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.     

Recombinant single-chain canine interleukin 12 induces interferon gamma mRNA expression in peripheral blood mononuclear cells of dogs with visceral leishmaniasis

Canine visceral leishmaniasis poses important concerns for public health and veterinary medicine in many areas of the world. Resistance to it seems to be associated with cellular specific immune responses of the so-called Th1 type. Interleukin-12 (IL-12) is one of the most potent inducers of Th1 type of immune responses to co-administered antigens. Herein, the cloning of canine IL-12, as a single-chain fusion protein (sccaIL-12), and its expression in biologically active form in COS-7 cells is reported. Supernatants from these cells stimulated the expression of comparable amounts of interferon gamma mRNA in peripheral blood mononuclear cells from dogs with natural visceral leishmaniasis. In addition, after stimulation with sccaIL-12, there was no difference between interferon gamma mRNA expressions in peripheral blood mononuclear cells of dogs with visceral leishmaniasis and from normal healthy control animals.     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.     

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.     

Control of immunoglobulin isotype production by porcine B-cells cultured with cytokines

Cytokines regulate immunoglobulin (Ig) isotype production following the Th1/Th2 paradigm, derived from studies of inbred mice. In pigs, it is not known which, if any, Ig isotypes may reflect a Th1/Th2 response. To evaluate this, purified porcine CD21(+) B-cells were co-cultured with Staphylococcus aureus Cowan strain 1 or Escherichia coli lipopolysaccharide as B-cell mitogens together with recombinant human IL-2, and recombinant porcine (rp) interferon (IFN)-gamma, IL-12 or IL-10. While the mitogens increased B-cell proliferation, cytokines had no additional effect. A quantitative competitive enzyme-immuno assay was used to measure concentrations of porcine IgM, IgG(1) and IgG(2) in B-cell culture supernatants. In vitro, porcine B-cells produced IgG(2), 106 +/- 17.3 microg/ml; IgG(1) 107 +/- 38.3 microg/ml and IgM 25.6 +/- 8.45 microg/ml. In some individuals, Th1 cytokines such as rpIFN-gamma and IL-12, enhanced IgG(2) in the face of low concentrations of IgG(1). Furthermore, individual responses, in some cases, tended to be diametrically opposed, reminiscent of previously documented categorical immune responses in pigs such that some individuals produced high concentrations of IgG(1) in response to the various doses of rp cytokines, while others produced lower concentrations. Pigs may generate a high IgG(1):IgG(2) ratio in response to rpIL-10, and possibly to other Th2-associated cytokines. However, B-cell response to rp cytokines in vitro exhibits marked variation by pig, a feature that is likely a function of highly variable individual genotypes and their interaction with complex environments.     

Longitudinal analysis of cytokine gene expression and parasite load in PBMC in Leishmania infantum experimentally infected dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, an intracellular protozoan parasite that causes a severe infectious disease. To evaluate the gene expression profile associated to CVL in vivo, we have measured monthly by real-time PCR over one year the IL-4, IL-10, IL-12, IL-13, IFN-gamma, TGF-beta and TNF-alpha mRNA levels in peripheral blood mononuclear cells in 6 experimentally infected dogs that exhibited different progressions of the illness. While in two dogs no parasite, or a very low number of parasites, was detected and the two dogs did not show any clinico-pathological abnormalities at the end of the study (L dogs), for the remaining dogs high parasite loads were detected and they developed clinical leishmaniasis (H dogs). The L dogs have null expression of both IL-4 and IL-13 for the first 4 months after the infection, whereas an early IL-4 and IL-13 expression occurs in this period of infection in most of the dogs that developed clinical leishmaniasis (H dogs). Furthermore, a higher IFN-gamma expression was associated with the increase of parasite load and clinical status in these dogs. Moreover, the high variability of expression at the pre-infection stage causes us to reject the possibility that the basal levels of these cytokines indicate the prognosis of the subsequent response against infection.     

B-1 and B-2 B-cells in the pig cannot be differentiated by expression of CD5

In a number of species, such as mice, humans and cattle, B-cells can be differentiated into two populations based on the surface expression of CD5, a marker normally found on T-cells. These B-cell subsets have been found to differ with regard to location, development and phenotypic characteristics. The B-1 (CD5(+)) B-cells have also been shown to have a more restricted immunoglobulin isotype expression profile, limited combinatorial diversity in immunoglobulin heavy chains and lower somatic hyper-mutation. They are potent producers of IL-10. In the pig, CD5(+) and CD5(-) B-cell populations have previously been described in this laboratory. Here, we show that B-cells isolated and separated into CD5(+) and CD5(-) populations do not differ with regard to immunoglobulin isotype or IL-10 RNA expression, nor do the immunoglobulin heavy chain V(D)J re-arrangements differ in terms of gene usage, CDR3 length and composition or the frequency of hyper-mutations. In conclusion, expression of CD5 cannot be used to differentiate between pig blood B-1 and B-2 B-cells.     

Interleukin-6 expression in gut of parasite challenged sheep

Following challenge with Trichosirongylus colubrifonizis, increased numbers of T-cells and immunoglobulin responses are seen in the intestine of sheep immunised by repeated infection with live worms. IL-6 mRNA expression in the small intestine from T. colubriformis-immunised and naive sheep was determined by in situ hybridisation, whereas CD4(+), IgA(+), IgG(+) cells in the gut were evaluated by immunohistochemistry. There was constitutive expression of IL-6 mRNA by cells in the naive gut, and the number of these cells was increased by parasite challenge. There were corresponding increases in numbers of CD4(+) and TCR gamma/delta(+) T-cells and IgG(+) B-cells. Our data are consistent with a role for IL-6, perhaps produced by CD4(+) and/or TCR gamma/delta(+) T-cells or B-cells, in B-cell terminal differentiation. Infiltration of B-cells, particularly IgG(+) B-cells, may reflect parasite immunity in the host.     

Effects of T lymphocytes, interleukin-1, and interleukin-6 on renal fibrosis in canine end-stage renal disease

Canine end-stage renal disease (ESRD) is defined as the almost complete failure of renal function or irreversible destruction and is characterized by extensive glomerular sclerosis, tubular atrophy, interstitial inflammation, and fibrosis. Renal fibrosis is a common pathway leading to kidney failure. Infiltrating immunocytes in the end-stage kidney and several related factors are involved in renal fibrogenesis. A total of 18 renal tissue samples were obtained from canine patients with ESRD using biopsy and necropsy procedures. The extent of renal fibrosis was histopathologically examined by Masson trichrome staining. T-cell and B-cell localization and macrophage lineages were determined by immunohistochemical staining. Additionally, interleukin-1 (IL-1), IL-2, and IL-6 levels in the canine ESRD kidney were immunohistochemically evaluated and compared with expression patterns in the normal kidney. Significant fibrosis and infiltrating immunocytes consistent with lymphocytes were observed. Although the B-cell count was increased in the end-stage kidney, immunostaining patterns disclosed a marked increase in the number of CD3(+) cells. Furthermore, the remarkable increase in IL-1 and IL-6 levels suggests that T cells in the kidneys of dogs with ESRD spontaneously express these cytokines. In this study, the correlation between the degree of renal fibrosis and cytokines in canine ESRD was examined. The present study shows that T lymphocytes and IL-6 play important roles in renal fibrosis.     

Cyclosporine A inhibits transcription of cytokine genes and decreases the frequencies of IL-2 producing cells in feline mononuclear cells

Cyclosporine A (CsA) has been widely used for suppression of transplant rejection and controlling pruritus in allergic dermatitis in humans, dogs and cats. CsA is known to suppress the expression of inflammatory cytokines, including IL-2, IL-4, IFN-gamma and TNF-alpha in humans, dogs and experimental mice. However, little is known about the immunomodulating effect of CsA in cats. The aim of this study was to evaluate the effects of CsA on the expression of inflammatory cytokines in feline peripheral blood mononuclear cells (PBMC). Real-time PCR analyses with Concanavalin A (ConA)-stimulated PBMC obtained from 5 cats revealed that the expression of mRNAs for IL-2, IL-4, IFN- gamma and TNF-alpha was inhibited by CsA in a dose-dependent manner. Moreover, an enzyme-linked immunospot (ELISPOT) assay, which is capable of detecting IL-2 secreting cells as single spots, revealed that the frequency of IL-2 secreting cells in ConA-stimulated feline PBMC was significantly reduced in the presence of CsA. These results might provide an explanation for the mechanisms of action of CsA in the suppression of transplant rejection and the control of pruritus in cats.     

Evaluation of L-asparaginase: Polyethylene Glycol Conjugate Versus Native L-asparaginase Combined with Chemotherapy: A Randomized Double-blind Study in Canine Lymphoma

L-asparaginase is an enzyme that inhibits protein synthesis by the depletion of sources of L-asparagine, which is necessary for transformed lymphoid cells to proliferate. L-asparaginase is used in the treatment of childhood acute lymphoblastic leukemia. A problem with L-asparaginase therapy is the immunogenicity of the enzyme and the development of anaphylactic reactions. Canine lymphoma is a predominantly B-cell tumor with widespread disease; without treatment, dogs with lymphoma usually survive 1-2 months. Canine lymphoma will respond to L-asparaginase therapy. A randomized double-blind study evaluated a polyethylene glycol (PEG) conjugate L-asparaginase combined with chemotherapy (vincristine, cyclophosphamide, doxorubicin, and prednisone). Thirty-five dogs were randomized to the PEG L-asparaginase group, and 34 dogs were randomized to the native L-asparaginase group. Thirty dogs (85.7%) achieved a complete remission (CR) with a median time to relapse of 217 days, and 32 (94.1%) dogs in the native L-asparaginase group achieved a CR with a median time to relapse of 214 days (P greater than 0.05). The asparaginase was well tolerated in both groups. Two dogs in the native L-asparaginase group had severe allergic reactions, and one dog in the PEG asparaginase group had a generalized urticarial reaction after repeated injections. This study indicates that PEG L-asparaginase has equal therapeutic efficacy to native L-asparaginase.     

Quantification of mRNA encoding cytokines and chemokines in nasal biopsies from dogs with sino-nasal aspergillosis

Canine sino-nasal aspergillosis is usually caused by Aspergillus fumigatus and is similar to human chronic erosive non-invasive fungal sinusitis. The pathogenesis of the disease is poorly understood. We investigated the nature of the local immune response mounted in canine sino-nasal aspergillosis. Quantitative RT-PCR was carried out on RNA isolated from nasal biopsies from diseased and control dogs, using specific assays designed to amplify mRNA encoding a panel of cytokines and chemokines. Canine sino-nasal aspergillosis was associated with significantly increased expression of mRNA encoding MCP-1, -2, -3 and -4, IL-8, IL-10, IL-18 and TNF-alpha relative to controls (P<0.01) but there was no difference between groups with respect to IL-4, IL-5, IL-6, IL-12, TGF-beta, and eotaxin-2 and -3. The up-regulation of proinflammatory cytokines and chemokines related to the influx of phagocytic cells might account for the localisation of this infection to the upper respiratory tract. The up-regulation of the expression of the immunomodulatory cytokine IL-10 in nasal tissue from affected dogs might be important in limiting the extent of local tissue destruction, but might also account for the fact that infected dogs are generally unable to clear this infection spontaneously.     

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.     

Gnotobiotics and immunopathology: the use of the gnotobiotic environment to study acquired and inherited immunodeficiency diseases

Gnotobiotic animals are highly valued for the study of infectious diseases wherein the clinical signs and lesions of disease can be directly related to host-pathogen interactions and not to the additive effects of environmental influences and other confounding factors. Gnotobiotic dogs have been used to study the pathogenesis of acquired immunodeficiencies associated with canine distemper virus (CDV). In recent years, the laboratory at OSU, in conjunction with University of Pennsylvania personnel have begun a series of long-term studies of dogs affected with the canine X chromosome-linked severe combined immunodeficiency (XSCID) syndrome. This fatal inherited defect is caused by mutation in the common gamma chain (IL2RG) gene and renders affected animals profoundly immunodeficient. XSCIDs dogs, raised within a gnotobiotic environment for up to 3 years remain clinically healthy and are, in every respect normal except for the persistent T-cell defect and the failure to develop lymph nodes. Bone marrow transplantation (unfractionated or enriched for CD34+ stem cells) is the treatment of choice for both the XSCID dogs and male human infants affected with this syndrome. In preliminary studies, we have shown that human CD34+ stem cells colonized XSCIDs-affected gnotobiotic dogs, migrated to the thymus and demonstrated post-thymic activation (CD45RA+ phenotype) in peripheral blood. While many issues are unresolved, these data suggest that, through the use of the gnotobiotic environment, xenotransplantation (human-to-dog) may yield a stable and immunologically functional human-dog chimera.     

Semi-quantitative analysis of cytokine expression in asymptomatic canine leishmaniasis

The dog is the main reservoir of Leishmania infantum, the parasite responsible for visceral leishmaniasis in Mediterranean countries. The infection in dogs shows different clinical presentations, from subclinical/asymptomatic to a fully developed disease, depending on the host's immune responses. The Th1/Th2 dichotomy is not clear in the different forms of canine leishmaniasis, since the data available from studies of immunity response in canine leishmaniasis are scarce and fragmented. The present work describes the cytokine expression in peripheral blood mononuclear cells (PBMC) obtained from asymptomatic dogs experimentally infected with L. infantum that present a cellular protective immune response. The results obtained from freshly isolated PBMC showed expressions of TNF-alpha, IL-2, IFN-gamma, IL-10 and IL-18 mRNA, similar to those from non-infected dogs. However, there was almost no expression of IL-4 mRNA detected in the asymptomatic infected dogs compared to the control dogs. Unspecific stimulation with ConA promoted the expression in a greater or lower degree of all the cytokines studied. In vitro stimulation of PBMC with soluble leishmanial antigen (SLA) promoted the expression of IL-2, IFN-gamma, TNF-alpha, IL-18, IL-4, IL-6 and IL-10 mRNA, with the two first being specifically induced. Although both Th1 and Th2 cytokines are produced, cell mediated immunity observed in these L. infantum-infected asymptomatic dogs depended on the preferential expression of Th1 cytokines.