消化法对犬皮肤成纤维细胞进行体外培养、传代。并对所培养的细胞进行倒置显微镜观察和苏木素-伊红染色,观察成纤维细胞形态,并对培养细胞行波形蛋白免疫荧光染色。结果倒置相差显微镜下可见长梭形细胞生长,苏木素-伊红染色可见细胞呈漩涡状、平行排列,第5代细胞免疫荧光检测波形蛋白(vimentin)表达阳性。结论建立了高效快速分离和稳定培养成纤维细胞的方法,为诱导犬心房纤维化提供了充足的种子细胞。     

Occurrence of the mucinous differentiation antigen CA125 in genital tract and conductive airway epithelia of diverse mammalian species (rabbit, dog, monkey)

CA125 is a human tumor-associated antigen of coelomic epithelial origin. In the present study, immunohistochemical analysis of normal rabbit, dog, and monkey tissues using monoclonal antibody OC125, revealed that in these animals positive staining for CA125 is found in all tissues that produce this mucin-like glycoprotein in man, i.e., the peritoneal and pleural mesothelium, the different Müllerian-duct-derived epithelia of the female genital tract, and the epithelium of trachea, bronchi, bronchioli, and mucoserous respiratory glands; CA125 was also detected in some ductal and acinar cells of the dog mammary gland. Without trypsin treatment of sections, staining was predominantly localized on the apical cell surface of all mentioned cell types. After treatment, mucin droplets inside respiratory mucous cells were also positively stained. In all cases, staining was associated with material positive for periodic acid-Schiff (PAS) and Alcian blue. In rats, its presence could not be demonstrated. Our results show that the CA125 epitope is not restricted to man and that its expression throughout different animal species is associated with well-defined tissue compartments. The expression of the mucous differentiation antigen CA125 in several common laboratory animals provides new opportunities for the experimental study of its biological significance.     

Toll-like receptor 4 in normal and inflamed lungs and other organs of pig, dog and cattle

Bacterial diseases, especially those of the lung caused by Gram-negative bacteria, inflict significant economic loss associated with mortality and morbidity in domestic animals. Toll-like receptor 4 (TLR4) has recently been recognized as a major receptor for cellular interactions with lipopolysaccharides derived from Gram-negative bacteria. However, there are no data on the expression of TLR4 in various organs of domestic animals. We performed immunohistochemistry and immuno-gold electron microscopy to localize TLR4 in lung and seven other organs from normal pig, dog and calf (n=2 each) and in inflamed lungs from calves (n=4) challenged with Mannheimia hemolytica. The data show TLR4 in macrophages in lung, small intestine, liver and spleen in all the species and pulmonary intravascular macrophages in calves and pigs. Epithelium in lung, small intestine, cornea and convoluted and straight renal tubules was stained for TLR4. Vascular endothelium of large blood vessels only in lungs and skin was positive, and skeletal muscles were negative for TLR4. In inflamed lungs, airway epithelium showed reduced staining for TLR4 while staining in macrophages remained unaltered. These are the first immunocytochemical data on TLR4 expression in domestic animal species and show similarity in TLR4 staining in macrophages, epithelium and vascular endothelium among dog, pig and cattle.     

Indirect immunohistochemistry on skin biopsy for the detection of persistently infected cattle with bovine viral diarrhoea virus in Italian dairy herds

Indirect immunohistochemistry (IHC) on skin biopsies for identification of persistently infected (PI) animals has been used as a parallel test to antigen and antibody ELISAs in a bovine viral diarrhoea (BVD) voluntary control program. The aim was to evaluate the reliability and feasibility of IHC on ear skin tissues to detect PI animals in field conditions, including both adult and calves under 6 months of age. In animals over 6 months of age skin biopsy and blood sample were collected at the same time, whereas in young calves blood sampling was performed when animals reached 6 months of age. One hundred and sixty-five animals were tested and immunohistochemical results were compared with those of antigen ELISA. In case of inconclusive results virus isolation and virus neutralization assays were performed. Agreement K value was 0.96. Immunohistochemical staining in positive animals was clearly detectable in the keratinocytes of the epidermis and adnexa.     

Hydrophobia, lectin binding, and molecular order in the stratum corneum of adult and neonatal rats and in human breast cells in culture.

A search for differences due to ANS staining (hydrophobia), Con A and PNA binding capacity, and birefringence was carried out on stratified epithelia of rat skin and human breast cells (HBC) in culture. Microfluorimetric measurements confirm that the ANS fluorescence of the stratum corneum from adults is higher than that of newborns. HBC exhibited an unexpected deep ANS-fluorescence. Differences in the binding capacity of the epithelial layers to Con A and PNA were detected with advancing age. Retardation measurements revealed that the form birefringence of the stratum corneum is higher in adult animals specially as revealed by the fact that its form birefringence curve branch from n = 1.414 to n = 1.479 is steeper, i.e. depict higher values. The strong birefringence of the cytoplasmic tonofilaments presented by cultured human breast cells was considered an unexpected finding and attributed to changes that the cells underwent following the in vitro conditions.     

Psychogenic alopecia in rhesus macaques presenting as focally extensive alopecia of the distal limb

Focally extensive alopecia affecting the distal limbs is a common clinical finding in rhesus macaque (Macaca mulatta) colonies and is both a regulatory and colony-health concern. We performed diagnostic examinations including physical exams, bloodwork, skin scrapes, surface cytology, and surface bacterial-fungal cultures on 17 rhesus macaques with this presentation of alopecia. Skin biopsies from alopecic skin obtained from each macaque were compared with those of normal skin from the same animal. Immunohistochemistry and metachromatic staining for inflammatory cells were performed to compare alopecic and normal skin. In addition, we compared these biopsies with those previously obtained from macaques with generalized alopecia and dermal inflammatory infiltrates consistent with cutaneous hypersensitivity disorders and with those from animals with normal haircoats. Bacterial and fungal cultures, skin scrapes, surface cytology, and bloodwork were unremarkable. Affected skin showed only mild histologic alteration, with rare evidence of trichomalacia and follicular loss. Numbers of mast cells and CD3+ lymphocytes did not differ between alopecic and normally haired skin from the same animal. The number of mast cells in alopecic skin from animals in the current cohort was significantly lower than that in skin of animals previously diagnosed with a cutaneous hypersensitivity disorder. Numbers of both mast cells and CD3+ lymphocytes in alopecic skin from the current cohort were similar to those from biopsies of animals with normal haircoats. Together, the clinical findings and pathology are consistent with a psychogenic origin for this pattern of alopecia in rhesus macaques.     

Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules

Summary Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.     

Immunolocalization of zona pellucida antigens in the ovarian follicle of dogs, cats, horses and elephants

A comparative evaluation of the location of immunoreactive porcine zona pellucida (pZP) glycoproteins was performed with polyclonal rabbit anti-pZP antibodies on ovarian sections of the dog, cat, horse, and elephant. For this, formalin (light microscopy) and glutaraldehyde (transmission electron microscopy [TEM]) fixed ovarian sections were incubated with antibodies raised against highly purified pZP. Staining patterns were determined with diaminobenzidine (DAB) at the light level. The dog ZP had a distinct staining distribution that is characterized by intense staining around the periphery of the ZP and the oolemma and less dense staining throughout the width of the ZP. In dog follicles that contained multiple oocytes, there were oocytes of identical and dissimilar stages. Cat ovarian sections showed uniform staining of the ZP. Horse results showed uniform staining of ZP and ooplasm, and granulosa cells (GC). Elephant sections showed staining of the ZP with dense staining at the oolemma, as well as staining of the ooplasm. In all species the staining of the ZP was not evident until GC differentiation. In all cases there was no staining of ovarian tissue with control normal rabbit serum. Specific staining patterns of ZP were evaluated by TEM and immunogold staining. The immunogold-linked anti-pZP antibodies stained the ZP matrix in all species. There was staining of ooplasm organelles suggesting that ZP secretion originates from the oocyte of the dog and cat. In addition, follicular and ZP measurements were taken that allowed accurate characterization of follicle stage. These findings suggest that in all four species the ZP is recognized by anti-pZP antibodies and there is also evidence to suggest the possible origins of ZP glycoproteins.