Immunologic surface markers on nonhuman primate lymphocytes.

Peripheral blood lymphocytes from 37 healthy rhesus macaques (Macaca mulatta) and thymocytes from 10 fetal and neonatal rhesus macaques were studied for membrane characteristics. Spontaneous rosette formation with sheep erythrocytes, a characteristic of human T lymphocytes, was evaluated. The presence of membrane-bound immunoglobulin and surface receptors for fixed complement was measured, using fluorescent antibody techniques and erythrocyte-antibody-complement rosettes, respectively. The mean percentages +/- 1 standard error of the lymphocyte markers in the peripheral blood lymphocytes from the macaques were: spontaneous rosettes, 63 +/- 1.0; erythrocyte-antibody-complement rosettes, 14.9 +/- 1.2; and membrane immunoglobulin-positive cells, 21.9 +/- 2.2. These values are very similar to values reported for human beings.     

Cell surface markers of the canine natural killer (NK) cell

Studies were performed to determine which of several cell surface markers are expressed on canine peripheral blood leukocyte (PBL) natural killer (NK) cells. Chromium-51 release assays showed a decrease in NK activity after depletion of PBL by carbonyl iron ingestion and adherence to IgG-antibody-coated ovine erythrocytes (EA gamma) and to IgM-antibody-complement-coated ovine erythrocytes (EA mu C). Effector cell adherence to and subsequent lysis of canine thyroid adenocarcinoma (CTAC) target cell monolayers provided direct visual identification of the putative canine NK cell. These surface immunoglobulin-negative cells, individually identified by their physical adherence to dead CTAC target cells, failed to form nonimmune rosettes with guinea pig erythrocytes or rosettes with EA mu or EA mu C. However, 39.0 +/- 4.2% of these adherent cells formed rosettes with EA gamma and 73.3 +/- 0.8% expressed the canine T-lymphocyte marker, Thy-1.     

Monoclonal antibodies: cell surface markers for canine keratinocytes

Plasma membranes were isolated from cultured canine keratinocytes by paraformaldehyde-induced membrane vesiculation. The isolated plasma membrane vesicles retained cell surface antigens (eg, a pemphigus vulgaris antigen). These membrane vesicles were used as an antigen source for the production of monoclonal antibodies. Eight antibodies that had specific reactivity to the cytoplasmic membrane of keratinocytes on frozen sections of canine esophagus were identified by use of an indirect immunoperoxidase method. The stratified squamous epithelium of the esophageal mucosa had 4 staining patterns. When applied to frozen sections of canine skin, lip, and tongue, the antibodies had different tissue specificities for differing stratified squamous epithelia. Using sodium dodecyl sulfate polyacrylamide-gel electrophoresis and the western blot technique, one of the antibodies was specific for a 60-kD cell surface molecule. Therefore, such monoclonal antibodies may be useful in defining heterogeneity between different stratified squamous epithelia, in identifying biologically important surface antigens, or in the diagnosis of tumors.     

Peanut agglutinin as a surface marker for canine T lymphocytes

Peanut agglutinin (PNA) and surface immunoglobulin (SIg) were investigated as markers for T and B lymphocytes in blood and lymphoid tissues of dogs of various ages. In the blood study, 4 age groups (n = 8 dogs/group) were used. The mean (+/- SD) percentages of PNA-positive (PNA+) cells were 68.4 +/- 8.6% (group 1, less than 1 year old), 70.3 +/- 9.2% (group 2, 1 to 2 years old), 72.0 +/- 3.7% (group 3, 5 to 6 years old), and 63.8 +/- 10.1% (group 4, 10 to 11 years old). The mean percentages of SIg-positive (SIg+) cells in blood were 32.1 +/- 10.6% (group 1), 43.2 +/- 7.0% (group 2), 34.3 +/- 4.8% (group 3), and 35.0 +/- 6.8% (group 4). The mean total percentages of PNA+ and SIg+ cells were 100 +/- 6.0% (group 1), 113.5 +/- 4.9% (group 2), 106.3 +/- 5.3% (group 3), and 98.9 +/- 9.2% (group 4). The proportions of PNA+ and SIg+ cells in dogs of group 2 were significantly (P less than 0.05) different from those in dogs of the other groups. Serial changes in PNA+ and SIg+ cells were investigated in blood of 6- to 29-week-old pups (n = 8). A significant (P less than 0.05) transient decrease in PNA+ cells and a corresponding increase in SIg+ cells was observed in pups between 14 and 17 weeks old. Lymphoid tissue specimens and blood samples were obtained from 2- to 6-month-old dogs (n = 11) and from 6- to 12-month-old dogs (n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)     

犬微卫星DNA遗传标记及其在犬繁育中的应用

遗传标记是指可追踪染色体、染色体某一节段、某个基因座在家系中传递的任何一种遗传特性。它具有两个基本特征,一是可遗传性,二是可识别性。因此,生物的任何有差异的突变型均可作为遗传标记。遗传标记主要包括形态标记、细胞学标记、生化标记和DNA分子标记四类,本文对犬微卫星     

Immunophysiological studies of interleukin-2 and canine lymphocytes.

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.     

Isolation of viable canine intestinal lymphocytes

A modification of a previously reported enzymatic technique was used to isolate 60% to 90% pure populations of viable lymphocytes from canine small intestinal mucosa. Mitogenesis assays were then simultaneously performed on these and isolated peripheral blood lymphocytes from the same dogs. A technique to isolate intraepithelial lymphocytes was not successful. Intestinal mucosal lymphocytes were simultaneously purified by 2 different techniques (suspensions B and C), which produced cell populations that responded similarly to phytohemagglutinin (PHA), concanavalin A, and pokeweed mitogens. The peripheral blood lymphocytes demonstrated greater response to PHA and concanavalin A mitogens than did the intestinal mucosal lymphocytes (P less than 0.05). Furthermore, peripheral blood lymphocytes had a significantly higher response to PHA than to pokeweed mitogen (P less than 0.05). The impure preparation of intraepithelial lymphocytes (suspension A) did not show any evidence of reactivity to mitogens. Further studies are needed to determine whether the EDTA or collagenase had any effect upon the intestinal mucosal lymphocyte responsiveness. The present study demonstrated that viable mucosal lymphocytes can be isolated from the canine intestine and that they can maintain their responsiveness to mitogens.     

Immunogenetic markers in canine paternity cases

Three cases of disputed paternity in dogs are reported. An identification or exclusion of a sire was possible in all families with the help of genetic markers. Attention is drawn to the lack of communication between dog-breeders, veterinarians and medico-biological research workers all of whom would benefit from an intensified co-operation in the field of dog genetics.     

Characterization of lymphocytes in canine gastrointestinal lymphoma

Primary canine gastrointestinal lymphoma has been believed to be of B-cell origin based on the morphology and behavior of the neoplastic cells and the evidence from the human medical field. However, the neoplasms have not to date been characterized as to the origin of the cell population. Forty-four cases diagnosed as canine gastrointestinal lymphoma were retrieved from the records of the Veterinary Teaching Hospitals at the University of Minnesota and the University of Wisconsin-Madison. Four of the cases have been previously identified as epitheliotropic T-cell gastrointestinal lymphoma. Twenty-three of the dogs were female, with 11 intact and 12 neutered, and 21 of the dogs were male, with 12 intact and nine neutered. Sixteen breeds as well as individuals of mixed breeding were represented. The Boxer and the sharpei were the most commonly represented breeds with six individuals each. The age range of the dogs was 1.5-14.66 years, with two dogs identified as adult and two of unknown age. Archived tissue blocks of gastrointestinal samples were sectioned in duplicate and prepared for immunohistochemical staining with CD3 (T-cell marker) and CD20 (B-cell marker). In 75% of the cases examined under light microscopy, 50-95% of the neoplastic cells stained positively with CD3 and exhibited marked epitheliotropic behavior. In three of the cases, from 10% up to 50% of the neoplastic cells stained positively with CD20, with widely scattered CD3(+) cells. In the remainder of the cases, few to none of the neoplastic cells stained with either of the markers. This retrospective study shows that canine primary gastrointestinal lymphoma is more commonly of T-cell origin, rather than B-cell origin.     

The canine lymphocyte: effect of streptolysin O on the proliferative response of canine lymphocytes

The effects of Streptolysin O (SLO) on canine peripheral blood lymphocytes were studied using the lymphocyte blastogenesis test (LBT). Canine lymphocytes stimulated with SLO produced a response that was similar to the response obtained with the commonly used phytomitogens: phytohemagglutinin (PHA), concanavalin A (CON A) and pokeweed mitogen (PWM). When SLO was added to any of the phytomitogens and incubated with canine lymphocytes, an additive or stimulatory response was obtained. However, mixing the phytomitogens in any combination did not produce a similar additive or stimulatory response. The results were interpreted to mean that in the canine system, the binding of SLO to lymphocytes does not sterically interfere with receptors for PHA, CON A, or PWM. Additionally, SLO may stimulate a population of lymphocytes that is distinct from that stimulated by the phytomitogens. Moreover, it would appear that the phytomitogens probably stimulate the same or an overlapping population of lymphocytes. Experiments using enriched lymphoid cell populations were used to characterize the lymphocytes stimulated by phytomitogens and SLO. Lipopolysaccharides from two different bacteria were unable to cause a significant lymphoproliferative response.     

Investigation of cross-reactivity between commercially available antibodies directed against human, mouse, and rat lymphocyte surface antigens and surface markers on canine cells

Using automated flow cytometry, 23 commercially available antibodies (all but one of them monoclonal) raised against surface antigens of specific populations of human, rat, and mouse lymphocytes were tested for cross-reactivity to peripheral blood lymphocytes from five clinically healthy adult dogs. Of all the antibodies tested, only the polyclonal anti-asialo GM1 directed against mouse NK cells, and the monoclonal antibodies anti-HLA-DR directed against the human class II antigen and anti-B1, a human pan B cell marker, consistently labeled subpopulations of canine lymphocytes.     

Oxidative stress markers in canine atopic dermatitis

There are no data in the veterinary literature relating to oxidative stress in canine atopic dermatitis (CAD). The study aimed to determine levels of oxidative stress markers, plasma malondialdehyde (MDA), total antioxidant capacity (TAC), whole blood glutathione peroxidase (GPX) and erythrocyte superoxide dismutase (SOD), in 15 CAD patients and 17 healthy dogs. A correlation between CADESI (Canine Atopic Dermatitis Extent and Severity Index) score and MDA was also determined. Significantly higher plasma MDA levels were found in patients than in healthy dogs. The significant, highly positive correlation determined between CADESI score and MDA in the patient group indicates an association between the severity of CAD and the extent of oxidative damage to membrane lipids. There were no significant differences in TAC, GPX and SOD between patients and healthy dogs. Our findings suggest that oxidative stress with increased lipid peroxidation could be involved in the pathogenesis of atopic dermatitis in dogs.     

Expression of neural markers on bone marrow-derived canine mesenchymal stem cells

OBJECTIVE: To evaluate cell surface markers of bone marrow-derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells. SAMPLE POPULATION: Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs. PROCEDURES: Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (beta III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation. RESULTS: Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed beta III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in beta III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Canine bone marrow-derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.     

FC-13 Molecular markers in canine T-cell lymphoma

A previous study described cutaneous lymphocytosis (CL) in 23 cats. The process resembles cutaneous pseudolymphoma in humans, a heterogeneous group of benign reactive proliferations of well-differentiated lymphocytes in the skin of humans. Morphological and immunophenotypic characteristics do not offer reliable criteria to accurately predict the clinical outcome of feline CL or pseudolymphoma in humans. Presence of clonal cell populations is more consistent with a neoplastic process. In a previous study, feline CL lesions (20 cats) were evaluated for clonality using PCR, and only two cats had monoclonal T-cell populations. Because false-negative results may occur, the purpose of this study was to repeat the PCR using a revised primer set based on analysis of additional feline T-cell receptor γ (TCRγ) sequences. DNA was isolated from 29 skin lesions and six internal organs of 20 cats. DNA integrity was assessed by glyceraldehyde-3-phosphate dehydrogenase PCR. Polymerase chain reaction clonality was performed using the revised primer set specific for feline TCRγ, and duplicate samples were evaluated. The PCR products were assessed by heteroduplex analysis. Clonal rearrangement of TCRγ was detected in 14 cats (24 of 35 tissues: 21 of 29 skin lesions and three of six internal organs); eight of these cats are still alive and six were euthanized. Monoclonal populations were seen in three of five cats that had involvement of internal organs. These findings indicate that feline CL is best considered as a slowly progressive process which may be reactive, but often evolves into a low-grade indolent lymphoma.
Funding: George H. Muller Fund for Research in Dermatology.     

Chemiluminescence of canine peripheral blood lymphocytes stimulated by mitogens

Luminol-dependent chemiluminescence of peripheral blood lymphocytes from dogs stimulated with concanavalin A (Con A) or phytohemagglutinin P (PHA) was measured with a Pico-Lite luminometer. 10 microliter of luminol gave optimal quantum yield from 1 X 10(6) lymphocytes sensitized with either 80 micrograms Con A or 160 micrograms PHA. Addition of superoxide dismutase did not influence the course of chemiluminescence. Whereas catalase produced 41% increase in quantum yield, mannitol caused a 51% inhibition of chemiluminescence. Lymphocytes exposed to varying doses of short term x-irradiation or lymphocytes isolated from dogs kept under continuous exposure through a gamma irradiation source showed dose-related depression of chemiluminescence. Membrane factors may be involved in lymphocyte stimulation to chemiluminescence as pulse experiments with Con A and PHA revealed. It is proposed that chemiluminescence measurements may be useful in monitoring early events in lymphocyte stimulation by antigens and mitogens.