Elevated serum gamma-glutamyltransferase activity and immunohistochemistry in two dogs with renal carcinoma.

During a 3-year time period, a 15-year-old male castrated Terrier mix (dog 1) and a 6-year-old female spayed Labrador Retriever (dog 2) presented to the North Carolina State Veterinary Hospital with similar blood work abnormalities and no significant physical examination findings. A CBC, chemistry panel, and urinalysis performed on both dogs were relatively unremarkable, other than a marked increase in serum gamma-glutamyltransferase (GGT) activity. Through imaging, both patients were diagnosed with a renal mass, and histopathology of both masses revealed a carcinoma. Immunohistochemical staining of the renal mass in both dog 1 and dog 2 were intensely positive for GGT. Dog 1 had the affected kidney removed, which normalized the GGT value. Dog 2 was euthanized, and metastasis to the lung was noted upon postmortem examination. There have been limited case studies documenting an elevation in serum GGT in dogs diagnosed with renal carcinoma. While renal carcinoma is uncommon in dogs, it is an important differential to keep in mind when there is a marked increase in serum GGT without accompanying increases in other measured liver enzymes. In addition, serum GGT can serve as a helpful biomarker for disease resolution and recurrence, as surgical removal of the renal mass (dog 1) led to the resolution of the elevated serum GGT. To our knowledge, this is the first report demonstrating IHC staining for GGT in a canine renal carcinoma.

Doxorubicin for treatment of histiocytic sarcoma in dogs: 31 cases (2003-2017).

OBJECTIVE: To evaluate the efficacy of doxorubicin for treatment of histiocytic sarcoma (HS) in dogs, whether administered as the sole treatment or as an adjunct to surgery or radiation therapy. ANIMALS: 31 client-owned dogs with localized or disseminated HS examined between 2003 and 2017. PROCEDURES: Medical records were reviewed retrospectively, and data were collected. The Kaplan-Meier method was used to estimate time-to-progression from the date of first doxorubicin administration and survival time from initial diagnosis. Factors that could be associated with poorer outcomes with doxorubicin treatment were analyzed with log-rank tests. RESULTS: The objective response rate (ORR) was 26%. When stratified by disease status, dogs with localized and disseminated forms experienced 43% and 21% ORRs, respectively. Median time to progression after initiating doxorubicin treatment (n = 30 dogs) was 42 days. Median survival time from initial diagnosis to death (n = 29 dogs) was 169 days. Complete responses were obtained in only 2 dogs that had localized disease and received multimodality therapy. CLINICAL RELEVANCE: Benefits of doxorubicin administration in canine HS are modest, with a limited ORR and delay in tumor progression, and are comparable to effects attained with other single-agent regimens.

Dog leukocyte antigen-88*034:01 presents nonamer peptides from canine distemper virus hemagglutinin, large polymerase, and matrix proteins.

Canine spontaneous cancers may offer greater fidelity than rodent models in advancing clinical immunotherapies. Boxers in particular are distinguished as study subjects by their popularity, and high incidence of human-relevant cancers. Further, the MHC class I allele DLA-88*034:01, with a known motif, dominates the breed, facilitating discovery of shared CTL responses against mutation-origin neoepitopes by standard prediction methods. We experimentally confirmed the allomorph's binding motif by developing an MHC surface stabilization assay. The assay validated four DLA-88*034:01-presented peptides from canine distemper virus, ubiquitously administered in routine vaccines, for positive controls in future CTL studies. In turn, these viral peptides substantiated motif-based prediction for DLA-88*034:01. The study adds new tools for studying neoepitope-specific CTL in Boxers to foster canine comparative oncology.

Cancer-testis antigens in canine histiocytic sarcoma and other malignancies.

Cancer-testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies, mostly solid tumours, due to epigenetic de-repression. Normally expressed only in fetal or gametogenic tissues, CTAs are tantalizing immunotherapy targets, since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers, and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this, the ligandome binding the dog leukocyte antigen (DLA)-88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA-derived peptides, which could serve as CD8+ T-cell epitopes. Twenty-two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumour and normal tissue by quantitative and end-point RT-PCR. The ortholog of an established CTA, IGF2BP3, had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis-enhanced proteins were also assessed. AKR1E2, SPECC1 and TPX2 were expressed variably in HS and T-cell lymphoma biopsies, but also at high levels in critical tissues, including kidney, brain and marrow, diminishing their utility. A more tissue-restricted candidate, NT5C1B, was detected in T-cell lymphomas, but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach, proceeding from identification of MHC class I-presented peptides to a comparative RNA expression survey of tumours and normal tissues.

Incidence and risk factors associated with development of clinical cardiotoxicity in dogs receiving doxorubicin.

BACKGROUND: Doxorubicin (DOX) can cause cumulative cardiotoxicity in dogs, but the incidence of clinical cardiotoxicity in dogs receiving DOX has not been determined. HYPOTHESIS/OBJECTIVES: To determine if the duration of DOX infusion influences the incidence of cardiotoxicity, to characterize the incidence of clinical cardiotoxicity in dogs during or after DOX chemotherapy, and to identify any risk factors associated with cardiotoxicity. ANIMALS: Four-hundred ninety-four dogs that received at least 1 dose of DOX for the treatment of cancer. METHODS: Retrospective study of dogs that received DOX from 2006 to 2015. RESULTS: Of 494 dogs, 20 (4.0%) developed clinical cardiotoxicity. The duration of DOX infusion was not significantly associated with clinical cardiotoxicity, whereas a higher cumulative dose of DOX, higher body weight, decreases in fractional shortening after 5 doses of DOX, and development of ventricular premature contractions were significantly associated with clinical cardiotoxicity. High-risk breeds for developing dilated cardiomyopathy had an incidence of 15.4%, whereas low-risk breeds had an incidence of 3.0%. CONCLUSIONS AND CLINICAL IMPORTANCE: Although the duration of DOX infusion did not influence the incidence of cardiotoxicity, premature contractions and decreases in fractional shortening should raise concern for the development of clinical cardiotoxicity. Overall, the incidence of clinical DOX-induced cardiotoxicity is low, but Boxers and other breeds at high risk for dilated cardiomyopathy may be at an increased risk.

The prevalent Boxer MHC class Ia allotype dog leukocyte antigen (DLA)-88*034:01 preferentially binds nonamer peptides with a defined motif.

Development of effective immunotherapy for chemoresistant malignancies can be advanced by studies in spontaneous cancer models, such as the dog. A crucial first step, T-cell epitope discovery, can be assisted by determination of binding motifs of common dog leukocyte antigen (DLA) class Ia allotypes. Boxers are popular, inbred dogs with increased risks of relevant target cancers and restricted MHC diversity. We sought to identify the motif of DLA-88*034:01, a breed-dominant allotype, to assist peptide prediction from tumor antigens. Mass spectrometry of eluted peptides showed a preference for nonamers with conserved amino acid preferences: basic at position (P)1; hydrophobic at P2; acidic at P4; histidine at P6; and phenylalanine at P9. This data should expedite finding epitopes restricted by this DLA-88 allotype.

JAK2 V617F-positive acute myeloid leukaemia (AML): a comparison between de novo AML and secondary AML transformed from an underlying myeloproliferative neoplasm. A study from the Bone Marrow Pathology Group.

The JAK2 V617F mutation is characteristic of most Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and occurs rarely in de novo acute myeloid leukaemia (AML). We sought to characterize AMLs that harbour this mutation and distinguish those that arise de novo (AML-DN) from those that reflect transformation of an underlying MPN (AML-MPN). Forty-five patients with JAK2 V617F-mutated AML were identified; 15 were AML-DN and 30 were AML-MPN. AML-MPN cases were more likely to have splenomegaly (P = 0·02), MPN-like megakaryocytes and higher mean JAK2 V617F VAF at diagnosis (P = 0·04). Mutations involving TET2 were exclusively identified in AML-DN patients. Mutations of genes affecting DNA methylation were more common in AML-DN (P < 0·01). A complex karyotype was more frequent in AML-MPN cases than in AML-DN (P < 0·01), with AML-DN more likely to display a normal karyotype (P = 0·02). Bone marrow histology after recovery from induction chemotherapy in AML-DN cases revealed no morphological evidence of any previously occult MPNs, while this was evident in most of the AML-MPN specimens (P < 0·01). These findings in this largest study of JAK2 V617F-mutated AMLs indicate that AML-DN is distinct from AML-MPN.

The canine MHC class Ia allele DLA-88*508:01 presents diverse self- and canine distemper virus-origin peptides of varying length that have a conserved binding motif.

Ideally, CD8+ T-cell responses against virally infected or malignant cells are defined at the level of the specific peptide and restricting MHC class I element, a determination not yet made in the dog. To advance the discovery of canine CTL epitopes, we sought to determine whether a putative classical MHC class Ia gene, Dog Leukocyte Antigen (DLA)-88, presents peptides from a viral pathogen, canine distemper virus (CDV). To investigate this possibility, DLA-88*508:01, an allele prevalent in Golden Retrievers, was expressed as a FLAG-tagged construct in canine histiocytic cells to allow affinity purification of peptide-DLA-88 complexes and subsequent elution of bound peptides. Pattern analysis of self peptide sequences, which were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), permitted binding preferences to be inferred. DLA-88*508:01 binds peptides that are 9-to-12 amino acids in length, with a modest preference for 9- and 11-mers. Hydrophobic residues are favored at positions 2 and 3, as are K, R or F residues at the C-terminus. Testing motif-matched and -unmatched synthetic peptides via peptide-MHC surface stabilization assay using a DLA-88*508:01-transfected, TAP-deficient RMA-S line supported these conclusions. With CDV infection, 22 viral peptides ranging from 9-to-12 residues in length were identified in DLA-88*508:01 eluates by LC-MS/MS. Combined motif analysis and surface stabilization assay data suggested that 11 of these 22 peptides, derived from CDV hemagglutinin, large polymerase, matrix, nucleocapsid, and V proteins, were processed and presented, and thus, potential targets of anti-viral CTL in DLA-88*508:01-bearing dogs. The presentation of diverse self and viral peptides indicates that DLA-88 is a classical MHC class Ia gene.

Canine scent detection of canine cancer: a feasibility study.

The scent detection prowess of dogs has prompted interest in their ability to detect cancer. The purpose of this study was to determine whether dogs could use olfactory cues to discriminate urine samples collected from dogs that did or did not have urinary tract transitional cell carcinoma (TCC), at a rate greater than chance. Dogs with previous scent training (n=4) were initially trained to distinguish between a single control and a single TCC-positive urine sample. All dogs acquired this task (mean =15±7.9 sessions; 20 trials/session). The next training phase used four additional control urine samples (n=5) while maintaining the one original TCC-positive urine sample. All dogs quickly acquired this task (mean =5.3±1.5 sessions). The last training phase used multiple control (n=4) and TCC-positive (n=6) urine samples to pro-mote categorical training by the dogs. Only one dog was able to correctly distinguish multiple combinations of TCC-positive and control urine samples suggesting that it mastered categorical learning. The final study phase evaluated whether this dog would generalize this behavior to novel urine samples. However, during double-blind tests using two novel TCC-positive and six novel TCC-negative urine samples, this dog did not indicate canine TCC-positive cancer samples more frequently than expected by chance. Our study illustrates the need to consider canine olfactory memory and the use of double-blind methods to avoid erroneous conclusions regarding the ability of dogs to alert on specimens from canine cancer patients. Our results also suggest that sample storage, confounding odors, and other factors need to be considered in the design of future studies that evaluate the detection of canine cancers by scent detection dogs.

Lymphatic function is required prenatally for lung inflation at birth.

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants.

Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance.

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.

Characterization and allelic variation of the transporters associated with antigen processing (TAP) genes in the domestic dog (Canis lupus familiaris).

The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8(+) T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs.

Comparison of automated versus manual neutrophil counts for the detection of cellular abnormalities in dogs receiving chemotherapy: 50 cases (May to June 2008).

OBJECTIVE: To determine the frequency of clinically relevant abnormalities missed by failure to perform a blood smear evaluation in a specific subset of dogs receiving chemotherapy and to compare automated and manual neutrophil counts in the same population. DESIGN: Retrospective case series. ANIMALS: 50 dogs receiving chemotherapy with a total nucleated cell count > 4,000 nucleated cells/µL. PROCEDURES: 50 blood smears were evaluated for abnormalities that have strong potential to change the medical plan for a patient: presence of blast cells, band neutrophils, nucleated RBCs, toxic change, hemoparasites, schistocytes, and spherocytes. Automated and manual neutrophil counts were compared. RESULTS: Blood smears from 10 (20%) patients had ≥ 1 abnormalities. Blast cells were identified on 4 (8%) blood smears, increased nucleated RBCs were identified on 5 (10%), and very mild toxic change was identified on 2 (4%). Correlation coefficient of the neutrophil counts was 0.96. Analysis revealed a slight bias between the automated and manual neutrophil counts (mean ± SD difference, -0.43 × 10(3)/µL ± 1.10 × 10(3)/µL). CONCLUSIONS AND CLINICAL RELEVANCE: In this series of patients, neutrophil count correlation was very good. Clinically relevant abnormalities were found on 20% of the blood smears. An automated CBC appears to be accurate for neutrophil counts, but a microscopic examination of the corresponding blood smear is still recommended; further studies are needed to determine whether the detection or frequency of these abnormalities would differ dependent on chemotherapy protocol, neoplastic disease, and decision thresholds used by the oncologist in the ordering of a CBC without a blood smear evaluation.

TAK1 (MAP3K7) signaling regulates hematopoietic stem cells through TNF-dependent and -independent mechanisms.

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1(+), c-Kit(+) (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.

A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.

Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine ß2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species.

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

The use of peptide-major-histocompatibility-complex multimers in type 1 diabetes mellitus.

Major histocompatibility complex (MHC) class I and MHC class II molecules present short peptides that are derived from endogenous and exogenous proteins, respectively, to cognate T-cell receptors (TCRs) on the surface of T cells. The exquisite specificity with which T cells recognize particular peptide-major-histocompatibility-complex (pMHC) combinations has permitted development of soluble pMHC multimers that bind exclusively to selected T-cell populations. Because the pathogenesis of type 1 diabetes mellitus (T1DM) is driven largely by islet-reactive T-cell activity that causes ß-cell death, these reagents are useful tools for studying and, potentially, for treating this disease. When coupled to fluorophores or paramagnetic nanoparticles, pMHC multimers have been used to visualize the expansion and islet invasion of T-cell effectors during diabetogenesis. Administration of pMHC multimers to mice has been shown to modulate T-cell responses by signaling through the TCR or by delivering a toxic moiety that deletes the targeted T cell. In the nonobese diabetic mouse model of T1DM, a pMHC-I tetramer coupled to a potent ribosome-inactivating toxin caused long-term elimination of a specific diabetogenic cluster of differentiation 8+ T-cell population from the pancreatic islets and delayed the onset of diabetes. This review will provide an overview of the development and use of pMHC multimers, particularly in T1DM, and describe the therapeutic promise these reagents have as an antigen-specific means of ameliorating deleterious T-cell responses in this autoimmune disease.

Making the most of major histocompatibility complex molecule multimers: applications in type 1 diabetes.

Classical major histocompatibility complex (MHC) class I and II molecules present peptides to cognate T-cell receptors on the surface of T lymphocytes. The specificity with which T cells recognize peptide-MHC (pMHC) complexes has allowed for the utilization of recombinant, multimeric pMHC ligands for the study of minute antigen-specific T-cell populations. In type 1 diabetes (T1D), CD8+ cytotoxic T lymphocytes, in conjunction with CD4+ T helper cells, destroy the insulin-producing ß cells within the pancreatic islets of Langerhans. Due to the importance of T cells in the progression of T1D, the ability to monitor and therapeutically target diabetogenic clonotypes of T cells provides a critical tool that could result in the amelioration of the disease. By administering pMHC multimers coupled to fluorophores, nanoparticles, or toxic moieties, researchers have demonstrated the ability to enumerate, track, and delete diabetogenic T-cell clonotypes that are, at least in part, responsible for insulitis; some studies even delay or prevent diabetes onset in the murine model of T1D. This paper will provide a brief overview of pMHC multimer usage in defining the role T-cell subsets play in T1D etiology and the therapeutic potential of pMHC for antigen-specific identification and modulation of diabetogenic T cells.

Safety and efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor.

OBJECTIVE: To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs. ANIMALS: 111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved. PROCEDURES: 58 dogs received an initial series of 4 injections of huTyr vaccine (102 µg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death. RESULTS: Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM. IMPACT FOR HUMAN MEDICINE: Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Delivery of progenitors to the thymus limits T-lineage reconstitution after bone marrow transplantation.

T-cell production depends on the recruitment of hematopoietic progenitors into the thymus. T cells are among the last of the hematopoietic lineages to recover after bone marrow transplantation (BMT), but the reasons for this delay are not well understood. Under normal physiologic conditions, thymic settling is selective and either CCR7 or CCR9 is required for progenitor access into the thymus. The mechanisms of early thymic reconstitution after BMT, however, are unknown. Here we report that thymic settling is briefly CCR7/CCR9-independent after BMT but continues to rely on the selectin ligand PSGL-1. The CCR7/CCR9 independence is transient, and by 3 weeks after BMT these receptors are again strictly required. Despite the normalization of thymic settling signals, the rare bone marrow progenitors that can efficiently repopulate the thymus are poorly reconstituted for at least 4 weeks after BMT. Consistent with reduced progenitor input to the thymus, intrathymic progenitor niches remain unsaturated for at least 10 weeks after BMT. Finally, we show that thymic recovery is limited by the number of progenitors entering the thymus after BMT. Hence, T-lineage reconstitution after BMT is limited by progenitor supply to the thymus.