Plasmodium Yoelii Uses the Murine Duffy Antigen Receptor for Chemokines As a Receptor for Normocyte Invasion and an Alternative Receptor for Reticulocyte Invasion

Blood. Apr, 2002  |  Pubmed ID: 11929753

Erythrocyte invasion by malaria parasites is a complex multistep process involving parasite and erythrocyte receptors. It is a critical stage in the parasite life cycle and, therefore, a logical step in which to intervene to prevent or ameliorate disease. Rodent models of malaria, commonly Plasmodium yoelii, are frequently used for studies of malaria pathogenesis. Little is known, however, about the invasion machinery of rodent malaria parasites. We have found previously that mice congenic for a region of chromosome 1, containing the Duffy antigen/receptor for chemokines (DARC), have different susceptibility to P yoelii infection. Because P vivax, a human parasite, and P knowlesi, a simian parasite, use DARC to enter human erythrocytes, we sought to identify the role of the murine DARC in P yoelii invasion. Using a novel in vivo invasion assay and DARC knock-out mice, we found that DARC knock-out normocytes (mature erythrocytes) had negligible levels of P yoelii invasion compared with wild-type normocytes, demonstrating that DARC is a receptor for invasion of murine erythrocytes. In contrast, DARC knock-out reticulocytes were invaded at a rate similar to that for wild-type reticulocytes. We conclude that there is a DARC- independent pathway for reticulocyte invasion. These findings represent the first identification of a murine malaria receptor on erythrocytes and the first determination that different pathways of invasion exist on normocytes and reticulocytes. Because we show conservation of host-receptor interactions between rodent and human malaria, we can now use this model to identify how immunity can interfere with the invasion process.

Antigen-presenting Cell Function During Plasmodium Yoelii Infection

Infection and Immunity. Jun, 2002  |  Pubmed ID: 12010983

Antigen-presenting cells (APC) play a key role in orchestrating immune responses. T-cell proliferative responses are inhibited during the erythrocyte stages of malaria infection, and a number of studies have suggested that APC are responsible for this phenomenon. In the present studies we examine individual components of the T-cell-activating function of APC: expression of costimulatory and major histocompatibility complex (MHC) class II proteins, the ability to process and present antigen to T cells, and the ability to support cytokine production. We find that during the acute phases of Plasmodium yoelii erythrocyte stage infection, APC upregulate the expression of class II MHC and CD80, maintain expression of CD86, process and present antigen, and support gamma interferon production. However the CD11b(+) subpopulation produces a soluble factor or factors that specifically inhibit interleukin-2 (IL-2) production by responding CD4 T cells. This factor is distinct from prostaglandin E(2), NO, or transforming growth factor beta. The data suggest that IL-2 suppression observed during malaria infection is not due to functional defects of APC but is triggered by production of a factor(s) that actively suppresses production of IL-2 by T cells.

Use of the Cell-Dyn 3500 to Predict Leukemic Cell Lineage in Peripheral Blood of Dogs and Cats

Veterinary Clinical Pathology / American Society for Veterinary Clinical Pathology. 2002  |  Pubmed ID: 12447779

Morphology and cytochemistry are the foundation for classification of leukemias in dogs and cats. Advances in automated hematology instrumentation, immunophenotyping, cytogenetics, and molecular biology are significantly improving our ability to recognize and classify spontaneous myeloproliferative and lymphoproliferative disorders.

Dendritic Cells from Malaria-infected Mice Are Fully Functional APC

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2004  |  Pubmed ID: 14688357

Malaria infection has long been associated with diminished T cell responses in vitro and more recently in experimental studies in vivo. Suppression of T cell-proliferative responses during malaria has been attributed to macrophages in a variety of murine and human systems. More recently, however, attention has been directed at the role of dendritic cells in this phenomenon, with several studies suggesting that maturation of dendritic cells is inhibited in vitro by the presence of malaria-infected E. In the studies reported here, we have examined the function of dendritic cells taken directly from infected mice. We found that they express high levels of costimulatory proteins and class II MHC, can activate naive T cells to produce IL-2 as efficiently as dendritic cells from uninfected mice, and support high levels of IFN-gamma production by naive T cells through an IL-12-dependent mechanism. Dendritic cells from infected mice also support higher levels of TNF-alpha production by naive T cells. These same dendritic cells present parasite Ag to a malaria-specific T cell hybridoma, a finding that demonstrates that dendritic cells participate in the generation of Ag-specific immunity during infection. Our findings challenge the contention that dendritic cell function is inhibited by malaria infection.

Detection of Neoplastic Lymphocytes in Peripheral Blood of Dogs with Lymphoma by Polymerase Chain Reaction for Antigen Receptor Gene Rearrangement

Veterinary Clinical Pathology / American Society for Veterinary Clinical Pathology. 2004  |  Pubmed ID: 15334349

Uniquely rearranged immunoglobulin and T-cell receptor gene sequences can be amplified and electrophoretically separated by size to detect a clonal population of lymphocytes.

Evolution of a B-cell Lymphoma to Multiple Myeloma After Chemotherapy

Journal of Veterinary Internal Medicine / American College of Veterinary Internal Medicine. Sep-Oct, 2004  |  Pubmed ID: 15515600

Molecular Methods to Distinguish Reactive and Neoplastic Lymphocyte Expansions and Their Importance in Transitional Neoplastic States

Veterinary Clinical Pathology / American Society for Veterinary Clinical Pathology. 2004  |  Pubmed ID: 15570556

Although lymphoma and leukemia usually can be diagnosed by routine cytology and histology, some cases present a diagnostic challenge for pathologists and clinicians. Often the dilemma lies in determining whether a population of lymphocytes is reactive or neoplastic. We review currently available methods for analyzing lymphocyte populations by immunophenotyping and by identifying clonally rearranged immunoglobulin and T-cell receptor genes and discuss how these tests can be used to clarify such diagnostic dilemmas. We also describe the detection of chromosomal abnormalities and methods on the horizon, such as gene expression profiling, to identify diagnostically useful oncogenes. Finally, we review the emerging concept of transitional neoplastic states, in which reactive lymphocytes transform to neoplastic lymphocytes in the presence of continued antigenic stimulation, such as that caused by infection with Helicobacter pylori. The existence of transitional neoplastic states underscores the need for an array of molecular diagnostic tools that would improve our ability to characterize lymphocyte populations in human and animal patients and enhance early detection of neoplastic lymphocytes such that eradication of the infectious or inflammatory stimulus could lead to cure.

Environmental Enrichment for Laboratory Rodents

ILAR Journal / National Research Council, Institute of Laboratory Animal Resources. 2005  |  Pubmed ID: 15775024

Modernization of housing and husbandry techniques for rodents has minimized confounding variables. The result has been vastly improved health maintenance and reproducibility of research findings, advances that have decreased the numbers of animals needed to attain statistically significant results. Even though not all aspects of rodent manipulation have been strictly defined, as housing and handling procedures have become increasingly standardized, many animal care personnel have recognized the lack of complexity of the rodents' environment. Concern for this aspect of animal well-being has led many research facilities to provide "environmental enrichment" for rodents. Additionally, regulatory agencies in the United States and Europe have also been increasingly concerned about this issue relative to laboratory animal husbandry. However, little is known about the influence such husbandry modifications may have on biological parameters. In this article, laws and guidelines relating to rodent enrichment are reviewed, the natural behaviors of select rodent species are discussed, and an overview of widely used types of enrichment in laboratory rodent management is provided. The literature evaluating effects of rodent enrichment is reviewed both in terms of neurological development and as an experimental variable, and results of a study evaluating the effect of enrichment on immune and physiological parameters are reported. Survey data on current enrichment practices in a large multi-institutional organization are presented, and practical aspects requiring consideration when devising a rodent enrichment program are discussed.

Cutting Edge: the Acquisition of TLR Tolerance During Malaria Infection Impacts T Cell Activation

Journal of Immunology (Baltimore, Md. : 1950). May, 2005  |  Pubmed ID: 15879082

An effective immune response to infection requires control of pathogen growth while minimizing inflammation-associated pathology. During malaria infection, this balance is particularly important. Murine malaria is characterized by early production of proinflammatory cytokines, which declines in the face of continuing parasitemia. The mechanism by which this occurs remains poorly understood. In this study, we investigated the role of dendritic cells (DCs) in regulating pro- and anti-inflammatory cytokine responses. As malaria infection progresses, DCs become refractory to TLR-mediated IL-12 and TNF-alpha production, while increasing their ability to produce IL-10 and retaining the capacity for activation of naive T cells. IL-12-secreting DCs from early infection stimulate an IFN-gamma-dominated T cell response, whereas IL-10-secreting DCs from later stages induce an IL-10-dominated T cell response. We suggest that phenotypic changes in DCs during Plasmodium yoelii infection represent a mechanism of controlling host inflammation while maintaining effective adaptive immunity.

Distinct B-cell and T-cell Lymphoproliferative Disease Prevalence Among Dog Breeds Indicates Heritable Risk

Cancer Research. Jul, 2005  |  Pubmed ID: 15994938

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor gamma chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using chi2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.

Utility of Polymerase Chain Reaction for Analysis of Antigen Receptor Rearrangement in Staging and Predicting Prognosis in Dogs with Lymphoma

Journal of Veterinary Internal Medicine / American College of Veterinary Internal Medicine. Mar-Apr, 2006  |  Pubmed ID: 16594590

In lymphoid neoplasia, molecular assays to confirm clonality rely on the fact that lymphoid cells normally contain DNA regions with unique sequences, resulting from recombination of the V, D, and J genes. The purpose of this study was to determine the utility of polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) for molecular staging and predicting prognosis in canine lymphoma. We hypothesized that the PARR assay would offer a sensitive method for detecting neoplastic cells in blood, and that the presence of such cells would be a negative prognostic finding compared with dogs with no detectable circulating tumor cells. Eighty-six patients with histologically or cytologically confirmed lymphoma were studied from initial diagnosis until death or euthanasia. All patients had PARR assays of a representative tumor-infiltrated lymph node and peripheral whole blood. Sixty-two patients had clonal rearrangements detected in the lymph node and were able to be staged by PARR. Seventeen patients (27%) had no detectable tumor in their blood and 45 (73%) were blood positive. Our findings showed that (1) PARR correlated with clinical stage in that the PARR assay was more likely to detect tumor cells in blood in stage 5 lymphomas, (2) PARR was more sensitive for detecting circulating tumor cells than visual assessment of blood or bone marrow because 80% of stage 3 lymphomas were blood-PARR-positive, and (3) PCR stage was not prognostic for disease-free interval (DFI) or survival.

Diagnosis of Mediastinal Masses in Dogs by Flow Cytometry

Journal of Veterinary Internal Medicine / American College of Veterinary Internal Medicine. Sep-Oct, 2006  |  Pubmed ID: 17063710

Biopsy of mediastinal masses can be invasive, but the procedure may be necessary if cytology of a mass aspirate is inconclusive. The 2 most common mediastinal masses, lymphoma and thymoma, may both be comprised of small lymphocytes. We investigated the ability of flow cytometry to distinguish between these 2 neoplasms.

Proteomics: a Review and an Example Using the Reticulocyte Membrane Proteome

Veterinary Clinical Pathology / American Society for Veterinary Clinical Pathology. Mar, 2007  |  Pubmed ID: 17311189

Proteomics is a rapidly expanding field of scientific study that combines techniques in protein solubilization and separation, mass spectrometry, and genome and protein database searching. The proteome is most commonly defined as the entire complement of proteins expressed in a given cell type or tissue under a given condition. A proteomics experiment may be as simple as identifying a single protein or as complex as identifying thousands of proteins in a cell lysate. In this review, we describe the general principles of proteomics and its analytic methods and present an example of an experiment to characterize the murine reticulocyte membrane proteome. A brief summary of proteomics applications and their clinical potential and relevance to clinical pathology is also presented.

Determining the Significance of Persistent Lymphocytosis

The Veterinary Clinics of North America. Small Animal Practice. Mar, 2007  |  Pubmed ID: 17336675

The authors provide a review of current knowledge of lymphocytosis in nonneoplastic conditions. They conclude that the list of major differentials for persistent nonneoplastic lymphocyte expansion in dogs and cats is short and that most of these conditions are relatively uncommon. Persistent lymphocytosis of small, mature, or reactive lymphocytes is most commonly the result of chronic lymphocytic leukemia or lymphoma. The first step in distinguishing nonneoplastic from neoplastic lymphocytosis is immunophenotyping by flow cytometry to determine the phenotypic diversity of the circulating cells. Clonality testing using the polymerase chain reaction for antigen receptor rearrangements assay is a useful second step in cases in which the phenotype data are equivocal. Once the diagnosis of malignancy has been established, the immunophenotype also provides prognostic information in dogs.

A Novel Canine Lymphoma Cell Line: a Translational and Comparative Model for Lymphoma Research

Leukemia Research. Dec, 2007  |  Pubmed ID: 17532464

A novel canine lymphoma cell line, OSW, was established from the malignant pleural effusion of a dog with peripheral T-cell lymphoma. The immunoprofile as determined by flow cytometry was as follows: positive for CD45, CD49d, CD18, CD11a; weakly positive for CD11b, CD11c, CD11d; and negative for CD45RA, CD1a, CD1c, CD3, TCRalphabeta, TCRgammadelta, CD4, CD5, CD8a, CD8b, CD90(Thy1), CD21, MHCII, CD14(TUK4), CD34, and MPO. Immunocytochemistry of cytospin preparations was negative for cytoplasmic CD3, CD79a, and MPO, but was positive for CD20. The cell line had an oligoclonal T-cell receptor gamma (TCRgamma) gene rearrangement. Array comparative genomic hybridization (aCGH) and single locus probe (SLP) analysis showed that there were copy number increases of loci on dog chromosome 13 (CFA 13), and copy number decreases were evident for regions of CFA 11, 22, 26, 30 and 32, which include several of the more common chromosomal aberrations reported previously in canine lymphoma. The OSW cell line grows rapidly in vitro and is tumorigenic as a xenograft in SCID/NOD mice. OSW represents one of only a few reported canine lymphoma cell lines and is the most thoroughly characterized. This cell line and xenograft represent significant in vitro and in vivo models, respectively, for comparative and translational lymphoma research.

Dendritic Cell Leukemia in a Golden Retriever

Veterinary Clinical Pathology / American Society for Veterinary Clinical Pathology. Jun, 2008  |  Pubmed ID: 18533919

An 8-year-old castrated male Golden Retriever was evaluated for decreased appetite, lethargy, and labored breathing of 1-week duration. Bilateral pulmonary infiltrates, hepatomegaly, and splenomegaly were present. Results of a CBC revealed marked leukocytosis (62,600/microL; reference interval 4000-15,500/microL) and large numbers of atypical cells (30,700/microL) with abundant cytoplasm. There was no concurrent anemia, neutropenia, or thrombocytopenia. Morphology of the atypical cells was most consistent with a histiocytic origin. Similar cells were identified in bone marrow aspirates, and were morphologically suggestive of the macrophage variant of disseminated histiocytic sarcoma. However, flow cytometry of the abnormal circulating cells revealed CD1c, CD11c, and major histocompatibility complex (MHC) Class II expression without expression of CD11d or lymphoid markers, consistent with myeloid dendritic antigen-presenting cells. At necropsy, the splenic architecture was effaced by neoplastic histiocytes that were also infiltrating lung, liver, an abdominal lymph node, myocardium, an bone marrow. Immunohistochemistry of the splenic neoplastic cells confirmed dendritic cell origin (CD1c+, CD11c+, MHC II+, no expression of CD11d and lymphoid markers). To the authors' knowledge, this is the first report of canine dendritic cell leukemia-in this instance accompanied by marked tissue infiltration.

Molecular Diagnostics of Hematologic Malignancies

Topics in Companion Animal Medicine. Aug, 2009  |  Pubmed ID: 19732733

Hematologic malignancies, particularly lymphoma and leukemia, are a diverse group of diseases with a myriad of different presentations. Although the diagnosis of these diseases can be straightforward, there are many cases in which the diagnosis is difficult to establish with conventional methods. Molecular diagnostic testing to identify oncogenes and clonal lymphocyte populations can aid in resolving ambiguous cases, and several of these tests are routinely available for canine patients. Sensitive polymerase chain reaction-based methods are also useful for answering a variety of research questions. Here, detection of mutations of the c-kit gene in mast cell tumors, the bcr-abl fusion gene in myelogenous leukemias, and clonality of lymphocyte populations for the diagnosis and monitoring of lymphoma and leukemia are discussed.

Monoclonal Gammopathy Without Hyperglobulinemia in 2 Dogs with IgA Secretory Neoplasms

Veterinary Clinical Pathology / American Society for Veterinary Clinical Pathology. Dec, 2010  |  Pubmed ID: 20969607

Two dogs, an 8.5-year-old intact male Golden Retriever and a 10-year-old spayed female English Springer Spaniel, each with varied clinical histories, were referred to the Colorado State University Veterinary Teaching Hospital for evaluation of hypercalcemia and severe anemia, respectively. In each dog, serum total protein and globulin concentrations were within reference intervals. Cytologic examination of bone marrow aspirates from both dogs revealed moderate to marked numbers of atypical lymphoid cells with plasma cell features. Using serum immunofixation and serum immunoglobulin (Ig) quantification, a monoclonal Ig protein was identified. In conjunction with other clinicopathologic and molecular findings, IgA secretory neoplasms, B-cell lymphoma with plasmacytoid features and multiple myeloma (MM), were diagnosed. To our knowledge, these cases represent the first descriptions of IgA-secreting neoplasms in dogs that lacked hyperglobulinemia. In cases of suspected B-cell lymphoma or MM in dogs, serum proteins should be fully evaluated for the presence of a monoclonal Ig even in dogs that lack characteristic hyperproteinemia or hyperglobulinemia. This evaluation will aid in the diagnosis of secretory B-cell lymphoma or MM leading to appropriate clinical and therapeutic case management.

Cloning and Tissue Expression of the Equine Transferrin Receptor

Veterinary Clinical Pathology / American Society for Veterinary Clinical Pathology. Dec, 2010  |  Pubmed ID: 21070303

Characterization of anemia in horses presents a challenge, as they do not release reticulocytes into peripheral blood. Transferrin receptor (TfR) expression is highest on erythroid cells in people and rats, and measurement of a soluble serum form (sTfR) is used to quantify erythropoiesis in these species. We hypothesized that equine TfR (eTfR) expression is similar in quantity and distribution to that in these other species and thus has potential for characterization of the regenerative response in anemic horses.

A Novel Unstable Duplication Upstream of HAS2 Predisposes to a Breed-defining Skin Phenotype and a Periodic Fever Syndrome in Chinese Shar-Pei Dogs

PLoS Genetics. Mar, 2011  |  Pubmed ID: 21437276

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (p(raw) = 2.3 × 10⁻⁶, p(genome) = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p < 0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.

Monoclonal Immunoglobulin Protein Production in Two Dogs with Secretory B-cell Lymphoma with Mott Cell Differentiation

Journal of the American Veterinary Medical Association. Dec, 2011  |  Pubmed ID: 22087724

A 9-year-old castrated male mixed-breed dog and a 7-year-old spayed female Boston Terrier, with clinical histories of a liver mass (dog 1) and bloody vomitus, diarrhea, and weight loss (dog 2), respectively, were referred for further evaluation.

CD40 Ligand is Necessary and Sufficient to Support Primary Diffuse Large B-cell Lymphoma Cells in Culture: a Tool for in Vitro Preclinical Studies with Primary B-cell Malignancies

Leukemia & Lymphoma. Feb, 2012  |  Pubmed ID: 22229753

Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development. However, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude that this system has broad applications for in vitro preclinical development for B-cell malignancies.

Molecular Diagnostics of Hematologic Malignancies in Small Animals

The Veterinary Clinics of North America. Small Animal Practice. Jan, 2012  |  Pubmed ID: 22285160

Environmental Enrichment During Rearing Alters Corticosterone Levels, Thymocyte Numbers, and Aggression in Female BALB/c Mice

Journal of the American Association for Laboratory Animal Science : JAALAS. 2012  |  Pubmed ID: 22330863

The goal of environmental enrichment for laboratory animals is to improve welfare, but some enrichment practices may affect research in unintended ways or even be harmful to the animals themselves. We previously found that mice raised at a commercial vendor then given multiple enrichment devices upon arrival at our facilities experienced thymic atrophy and greater variation in measured parameters than did their unenriched counterparts, suggesting that enrichment conditions affected corticosteroid expression in mice. The current study verified and expanded these results, examining 120 female BALB/c mice raised with or without nesting material at a commercial vendor (n = 60 per group) and allocated (n = 20 per group) to receive no enrichment, nesting material, or 'superenrichment' on arrival at our facilities. Nesting material provided prior to weaning was associated with higher levels of urinary corticosteroid, whereas superenrichment and nesting material during the adult period both led to increased thymic atrophy. Paradoxically, mice that never received enrichment, despite having the lowest corticosterone levels and least thymic atrophy, had increased tail wounds resulting from aggressive interactions. Therefore, enrichment devices that are as seemingly innocuous as nesting material, even if only provided in the preweaning period, may lead to significant, lasting changes in behavioral, physical, or immunologic measures with the potential to alter research outcomes.