Observations of apparent C-type particles in baboon (Papio cynocephalus) placentas.

Ultrastructural investigations have revealed the presence of apparent endogenously derived C-type particles in the placental villi of each of 13 baboons studied. Both budding and mature forms were observed in the syncytiotrophoblast of these animals at various stages of pregnancy.

Optimized transduction of canine paediatric CD34(+) cells using an MSCV-based bicistronic vector.

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Canine X-linked severe combined immunodeficiency. A model for investigating the requirement for the common gamma chain (gamma c) in human lymphocyte development and function.

Our laboratory has identified and characterized an X-linked severe combined immunodeficiency (XSCID) in dogs that is due to mutations in the common gamma (gamma c) subunit of the interleukin-2 (IL2), IL4, IL7, IL9, and IL15 receptors. Canine XSCID, unlike genetically engineered gamma c-deficient mice, has a clinical and immunologic phenotype virtually identical to human XSCID. It appears that species-specific differences exist in the role of the gamma c and its associated cytokines in mice compared to their role in humans and dogs, suggesting gamma c-deficient dogs may be a more relevant model for studying the role of the gamma c in humans. We are utilizing this model for a variety of studies to address: 1. Fundamental questions concerning the role of the gamma c in cytokine regulation and lymphocyte development. 2. The pathogenesis of XSCID. 3. Strategies for improving bone marrow transplantation outcome. 4. Development and evaluation of strategies for gene therapy. 5. Human hematopoietic stem cell development.

Development and functional characterization of T cell lines from canine Peyer's patches.

We have propagated concanavalin A-stimulated cells from canine Peyer's patches in vitro in the presence of interleukin-2 (IL-2). The cells were characterized as T cells by determination of their phenotype and by functional assays. They are IL-2 dependent and respond to IL-2 of murine, primate and canine origin. The long-term cultured cells provided help for immunoglobulin production by purified autologous B cells and suppressed IgG production by nonseparated autologous peripheral blood mononuclear cells.

Isolation and phenotypic and functional characterization of cells from Peyer's patches in the dog.

Cells were isolated from canine Peyer's patches (PPs) and their phenotype and capacity to secrete immunoglobulins in vitro were determined. Cells isolated from duodenal and jejunal PPs of adult dogs consisted of 91.4% lymphocytes and 1.6% macrophages with 55.4% mIg(+)-cells and 35.6% Thy-1(+)-cells. In vitro IgA secretion by pokeweed mitogen (PWM)-stimulated PP cells exceeded that by cells from other lymphoid tissues and was specifically increased by concanavalin A, suggesting a role for isotype-specific T-cells. Comparison of duodenal and jejunal (proximal) PPs and the ileal PP revealed that the ileal PP contained fewer T-cells, fewer mIgA(+)-cells and more mIgM(+)-cells. Cells from the ileal PP produced very little IgA and IgG, but abundant IgM in vitro. These data suggest that the proximal PPs of dogs are important in the generation of IgA B-cells, similar to PPs of rodents. The ileal PP of dogs may have a function in the early development of the B-cell system of the dog.

Immunohistology of Peyer's patches in the dog.

Canine Peyer's patches were examined by light microscopy and immunohistochemistry for possible variations depending on the location within the small intestine and for similarities and dissimilarities to PPs from other species. The duodenal and jejunal PPs were characterized by relatively large domes and interfollicular areas. In contrast, the ileal PP had small domes and poorly developed interfollicular areas and very large follicles. T cells were found in the interfollicular area and corona and in lesser numbers in the dome and germinal centers. The ileal PP contained far fewer T cells than the proximal PPs. Domes of canine PPs contained some cytoplasmic IgA+ (cIgA+) and many cIgG+ cells. Peanut agglutinin (PNA) stained germinal center cells in a selective but not-uniform way and did not stain T cells.

Ultrastructure and alkaline phosphatase activity of the dome epithelium of canine Peyer's patches.

The dome epithelium of Peyer's patches from different parts of the intestine of four dogs was examined by scanning and transmission electron-microscopy and by alkaline phosphatase histochemistry on glycol-methacrylate embedded sections. M cells were scattered among more numerous enterocytes in duodenal and jejunal Peyer's patches, but constituted the major cellular component of dome epithelia of the ileal Peyer's patches. Alkaline phosphatase histochemistry demonstrated low enzyme activity in the brush border of M cells, as compared to enterocytes, in the duodenum and jejunum, allowing identification of M cells at the light microscopic level. Alkaline phosphatase activity was too low in ileal enterocytes to permit visualization of M cells. The presence of intrafollicular invaginations of dome epithelium is a consistent finding in duodenal Peyer's patches of the dog and these invaginations were characterized by few M cells, many intraepithelial lymphocytes and strong alkaline phosphatase activity.

An improved method for the isolation of enriched canine peripheral blood mononuclear cell and peripheral blood lymphocyte preparations.

A two-step purification method was developed for obtaining (1) peripheral blood mononuclear cell preparations of greater than 97% purity and (2) peripheral blood lymphocyte preparations of greater than 95% purity from canine whole blood with yields similar to or greater than those obtained by conventional techniques.

Short-term effect of cyclophosphamide and azathioprine on selected aspects of the canine blastogenic response.

The short-term, in vitro responses of canine peripheral blood lymphocytes to mitogenic stimulation and serum immunoglobulin concentrations were evaluated following treatment with currently recommended doses of cyclophosphamide and azathioprine. Cyclophosphamide had no significant effect on either the serum immunoglobulin concentrations or the blastogenic response of lymphocytes to mitogenic stimulation. Serum immunoglobulin concentrations remained unchanged following azathioprine treatment. The blastogenic response was significantly suppressed following one week of azathioprine therapy and returned to baseline values one week following cessation of treatment. The response to phytohemagglutinin was most suppressed, followed, in order, by the response to concanavalin A, and to pokeweed mitogen. These results suggest that the short-term use of azathioprine, but not cyclophosphamide, in clinically used dosages, does suppress selective aspects of the canine immune system, and the T cells appear to be more susceptible than B cells to the immunosuppressive effect of this drug.

Detection of canine interleukin-2 receptors by flow cytometry.

This study describes a method for detecting canine interleukin-2 receptors (IL-2R) by flow cytometry, using human recombinant IL-2 labeled with phycoerythrin (IL-2-PE). Peripheral blood mononuclear cells from four normal dogs were washed, incubated with IL-2-PE, and then washed to remove any unbound IL-2-PE. Flow cytometric analysis of the cells was performed with a 488 nm argon laser while gating on lymphocytes. Cells expressing the IL-2R were identified by their fluorescence as compared to cells stained with an anti-mouse immunoglobulin-G conjugated to phycoerythrin. The average percentage of resting cells expressing the IL-2R was found to be 21%. The addition of unlabeled human recombinant IL-2 to Day 3 phytohemagglutinin (PHA)-stimulated cells reduced the fluorescence intensity four-fold, thereby demonstrating the specificity of IL-2-PE for canine IL-2R. Following stimulation with optimal concentrations of PHA, the percentage of cells expressing the IL-2R increased daily and reached a maximum on Day 3 (76.4%). IL-2R density, as measured by mean fluorescence intensity, also increased and reached maximal levels on Days 2-3 (twenty-fold greater than resting cells). The binding, inhibition, and kinetic experiments provide evidence that human recombinant IL-2-PE is a useful tool for studying canine IL-2R expression. Thus, a one-step direct method for the flow cytometric detection and quantification of the canine IL-2R is now available.

A rapid microtechnique for in vitro stimulation of canine lymphocytes using whole blood.

A simple, rapid microtechnique utilizing whole blood has been developed for evaluating the in vitro stimulation of canine lymphocytes. The test uses 5 ul. of heparinized whole blood per culture in a microtiter plate and requires no serum supplement. Cultures are harvested by an automated multiple sample cell harvester. This technique permits large numbers of replicate samples to be tested on individual animals with minimal amounts of blood and minimal technician time.

Modulation of canine lymphocyte blastogenesis via histamine.

The effect of histamine on in vitro T cell blastogenic responses of canine peripheral blood lymphocytes to phytohemagglutinin-P (PHA-P) was investigated. A dose dependent inhibition of blastogenesis was observed; an effect which could be blocked by cimetidine, a type II histamine receptor antagonist, but not by diphenhydramine, a type I receptor antagonist, suggesting that histamine's inhibitory effect is mediated through a type II histamine receptor. The inhibitory effect of histamine on blastogenesis was also reversible by indomethacin, a prostaglandin synthetase inhibitor, implicating prostaglandin involvement in histamine suppression. Histamine release at sites of inflammation may result in down regulation of local immune responses by activation of specific immunoregulatory cells. This could permit the escape of certain neoplasia from local immunosurveillance mechanisms. Cimetidine may block activation of histamine responsive regulatory cells bearing type II receptors, which may help explain the beneficial effect cimetidine therapy has on regression of certain human tumors (i.e., malignant melanomas).

The effect of general anesthesia on canine lymphocyte function.

The in vitro response of canine peripheral blood lymphocytes to mitogenic stimulation was evaluated following general anesthesia for a relatively minor diagnostic procedure. A marked suppression in the blastogenic response to phytohemagglutinin and concanavalin A was observed 4 hours postinduction which persisted through the first 24 hours and was normal by 4 days. A mild suppression to stimulation with pokeweed mitogen was observed at 4 hours postinduction but the response was back to normal by 24 hours. These results suggest general anesthesia has a transient immunosuppressive effect on canine lymphocytes and T cells are more susceptible to the immunosuppressive effect than B cells.

Polyclonal activation of canine B lymphocytes evaluated by a protein A reverse hemolytic plaque assay.

This paper describes the optimal culture and assay conditions for the polyclonal activation of canine lymphocytes with pokeweed mitogen and the quantitation of immunoglobulin secreting plaque-forming cells (PFC) using a staphylococcal protein A-reverse hemolytic plaque assay. The assay permits the quantitation of total immunoglobulin secreting PFC as well as class-specific immunoglobulin secreting PFC. On the optimal day of culture, a mean of 176 IgA PFC/10(6), 575 IgM PFC/10(6), 1276 IgG PFC/10(6), and 2158 total PFC/10(6) cells were generated following polyclonal activation. This study provides a simple and reproducible assay for the delineation of the immunoregulatory mechanisms involved in the differentiation of canine B lymphocytes.

Development of monoclonal antibodies against canine glomerular antigens.

Monoclonal antibody producing hybridomas were developed by fusing spleen cells from BALB/c mice immunized against canine glomeruli with SP2 myeloma cells. Monoclonal antibody reactivity was tested using an indirect immunofluorescence assay on various normal canine tissues and canine kidney affected with glomerulonephritis. Two of the hybridomas developed (3H2 and 3A5) reacted with glomeruli and not with renal tubules. Antibody produced by hybridoma 3A5 also reacted with smooth muscle of all other tissues tested and 3H2 with lung tissue. Antigens recognized by monoclonal antibodies were studied by assessing their heat stability and susceptibility to proteolysis and neuraminidase digestion. Antigen and antibody molecular weights were determined by using a western blotting technique. Glomerular proteins that reacted with antibody produced by hybridoma 3H2 had molecular weights ranging from approximately 92,500 daltons to 200,000 daltons. Antigens reacting with both monoclonal antibodies were likely protein antigens. It was concluded that monoclonal antibodies would be useful in the study of glomerular antigens in normal dogs and dogs with glomerulonephritis.

B-cell function in canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gammac) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors and has a similar clinical phenotype to human XSCID. We have previously shown that the block in T-cell development is more profound in XSCID dogs than in genetically engineered gamma c-deficient mice. In this study we evaluated the B-cell function in XSCID dogs. In contrast to the marked decrease in peripheral B-cells in gamma c-deficient mice, XSCID dogs have increased proportions and numbers of peripheral B-cells as observed in XSCID boys. Canine XSCID B-cells do not proliferate following stimulation with the T-cell-dependent B-cell mitogen, pokeweed mitogen (PWM); however, they proliferate normally in response to the T-cell-independent B-cell mitogen, formalin-fixed, heat-killed Staphylococcus aureus. Canine XSCID B-cells are capable of producing IgM but are incapable of normal class-switching to IgG antibody production as demonstrated by in vitro stimulation with PWM and immunization with the T-cell-dependent antigen, bacteriophage PhiX174. Similar results have been reported for XSCID boys. Thus, it appears that gamma c-dependent cytokines have differing roles in human and canine B-cell development than in the mouse making the XSCID dog a valuable model for studying the role of these cytokines in B-cell development and function.

A single nucleotide insertion in the canine interleukin-2 receptor gamma chain results in X-linked severe combined immunodeficiency disease.

The immunologic and genetic analysis of a 14-week-old-male cardigan Welsh corgi puppy that presented with failure to thrive, diarrhea, and intermittent vomiting are described. The lack of palpable lymph nodes, the premature death of a male sibling, and similar clinical signs in a male cousin suggested that a primary immunodeficiency disease might be responsible for his poor clinical condition. Quantitation of serum immunoglobulins revealed low concentrations of IgG and undetectable IgA, yet normal concentrations of IgM. A complete blood cell count showed a slight anemia and lymphopenia. Although the peripheral blood contained a normal percentage of T cells, with an increased CD4:CD8 ratio, they were unable to proliferate in response to phytohemagglutinin (PHA) and/or interleukin 2 (IL-2). Furthermore, following PHA activation, the peripheral blood lymphocytes (PBL) demonstrated a nearly complete lack of IL-2 binding. All of these laboratory findings were identical with our previous findings from dogs with X-linked severe combined immunodeficiency (XSCID) that is due to a mutation in their IL-2 receptor gamma (IL-2R gamma) chain. Examination of the corgi's IL-2R gamma cDNA revealed an insertion of a cytosine following nucleotide 582, resulting in a premature stop codon prior to the transmembrane domain. The insertion also created an EcoO109 restriction enzyme site that enabled us to detect the mutation in the patient's genomic DNA. This new mutation in the IL-2R gamma chain discovered in a cardigan Welsh corgi puppy results in XSCID with similar immunologic abnormalities as observed in dogs with the same disease resulting from a different IL-2R gamma chain mutation.

Canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gamma c) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. The most striking clinical feature is a failure to thrive or 'stunted' growth. Recurrent or chronic infections begin at the time of decline of maternal antibody, usually between six and eight weeks of age. Affected dogs rarely survive past three to four months of age. The major pathologic feature of canine XSCID is a small, dysplastic thymus. Grossly identifiable lymph nodes, tonsils, and Peyer's patches are absent in XSCID dogs. During the neonatal period, XSCID dogs have few, if any, peripheral T cells and increased number of peripheral B cells. Some XSCID dogs do develop phenotypically mature, nonfunctional T cells with age, however, the absolute number of peripheral T cells remain significantly decreased compared to age-matched normal dogs. An interesting finding is that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA+ (naive) phenotype to a CD45RA- (activated or memory phenotype). One of the characteristic findings in XSCID dogs is an absent or markedly depressed blastogenic response of T cells in response to stimulation through the T cell receptor and when the necessary second messengers for cellular proliferation are directly provided that by-pass signals delivered through ligand-receptor interaction. The proliferative defect is due to the inability of T cells to express a functional IL-2 receptor. Canine XSCID B cells do not proliferate following stimulation with T cell-dependent B cell mitogens, however, they proliferate normally in response to T cell-independent B cell mitogens. Canine XSCID B cells are capable of producing IgM but are incapable of class-switching to IgG antibody production following immunization with the T cell-dependent neoantigen, bacteriophage phiX174. The number of thymocytes in the XSCID thymus is approximately 0.3% of the thymocytes present in the thymus of age-matched normal dogs. The proportion of CD4-CD8- thymocytes in XSCID dogs is increased 3.5-fold and the CD4+CD8+ population is decreased 2.3-fold. These findings demonstrate that (1) a functional gamma c is required for normal B and T cell function, (2) early T cell development is highly dependent upon a functional gamma c, and (3) B cell development can occur through a gamma c-independent pathway.

Bone marrow transplantation for canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain which is a subunit of the receptors of IL-2, IL-4, IL-7, IL-9 and IL-15. Bone marrow transplantation (BMT) of human XSCID patients without pretransplant conditioning (cytoablation) results in engraftment of donor T-cells and reconstitution of T-cell function but engraftment of few, if any, donor B cells with resultant poor reconstitution of humoral immune function. In this study, we show that XSCID dogs can be transplanted with allogeneic bone marrow cells resulting in engraftment of both donor B and T cells and reconstitution of full systemic immune function including normal humoral immune function without the need for cytoablation.

Overview of immune system development in the dog: comparison with humans.

Dogs play an important role in toxicology because of their importance as a large animal, pre-clinical model for evaluating potential toxicity in human drug development including the effects of investigational drugs on the immune system. The purpose of this paper is to review the development of the canine immune system during the fetal, neonatal and postnatal periods and to compare it with that of the human immune system. Unlike rodents, the development of the canine immune system shares many similarities to that of the human. In both dogs and humans, the immune system, including the mucosal immune system, is fully developed before birth although the maturity of the immune response may continue into the postnatal period.