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A novel canine papillomavirus, CfPV-2, was cloned from a footpad lesion of a golden retriever. Unlike the known canine oral papillomavirus (COPV), which has a double-stranded DNA genome size of 8607 bps, the genome of CfPV-2 is 8101 bps. Some of this size difference is due to an abbreviated early-late region (ELR), which is 1200 bps shorter than that of COPV. However, CfPV-2 has other differences from COPV, including the presence of an E5 ORF between the E2 gene and the ELR and an enlarged E4 ORF (one of the largest PV E4 open reading frames). The genome of CfPV-2 shares low homology with all the other papillomaviruses and, even in the most highly conserved ORF of L1, the nucleotide sequence shares only 57% homology with COPV. Due to this highly divergent DNA sequence, CfPV-2 establishes a new PV genus, with its closest phylogenetic relatives being amongst the Xi and Gamma genuses. CfPV-2 also has unique biological features; it induces papillomas on footpads and interdigital regions which, if infection is persistent, can progress to highly metastatic squamous cell carcinoma. CfPV-2 does not induce oral papillomas in immunocompetent animals and antibodies generated against COPV and CfPV-2 are type-specific. The availability of a new canine papillomavirus with differing genetic and biological properties now makes it possible to study type-specific host immune responses, tissue tropism and the comparative analysis of viral gene functions in the dog.

X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor γ chain (γc). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term–surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.

Since the occurrence of T cell leukemias in the original human γ-retroviral gene therapy trials for X-linked severe combined immunodeficiency (XSCID), considerable effort has been devoted to developing safer vectors. This review summarizes gene therapy studies performed in a canine model of XSCID to evaluate the efficacy of γ-retroviral, lentiviral, and foamy viral vectors for treating XSCID and a novel method of vector delivery. These studies demonstrate that durable T cell reconstitution and thymopoiesis with no evidence of any serious adverse events and, in contrast to the human XSCID patients, sustained marking in myeloid cells and B cells with reconstitution of normal humoral immune function can be achieved for up to 5 years without any pretreatment conditioning. The presence of sustained levels of gene-marked T cells, B cells, and more importantly myeloid cells for almost 5 years is highly suggestive of transduction of either multipotent hematopoietic stem cells or very primitive committed progenitors.

Objective–To investigate the potential cell-mediated immune response of atopic dogs to the yeastMalassezia pachydermatisand to correlate it with the type-1 hypersensitivity (humoral) response of the same population of dogs.Animals–16 clinically normal dogs, 15 atopic dogs withMalasseziadermatitis, 5 atopic dogs withMalasseziaotitis, and 7 atopic control (ie, withoutMalasseziadermatitis or otitis) dogs.Procedure–A crude extract ofM pachydermatiswas extracted for use as an intradermal allergy testing reagent and for stimulation of isolated peripheral blood mononuclear cells in vitro. Flow cytometry was also used to assess cell surface antigenic determinants (CD3, CD4, CD8, CD14, CD21, CD45RA, surface immunoglobulin) on peripheral blood mononuclear cells.Results–Atopic dogs with cytologic evidence ofMalasseziadermatitis had an increased lymphocyte blastogenic response to crudeM pachydermatisextract, compared with clinically normal dogs and dogs withMalasseziaotitis. Atopic control dogs did not differ significantly in their responses from atopic dogs withMalasseziadermatitis or otitis. A significant correlation was not found between the lymphocyte blastogenic response and the type-1 hypersensitivity response toM pachydermatiswithin any of the groups.Conclusions and Clinical Relevance–Cell-mediated and humoral reactivities toM pachydermatiscontribute to the pathogenesis of atopic dermatitis in dogs but are not directly correlated. Modification of the dysregulated immune response towardM pachydermatismay assist in the reduction of pathologic changes associated with an atopic dermatitis phenotype in dogs. (Am J Vet Res2002;63:358–362)

X-linked severe combined immunodeficiency (XSCID) is caused by a genetic mutation within the common gamma chain (γc), an essential component of the cytokine receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21. XSCID patients are most commonly treated with bone marrow transplants (BMT) to restore systemic immune function. However, BMT-XSCID humans and dogs remain at an increased risk for development of cutaneous papillomavirus (PV) infections and their associated neoplasms, most typically cutaneous papillomas. Since basal keratinocytes are the target cell for the initial PV infection, we wanted to determine if canine XSCID keratinocytes have a diminished antiviral cytokine response to poly(dA:dT) and canine papillomavirus-2 (CPV-2) upon initial infection. We performed quantitative RT-PCR for antiviral cytokines and downstream interferon stimulated genes (ISG) on poly(dA:dT) stimulated and CPV-2 infected monolayer keratinocyte cultures derived from XSCID and normal control dogs. We found that XSCID keratinocytes responded similarly to poly(dA:dT) as normal keratinocytes by upregulating antiviral cytokines and ISGs. CPV-2 infection of both XSCID and normal keratinocytes did not result in upregulation of antiviral cytokines or ISGs at 2, 4, or 6 days post infection. These data suggest that the antiviral response to initial PV infection of basal keratinocytes is similar between XSCID and normal patients, and is not the likely source for the remaining immunodeficiency in XSCID patients.

Current approaches to hematopoietic stem cell (HSC) gene therapy involve the collection and ex vivo manipulation of HSCs, a process associated with loss of stem cell multipotency and engraftment potential. An alternative approach for correcting blood-related diseases is the direct intravenous administration of viral vectors, so-called in vivo gene therapy. In this study, we evaluated the safety and efficacy of in vivo gene therapy using a foamy virus vector for the correction of canine X-linked severe combined immunodeficiency (SCID-X1). In newborn SCID-X1 dogs, injection of a foamy virus vector expressing the human IL2RG gene resulted in an expansion of lymphocytes expressing the common γ chain and the development of CD3+ T lymphocytes. CD3+ cells expressed CD4 and CD8 coreceptors, underwent antigen receptor gene rearrangement, and demonstrated functional maturity in response to T-cell mitogens. Retroviral integration site analysis in 4 animals revealed a polyclonal pattern of integration in all dogs with evidence for dominant clones. These results demonstrate that a foamy virus vector can be administered with therapeutic benefit in the SCID-X1 dog, a clinically relevant preclinical model for in vivo gene therapy.

A mutation in the canine Kindlin-3 geneassociated with increased bleeding risk and susceptibility to infections

Foamy viruses (FV) are ideal vectors for in vivo gene therapy because FV infection is non-pathogenic in people, FV are more resistant to serum inactivation than lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and FV exhibit favorable proviral integration patterns compared to gammaretroviruses. In vivo gene therapy with a FV vector expressing the human common gamma chain (γC) under the short EF1α promoter results in a functional immune reconstitution in X-SCID dogs.

Two, male, Cardigan Welsh corgi puppies, one of which was diagnosed with X-linked severe combined immunodeficiency (XSCID), are described in this report. The first puppy was euthanized before definitive immunological testing could be performed. When the second puppy was presented and the relationship between the two was discovered, immunological testing was pursued immediately due to this puppy's rapid deterioration. The immunological test results and genetic studies were compared to the XSCID basset hounds and found to be similar. By unveiling the mutation, the pedigree could be analyzed and the carrier females removed from the breeding population.

In both humans and canines, X-linked severe combined immunodeficiency disease (XSCID) is caused by mutations in the interleukin-2 receptor gamma chain gene (IL2RG) which results in a lack of response to common gamma-chain (gammaC) dependent cytokines and abnormal development of T and B lymphocytes, and natural killer (NK) cells. Death from infections usually occurs before 1 year of age unless allogeneic hematopoietic cell transplantation (HCT) is performed. While HCT is successful if an HLA-matched sibling donor is available, transplants from mismatched and unrelated donors are associated with greater morbidity and overall survival can be as low as 50%. To circumvent these complications, several clinical trials are testing the possibility of utilizing blood and marrow stem cells from the patient for ex vivo gene therapy to treat X-SCID. Although these trials show promising results, they require expensive GMP cell manufacturing that are not accessible to many patients, and may also necessitate low level of conditioning to improve engraftment of gene-corrected cells. With these limitations in mind, we have explored in vivo gene therapy as a treatment for X-SCID. We previously showed that foamy virus vectors (FVs), exhibit a potentially more favorable integration profile compared to lenti- and gamma-retroviral vectors. In vivo delivery of a gammaC-FV in dogs resulted in immune reconstitution with gene-corrected T cells in dogs but the treated animals still developed infections and had low levels of immunoglobulin levels. We hypothesized that an increased transduction of hematopoietic stem/progenitor cells in vivo might result in more rapid and sustained immune reconstitution. Thus, in the current study, we used cG-CSF and AMD3100 to mobilize hematopoietic stem/progenitor cells into the peripheral blood prior to in vivo injection with a FV expressing the gammaC gene driven by a PGK promoter (PGK-gammaC-FV). We mobilized two X-SCID dogs at ~3 weeks of age with 5ug/kg of cG-CSF bi-daily from day -4 to -1 prior to FV injection, and with 4mg/kg of AMD3100 on the morning of the injection with 4x10e8 IU of PGK-gammaC-FV. Our mobilization protocol resulted in a 10-fold increase in CD34+ cells in the peripheral blood of mobilized X-SCID dogs as compared to a unmobilized normal littermate control (Figure 1 A). Lymphocyte recovery and gene marking in the mobilized animals was significantly improved as compared to animals that were previously injected with similar doses of either PGK-gammaC-FV or EF1a-gammaC-FV but without mobilization. As illustrated in Figure 1B-C, lymphocyte counts expanded to ~3000 cells/uL with ~75% gene marking in the mobilized animals treated with PGK-gammC-FV within 30 days, as compared to Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Scharenberg:bluebird bio: Consultancy, Equity Ownership, Research Funding; Alpine Immune Sciences: Consultancy. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

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.

X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the common gamma chain, γC (interleukin-2 receptor subunit gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)-yC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the gene-corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.

Parallel genetic analysis of animal and human genetic diseases can facilitate the identification and characterization of the causative gene defects. For example, canine X-linked severe combined immunodeficiency (SCID) is characterized by clinical, pathological, and immunological manifestations similar to the most common form of human SCID. To derive a canine syntenic map including genes that in humans are located in proximal Xq, near human X-linked SCID, poly(TG) polymorphisms were identified at the canine phosphoglycerate kinase (PGK) and choroideremia (CHM) loci. These plus a polymorphic poly(CAG) sequence in exon 1 of the canine androgen receptor gene (AR) were used to genotype members of the colony informative for X-linked SCID. No recombinations among SCIDX1, AR, PGK, or CHM were observed. Fluorescence in situ hybridization localized PGK and CHM to proximal Xq in the dog, in the same chromosomal location occupied by the human genes. Somatic cell hybrid analysis and methylation differences at AR demonstrated that female dogs carrying X-linked SCID have the same lymphocyte-limited skewed X-chromosome inactivation patterns as human carriers. These genetic and phenotypic findings provide evidence that mutations in the same gene, now identified as the gamma chain of the IL-2 receptor, cause canine and human X-linked SCID. This approach is an efficient method for comparative gene mapping and disease identification.

The principal role of the immune system is the protection of the host against invasion by infectious disease agents and other substances considered foreign to the host. The term "immunity" refers to all the mechanisms that the body uses to protect itself against environmental antigens. This article gives an overview of the immune system and its defense immunodeficient diseases.

We describe the occurrence of acute monocytic leukemia in a dog with X-linked severe combined immunodeficiency (XSCID) that had been raised in a gnotobiotic environment for 20 months. This case represents the first reported instance of malignancy in canine XSCID, the first case of acute monocytic leukemia in any species with severe combined immunodeficiency, and the first documented malignancy in any species with XSCID that was not associated with immunotherapy.

This study describes the thymic morphology in 52 dogs (ranging in age from 1 to 79 days) with X-linked severe combined immunodeficiency disease (XSCID). The thymuses from the XSCID dogs and age-matched controls were evaluated histotogically for the presence of Hassall's corpuscles and branchial duct remnants, the degree of corticomedullary differentiation, and lymphoid development and organization. Within this population of XSCID dogs with the same genetic defect, three histologic patterns of thymic dysplasia were recognized. Simple dysplasia, noted in 27 XSCID thymuses, was characterized by varying numbers of lymphocytes, no corticomedullary demarcation, and an absence of Hassall's corpuscles. Dysplasia with Hassall's corpuscles was noted in 21 dogs and consisted of varying numbers of lymphocytes, no corticomedullary demarcation, and varying numbers of Hassall's corpuscles. Dysplasia with partial corticomedullary demarcation, noted in 4 dogs, consisted of relatively normal-looking thymuses with well-defined corticomedullary demarcation and numerous Hassall's corpuscles; however, the lobules were extremely small and the subcapsular cortical region was devoid of lymphocytes. Cystic branchial duct remnants were present in 46 of the 52 XSCID thymuses and were more numerous in those thymuses with the pattern of simple dysplasia. The thymuses of XSCID pups less that 4 weeks of age were of the simple dysplastic type and thymuses of XSCID dogs greater than 4 weeks of age were more developed, as evidenced by increased numbers of Hassall's corpuscles and greater corticomedullary demarcation. In conclusion, the thymic dysplasia and lymphoid hypoplasia associated with X-linked severe combined immunodeficiency disease in the dog does not appear to be due to a developmental arrest but rather due to an active process dependent on factors probably related to the overall genetic defect.

We have previously shown that in vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency (XSCID) results in sustained T cell reconstitution and sustained marking in myeloid and B cells for up to 4 years with no evidence of any serious adverse effects. The purpose of this study was to determine whether ex vivo γ-retroviral gene therapy of XSCID dogs results in a similar outcome. Eight of 12 XSCID dogs treated with an average of dose of 5.8 × 10(6) transduced CD34(+) cells/kg successfully engrafted producing normal numbers of gene-corrected CD45RA(+) (naïve) T cells. However, this was followed by a steady decrease in CD45RA(+) T cells, T cell diversity, and thymic output as measured by T cell receptor excision circles (TRECs) resulting in a T cell lymphopenia. None of the dogs survived past 11 months post treatment. At necropsy, few gene-corrected thymocytes were observed correlating with the TREC levels and one of the dogs was diagnosed with a thymic T cell lymphoma that was attributed to the gene therapy. This study highlights the outcome differences between the ex vivo and in vivo approach to γ-retroviral gene therapy and is the first to document a serious adverse event following gene therapy in a canine model of a human genetic disease.

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.

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.

Dogs with X-linked severe combined immunodeficiency (XSCID) can be successfully treated by bone marrow transplants (BMT) resulting in full immunologic reconstitution and engraftment of both donor B and T cells without the need for pre-transplant conditioning. In this study, we evaluated the T cell diversity in XSCID dogs 4 months to 10 1/2 years following BMT. At 4 months post transplantation, when the number of CD45RA+ (naïve) T cells had peaked and plateaued, the T cells in the transplanted dogs showed the same complex, diverse repertoire as those of normal young adult dogs. A decline in T cell diversity became evident approximately 3 1/2 years post transplant, but the proportion of Vβ families showing a polyclonal Gaussian spectratype still predominated up to 7 1/2 years post transplant. In two dogs evaluated at 7 1/2 and 10 1/2 years post transplant, >75% of the Vβ families consisted of a skewed or oligoclonal spectratype that was associated with a CD4/CD8 ratio of

In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy because they are non-pathogenic in humans, they exhibit increased serum stability and they integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of functional CD3+ lymphocytes in canine X-SCID (Burtner CR et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy that include different promoters and different fluorophores. We performed a head-to-head comparison of two promoters, our previously used EF1α promoter and the human phosphoglycerate kinase (PGK) promoter, by simultaneously injecting three X-SCID pups with equal titers of two therapeutic, human γC-encoding FV vectors. These vectors expressed the fluorophores GFP or mCherry to allow for tracking of transduced cells. Each dog received between 3 and 4 x 108 infectious units of each FV vector. In all treated dogs, lymphocyte marking in the PGK arm reached 50% between day 60 and day 110 post-injection and continued to expand over time, while the EF1α arm peaked at day 42 and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the EF1α arm, suggesting competition within the expanding T cell niche. The development of total CD3+ T cells achieved therapeutic levels (1000 cells/μL of blood) in all three dogs between day 70 and day 130 post-treatment (Fig 1B). We further validated the functionality of these cells by showing that they express a diverse T cell receptor repertoire using spectratyping analysis. In addition, peripheral blood mononuclear cells from the treated animals could be activated in vitro by exposure to the mitogen Phytohemagglutinin A at a level comparable to normal cells. Immunization of the treated dogs with bacteriophage ΦX174 showed production of specific IgG antibodies, suggesting the ability of B lymphocytes to undergo isotype switching. Finally, retroviral integration site analysis revealed polyclonal contribution to the reconstituting T cells. In summary, our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. The outcome in dogs was substantially improved compared to our previous study using EF1α, where robust lymphocyte marking was achieved in only 2 of 5 dogs, and where clonal dominance was observed. Ongoing work will determine whether the superior performance of the PGK vector is due to higher γC expression in PGK vs. EF1α corrected cells. Figure 1. T-cells expansion in X-SCID dogs following FV treatment. A) Percent of gene-modified peripheral blood lymphocytes in each experimental arm after in vivo gene therapy. B) Absolute CD3+ count per μL peripheral blood in all treated animals. Dotted line indicates therapeutic counts of CD3+ cells. Figure 1. T-cells expansion in X-SCID dogs following FV treatment. A) Percent of gene-modified peripheral blood lymphocytes in each experimental arm after in vivo gene therapy. B) Absolute CD3+ count per μL peripheral blood in all treated animals. Dotted line indicates therapeutic counts of CD3+ cells. Disclosures No relevant conflicts of interest to declare.

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain (γc) gene and is identical clinically and immunologically to human XSCID, making it a true homologue of the human disease. Bone marrow-transplanted (BMT) XSCID dogs not only engraft donor T cells and reconstitute normal T-cell function but, in contrast to the majority of transplanted human XSCID patients, also engraft donor B cells and reconstitute normal humoral immune function. Shortly after our initial report of successful BMT of XSCID dogs, it soon became evident that transplanted XSCID dogs developed late-onset severe chronic cutaneous infections containing a newly described canine papillomavirus. This is analogous to the late-onset cutaneous papillomavirus infection recently described for human XSCID patients following BMT. Of 24 transplanted XSCID dogs followed for at least 1 year post-BMT, 71% developed chronic canine papillomavirus infection. Six of the transplanted dogs that developed cutaneous papillomas were maintained for >3 1/2 years post-BMT for use as breeders. Four of these six dogs (67%) developed invasive squamous cell carcinoma (SCC), with three of the dogs (75%) eventually developing metastatic SCC, an extremely rare consequence of SCC in the dog. This finding raises the question of whether SCC will develop in transplanted human XSCID patients later in life. Canine XSCID therefore provides an ideal animal model with which to study the role of the γc-dependent signaling pathway in the response to papillomavirus infections and the progression of these viral infections to metastatic SCC.

AIM: Recent studies demonstrating the direct involvement of dendritic cells (DC) in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. METHODS: Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient (IL2-/-) mice that develop colitis. RESULTS: In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type 1 myeloid (CD11c+, CD11b+, B220-, CD8α-) DC made up the largest (40-45%) population and all DC expressed low levels of CD80, CD86, and CD40, and had high endocytic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes (MLN). The majority (>85%) of DC in the colon and MLN of IL2-/- mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. CONCLUSION: These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.

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.

As in many human patients with X-linked hypohidrotic ectodermal dysplasia (XHED), XHED dogs are at an increased risk for pulmonary disorders. Localized immune system defects had been suspected previously in affected dogs because of frequent infections and unexpected deaths due to opportunistic respiratory tract infections. Experiments were designed to examine systemic and localized humoral and cellular responses, development and function of T cells, and thymic morphology. All dogs used in these experiments were clinically healthy at the time of examination and their immune responses were compared to normal littermates. Serum immunoglobulin concentrations differed somewhat between normal dogs and dogs affected with XHED but they were all within normal ranges. The XHED dogs responded appropriately to vaccination with tetanus toxoid suggesting normal systemic B and plasma cell function. Thymic morphology was compared between normal and affected dogs and T cells were assessed for functionality. Numbers and phenotypes of T and B cells in blood and thymus of affected dogs were within normal limits suggesting normal development of T cells. Cytotoxic and phagocytic ability of macrophages and neutrophils was also normal in affected dogs. In contrast, the secretory IgA concentrations found in affected dogs were significantly higher than in normal dogs, while lacrimal secretions were significantly decreased. These results suggest a compensatory mechanism for secretory IgA, so that the total amount equals that in normal dogs. The results presented in this study indicate that the XHED dogs have a relatively intact immune system and suggest that the same is true for humans with the homologous form of XHED.

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.

The gut maintains a delicate balance between the downregulation of inflammatory reactions to commensal bacteria and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. Intestinal epithelial cells, including colonic epithelial cells (CECs) possess many properties of cells of the innate immune system, in particular the ability to recognize and respond to microbial antigens. Recognition of microorganisms by CECs is based upon their recognition of signature molecules, called microbe-associated molecular patterns (MAMP), by pattern recognition receptors (PRR) including membrane toll-like receptors (TLR) and cytosolic Nod2, an intracellular counterpart of TLRs. The purpose of this study was to determine whether primary CECs from normal dogs express a functional TLR2, TLR4, and Nod2 and whether they are regulated by inflammatory mediators. We show that canine primary CECs express TLR2, TLR4, and Nod2 that can be modulated in response to their respective MAMPs, lipopolysaccharides (LPS) or peptidoglycans (PGN). Furthermore, we demonstrate that these receptors are functional as evidenced by the induction of cytokine gene expression in response to LPS or PGN.

Background and aims: As the first point of contact with enteric antigens, intestinal epithelial cells (IEC) may be key in regulating mucosal immune responses. We determined therefore if murine colonic epithelial cells (CEC) have tolerogenic or activating effects on CD4 T cells. Methods: Using a novel CEC, macrophages, and CD4 T cell coculture system, mitogen and antigen specific responses of naive and antigen primed CD4 T cells were assessed. Results: Although a proportion of CEC express the costimulatory molecules B7.1, B7.2, CD40, and CD54, they were unable to promote mitogen or antigen driven activation of CD4 T cells, even in the presence of exogenous costimulatory signals. CD4 T cells cocultured with CEC were CD25 lo and CD45RB lo and remained in the G1 phase of the cell cycle. CEC were also able to prevent CD4 T cell activation by professional antigen presenting cells. CEC mediated suppression of T cell activation was cell contact dependent and transforming growth factor β independent. Conclusions: These observations suggest that CEC contribute to the maintenance of T cell tolerance in the gut by preventing inappropriate activation of CD4 T cells.

Our laboratory has identified an X-linked severe combined immunodeficiency (XSCID) in dogs that is the result of mutations in the common gamma chain (gammac) subunit of the interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. Canine XSCID, unlike genetically engineered gammac-deficient mice, has a clinical and immunologic phenotype virtually identical to human XSCID, suggesting species-specific differences exist in the role of the gammac and its associated cytokines in mice in comparison to their role in humans and dogs. This review compares and contrasts thymopoiesis and postnatal T cell development in gammac-deficient (XSCID) dogs raised in a conventional environment, with gammac-deficient dogs raised in a gnotobiotic environment. Therapy to accelerate T cell regeneration following hematopoietic stem cell transplantation or gene therapy is also discussed.

X-linked severe combined immunodeficiency (X-SCID) is the most common form of human SCID and is caused by mutations in the common gamma chain (gammac), a shared component of the interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. BMT for human X-SCID results in engraftment of donor T-cells and reconstitution of normal T-cell function but engraftment of few, if any, donor B-cells and poor reconstitution of humoral immune function. Canine X-SCID is also caused by mutations in the yc and has an immunological phenotype identical to that of human X-SCID. We have previously reported that transplantation of nonconditioned X-SCID dogs with unfractionated histocompatible bone marrow results in engraftment of both donor B- and T-cells and reconstitution of normal T-cell and humoral immune function. In this study, we assessed the ability of purified canine CD34+ bone marrow cells to reconstitute lymphoid populations after histocompatible BMT in 6 nonablated X-SCID dogs. All dogs showed engraftment of donor T-cells, with T-cell regeneration occurring through a thymic-dependent pathway, and had reconstituted normal T-cell function. In contrast to our previous studies, only 3 dogs had engraftment of donor B-cells and reconstituted normal antigen-specific B-cell function post-BMT. The variable donor B-cell engraftment and reconstitution of normal humoral immune function observed in this study are similar to the outcomes observed in the majority of human X-SCID patients following BMT. This study demonstrates that canine CD34+ cells contain progenitors capable of immune reconstitution and is the first study to document the ability of CD34+ bone marrow cells to reconstitute normal B- and T-cell function in a nonablated large-animal model of BMT. This study also demonstrates that the quality of immune reconstitution following CD34+ BMT may be dosage dependent Thus canine X-SCID provides a large-animal preclinical model that can be used not only to determine the optimal conditions for both donor B- and T-cell engraftment following CD34 BMT, but also to develop and evaluate strategies for gene therapy protocols that target CD34 cells. Biol Blood Marrow Transplant 2002;8(4):188-97.

In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy for 3 primary reasons: (1) FV are non-pathogenic in humans, (2) they exhibit enhanced serum stability as compared to lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and (3) FV integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of CD3+ lymphocytes in canine X-SCID, which undergo T cell receptor rearrangement and exhibit a functional signaling response to T cell activating mitogens (Burtner CR, Beard BC, Kennedy DR, et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy, including different promoters, using injections of vectors marked with different fluorophores. Preliminary data indicated that ex vivo transduction of canine CD34+ cells with a FV vector expressing human γC and a fluorescent reporter under the human phosphoglycerate kinase (PGK) promoter resulted in higher transduction efficiencies and increased mean fluorescence intensity, compared to that of an identical vector containing the EF1α promoter. We therefore performed a head-to-head comparison of the two promoters by simultaneously injecting X-SCID pups with equal titers of 2 therapeutic, human γC-encoding FV vectors that differed only in the promoter used to drive human γC expression and in the fluorophore color to distinguish gene-marked cells (GFP and mCherry). Each dog received 4 x 108 infectious units of each FV vector. A significant population of gene-marked lymphocytes appeared in the PGK arm 42 days post in vivo gene therapy, which continued to expand over the next two months of follow-up (Fig 1A). By 84 days post injection, lymphocyte gene marking in the competitive PGK arm reached 60% in both dogs. For comparison, this robust level of lymphocyte gene marking was achieved in only 2 of 5 dogs after 122 and 160 days, respectively, in our previous EF1α virus treated cohort. In contrast, the EF1α arm peaked at 42 days after in vivo gene therapy and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the by the EF1α arm, suggesting competition within the expanding T cell niche. The expansion of gene-marked lymphocytes was followed by the development of CD3+ T cells, leading to a therapeutic level of CD3+ cells (1000 cells/μl of blood) in both dogs (Fig 1B). Additionally, our data indicate low but persistent gene marking in other blood cells, including granulocytes and B cells, with B cell marking in one animal exceeding 2% in the PGK arm. Our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. These data also highlight the utility of the in vivo approach to explore key parameters of vector design in competitive repopulation experiments that may be useful for other diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

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.

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.

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.

Juvenile polyarteritis syndrome (JPS) is an idiopathic febrile disease in dogs. Elevated serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) have been reported in human patients with vasculitis. We investigated whether these cytokines are also elevated in serum of dogs with JPS using sensitive bio-assays. Increased levels of IL-6 activity were detected in the serum of 12 acutely ill dogs, whereas the IL-6 activity decreased to low or undetectable levels during convalescence. Treatment of 5 acute JPS dogs with prednisone resulted in a rapid clinical improvement accompanied by a decrease of IL-6 activity. Withdrawal of prednisone treatment caused reappearance of clinical symptoms and high serum IL-6 activity within a few days. TNF activity could not be detected in the samples of normal dogs, convalescent JPS, or acute JPS dogs. These studies support a role for IL-6 in the pathogenesis of JPS.

Eighteen young Beagle dogs (eight males and 10 females), ages 6-40 months, with canine juvenile polyarteritis syndrome (CJPS), a naturally occurring vasculitis and perivasculitis of unknown etiology, were necropsied, and their tissues were examined by histopathologic and histochemical methods. The condition is characterized by recurring episodes of an acute onset of fever (>40 C) and neck pain that persist for 3-7 days. The major histopathologic alterations were a systemic vasculitis and perivasculitis. During the febrile, painful period of CJPS, the vascular lesions ranged from a histiocytic-lymphocytic periarterial infiltration to transmural arterial inflammation with concomitant fibrinoid necrosis and vascular thrombosis. Massive periarterial accumulations of inflammatory cells were common and often extended into adjacent tissues. The small- to medium- sized muscular arteries of the heart, cranial mediastinum, and cervical spinal meninges were consistently involved. Vasculitis occasionally occurred in other organ systems. The vascular lesions in dogs examined during clinically normal periods consisted of intimai and medial fibrosis, ruptured elastic laminae, and mild perivasculitis; these lesions were probably related to previous episodes of vasculitis. Eight dogs that had experienced repeated acute episodes also developed splenic, hepatic, and renal amyloidosis. The clinical signs, laboratory abnormalities, and the vascular lesions suggest that the condition may be immune-system mediated. CJPS may serve as a naturally occurring animal model of human immune-system-mediated vasculitides such as polyarteritis nodosa, infantile polyarteritis, and Kawasaki disease.

X-linked severe combined immunodeficiency (SCID) is characterized by profound defects in cellular and humoral immunity and, in humans, is associated with mutations in the gene for the gamma chain of the IL-2 receptor (IL-2R gamma). We have examined this gene in a colony of dogs established from a single X-linked SCID carrier female. Affected dogs have a 4-bp deletion in the first exon of the IL-2R gamma gene, which precludes the production of a functional protein, demonstrating that the canine disease is a true homologue of human X-linked SCID.

This study describes the immunologic abnormalities during the acute phase of juvenile polyarteritis syndrome (JPS), a multisystem necrotizing vasculitis of young dogs with a predilection for the coronary arteries. JPS has striking clinical, laboratory, and pathologic similarities to Kawasaki disease (KD), the most common cause of acquired heart disease in children in the United States. The immunologic abnormalities include an increase in serum IgA, an increase in the percentage of peripheral B cells and a decrease in the percentage of total peripheral T cells, a marked suppression of the blastogenic response to mitogenic stimulation, an inability to generate immunoglobulin-secreting plasma cells following polyclonal activation, the presence of antineutrophil cytoplasmic antibodies, and evidence of monocyte/macrophage activation. These immunoregulatory abnormalities are similar to those observed in children during the acute phase of KD. This unique, naturally occurring animal model of necrotizing vasculitis may prove useful for investigating novel therapeutic interventions in the treatment of necrotizing vasculitis and may yield insight into the immunopathology and etiology of KD.

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.

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.

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.

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).

X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency (dysfunctional B cells; absence of T and NK cells) and early mortality, caused by mutations in the IL2RG gene encoding the common gamma chain (γc) of receptors for interleukins (IL)-2,-4,-7,-9,-15 and -21. The standard therapy for XSCID in infants is a T cell depleted bone marrow transplant with either none or very modest conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution that may be limited to only the T cell lineage. Ex vivo autologous stem cell gene therapy has emerged as an alternate treatment capable of achieving substantial to complete immune reconstitution in infants without a sibling donor. However, some children have developed lymphocytic leukemia, which appears in part to be related to vector insertional mutagenesis. The gammaretroviruses currently in use have potent enhancers in the LTR and have a predilection to insert at the 5′ end of genes. Self-inactivating (SIN) lentivirus vectors may be advantageous because they do not show this property and can be constructed with internal promotors that have less enhancer activity together with insertion of insulators. We have constructed SIN simian immunodeficiency viral vectors (SIVmac) encoding human γc (hγc), with or without a double copy chicken insulator core element in the 3′ LTR. In addition we have pseudotyped the vector with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicity of the VSV-G envelope traditionally used with lentivectors. We have previously shown that it is possible to correct a dog model of XSCID using a RD114-pseudotyped gammaretroviral vector encoding the dog γc for in vivo gene therapy. Unlike the mouse model of XSCID which lacks B cells, the dog model more closely resembles the phenotype in humans. We decided to use our in vivo dog model for preclinical testing of safety and efficacy of our SIV hγc vectors. Viral particles were produced by transient transfection of 293T cells with a 4-plasmid system and concentrated by high-speed centrifugation. 30 mls (average 2.4–3×107 viral particles in total) was injected IV into 2–5 day-old pups. Transgene marking in blood lymphocytes was detected as early as 2 wks after treatment, increased within the first 6–8 wks and became relatively stable thereafter. The absolute lymphocyte count was normalized in one dog (injected on the second day after birth) by wk 6 (4000, 76% hγc+), and improved in two other dogs (422, 29% hγc+ at 18 wks; 498, 27% hγc+, at 16 wks) which had received the same amount of virus over 2 days. Furthermore, up to 5% of the myeloid lineage showed gene marking at 10–18 wks after viral delivery, indicating that early committed progenitors or HSCs had been transduced. It is noteworthy that our finding that the hγc can improve the disease phenotype in XSCID dogs makes it an excellent large animal model for preclinical evaluation of vectors. In summary, we have demonstrated that in vivo delivery of SIV lentiviral vectors expressing hγc efficiently reconstitute the T-lymphoid compartment in the XSCID canine model. We plan to monitor these dogs closely for potential adverse events.

AIM: To determine if primary murine colonic epithelial cells (CEC) respond to commensal bacteria and discriminate between different types of bacteria. METHODS: A novel CEC: bacteria co-culture system was used to compare the ability of the colonic commensal bacteria, Bacteroides ovatus, E. coli (SLF) and Lactobacillus rhamnosus (LGG) to modulate production of different cytokines (n = 15) by primary CEC. Antibody staining and flow cytometry were used to investigate Toll-like receptor (TLR) expression by CEC directly ex vivo and TLR responsiveness was determined by examining the ability of TLR ligands to influence CEC cytokine production. RESULTS: Primary CEC constitutively expressed functional TLR2 and TLR4. Cultured in complete medium alone, CEC secreted IL-6, MCP-1 and IP-10 the levels of which were significantly increased upon addition of the TLR ligands peptidoglycan (PGN) and lipopolysaccharide (LPS). Exposure to the commensal bacteria induced or upregulated different patterns of cytokine production and secretion. E. coli induced production of MIP-1α/β and β defensin3 whereas B. ovatus and L. rhamnosus exclusively induced MCP-1 and MIP-2α expression, respectively. TNFα, RANTES and MEC were induced or up-regulated in response to some but not all of the bacteria whereas ENA78 and IP-10 were up-regulated in response to all bacteria. Evidence of bacterial interference and suppression of cytokine production was obtained from mixed bacterial: CEC co-cultures. Probiotic LGG suppressed E. coli- and B. ovatus-induced cytokine mRNA accumulation and protein secretion. CONCLUSION: These observations demonstrate the ability of primary CEC to respond to and discriminate between different strains of commensal bacteria and identify a mechanism by which probiotic bacteria (LGG) may exert anti-inflammatory effects in vivo.