Christine S. Olver

Cutting Edge: The Acquisition of TLR Tolerance during Malaria Infection Impacts T Cell Activation

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-α 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-γ-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.

Dendritic Cells from Malaria-Infected Mice Are Fully Functional APC

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-γ production by naive T cells through an IL-12-dependent mechanism. Dendritic cells from infected mice also support higher levels of TNF-α 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.

Proteomic analysis of secreted exosomes

This chapter focuses on the contribution of proteomic analysis to the understanding of the process of exosome secretion and the mechanism and function of exosomes. It also describes the potential of exosome proteomic analysis to aid in the development of exosomes for therapeutic use.

Plasmodium yoelii 17XL infection up-regulates RANTES, CCR1, CCR3 and CCR5 expression, and induces ultrastructural changes in the cerebellum

BackgroundMalaria afflicts 300–500 million people causing over 1 million deaths globally per year. The immunopathogenesis of malaria is mediated partly by co mplex cellular and immunomodulator interactions involving co-regulators such as cytokines and adhesion molecules. However, the role of chemokines and their receptors in malaria immunopathology remains unclear. RANTES (Regulated on Activation Normal T-Cell Expressed and Secreted) is a chemokine involved in the generation of inflammatory infiltrates. Recent studies indicate that the degradation of cell-cell junctions, blood-brain barrier dysfunction, recruitment of leukocytes and Plasmodium-infected erythrocytes into and occlusion of microvessels relevant to malaria pathogenesis are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, thus suggesting a unique role for RANTES in the generation and maintenance of the malaria-induced inflammatory response. The hypothesis of this study is that RANTES and its corresponding receptors (CCR1, CCR3 and CCR5) modulate malaria immunopathogenesis. A murine malaria model was utilized to evaluate the role of this chemokine and its receptors in malaria.MethodsThe alterations in immunomodulator gene expression in brains of Plasmodium yoelii 17XL-infected mice was analysed using cDNA microarray screening, followed by a temporal comparison of mRNA and protein expression of RANTES and its corresponding receptors by qRT-PCR and Western blot analysis, respectively. Plasma RANTES levels was determined by ELISA and ultrastructural studies of brain sections from infected and uninfected mice was conducted.ResultsRANTES (p < 0.002), CCR1 (p < 0.036), CCR3 (p < 0.033), and CCR5 (p < 0.026) mRNA were significantly upregulated at peak parasitaemia and remained high thereafter in the experimental mouse model. RANTES protein in the brain of infected mice was upregulated (p < 0.034) compared with controls. RANTES plasma levels were significantly upregulated; two to three fold in infected mice compared with controls (p < 0.026). Some d istal microvascular endothelium in infected cerebellum appeared degraded, but remained intact in controls.ConclusionThe upregulation of RANTES, CCR1, CCR3, and CCR5 mRNA, and RANTES protein mediate inflammation and cellular degradation in the cerebellum during P. yoelii 17XL malaria.

Prolonged remission of advanced bronchoalveolar adenocarcinoma in a dog treated with autologous, tumour-derived chaperone-rich cell lysate (CRCL) vaccine

Abstract Purpose: This paper presents the treatment of a 12-year-old female spayed Great Dane who presented with vestibular signs (ataxia, nystagmus, hind end collapse). Thoracic radiographs revealed a discrete pulmonary nodule in the right cranial lung lobe. Ultrasound-guided fine needle aspirate detected primary bronchoalveolar adenocarcinoma, verified via computed tomography, with a second smaller nodule discovered in the right cranial lung lobe. Materials and methods: A lateral thoracotomy with right cranial lung lobectomy was performed. Histopathological analysis of the nodules and an excised lymph node identified grade III bronchoalveolar adenocarcinoma with vascular infiltration and lymph node metastasis – a grim diagnosis with a reported median survival time of 6–27 days. A 10-g sample of the tumour was processed into a chaperone-rich cell lysate (CRCL) vaccine, which was administered weekly to the patient. Imiquimod – a Toll-like receptor 7 (TLR7) agonist – was applied topically for the first 12 treatments to stimulate local Langerhans cells. A single injection of bacillus Calmette-Guerin (BCG) was administered for additional immune stimulation at week 30 of treatment. Results: The dog remained stable and in otherwise good health until diffuse relapse occurred 44 weeks after the initial treatment; following gastrointestinal bleeding, the dog was euthanised 50+ weeks post diagnosis. Conclusion: To the authors’ knowledge, this is the first report of significantly prolonged survival following a diagnosis of grade III/stage III bronchoalveolar adenocarcinoma in a canine patient. This case report suggests that CRCL vaccine combined with topical imiquimod is a safe, effective treatment for canine tumours.

Thrombelastographic characterization of coagulation/fibrinolysis in horses: role of carboxyheme and metheme states.

Carboxyheme and metheme states modulate hemostasis in humans and other species. Further, carbon monoxide and/or nitric oxide production increase in inflammatory disorders involving the gastrointestinal tract, with associated hypercoagulability or hypocoagulability. In particular, the horse suffers both thrombotic or coagulopathic complications during acute gastrointestinal disease. This investigation characterized the thrombelastographic response to carboxyheme (via CORM-2) or metheme (via phenylhydroxylamine, PHA) states without/with addition of tissue type plasminogen activator. Citrated plasma was obtained from 14 normal mares and three horses with enteritis. In normal horses, a carboxyheme state did not enhance the velocity of clot growth and minimally enhanced clot strength (9%). In contrast, a metheme state was associated with a decrease in the velocity of clot formation (54%) and clot strength (47%). During fibrinolysis, a carboxyheme state significantly decreased the onset (113%) and velocity (27%) of fibrinolysis; however, in contrast, a metheme state more markedly increased the onset (84%) and velocity (133%) of fibrinolysis. These data support a carbon monoxide-dominant modulation of hemostasis in normal horses. In contrast, an increase in the severity of acute gastrointestinal disease was associated with a likely nitric oxide-mediated, metheme state-induced hypocoagulable/hyperfibrinolytic state. Additional investigation is warranted to determine the role played by carbon monoxide and nitric oxide in equine thrombotic and coagulopathic disease.

Preliminary characterization of the murine membrane reticulocyte proteome

The maturation from reticulocyte (immature red blood cell) to erythrocyte (mature red blood cell) includes the loss or decreased expression of cell surface molecules through exosome formation and secretion. Identifying the molecules lost and the molecular events involved is important to our understanding of this final stage of erythropoiesis and of diseases where it is deranged. Also, the presence of certain cell surface molecules is likely responsible for the invasion of certain malaria parasites into reticulocytes. Using a global proteomics approach, we identified proteins potentially lost during and/or involved in the reticulocyte maturation process. The reticulocyte proteome has not yet been published, as previous such studies have focused on the mature erythrocyte. Membrane-rich fractions were fractionated by electrophoresis followed by analysis with tandem mass spectrometry. Seven hundred forty four proteins were identified in the reticulocyte-rich membrane fraction, 192 proteins in the erythrocyte-rich membrane fraction, with 157 common to both fractions. Many of the proteins found uniquely in the reticulocyte were associated with structures known to be in reticulocytes (mitochondria, Golgi). Additional proteins detected are or may be specifically involved in vesicle trafficking, a process important in the maturation process. A number of unique plasma membrane proteins were also identified. These results provide the groundwork for future targeted studies to improve our understanding of the mechanism of reticulocyte maturation and the role of reticulocytes in disease.

Acute myeloid leukemia with multilineage dysplasia in an alpaca

An 18-year-old female alpaca was presented to the Colorado State University Veterinary Teaching Hospital for chronic ill thrift over a 1-year period. Six weeks previously, an infected left mandibular cheek tooth was removed by oral extraction. On physical examination the patient was cachectic, lethargic, and weak. Abnormalities on the CBC included neutropenia, thrombocytosis, and severe nonregenerative, macrocytic, hypochromic anemia. Dysplastic nucleated erythrocytes and micromegakaryocytes were observed on the peripheral blood smear. Neutrophils, bands, and metamyelocytes appeared markedly toxic. Numerous blasts containing variable numbers of fine azurophilic granules were also observed. Based on their morphology, the cells were interpreted to be progranulocytes and myeloblasts, and a presumptive diagnosis of acute myeloid leukemia (AML) was made. The blast cells accounted for 60% of the nucleated cell population on bone marrow aspirates, further supporting a diagnosis of AML with multilineage dysplasia. Post mortem examination showed infiltration of the neoplastic cells into spleen, liver, kidney, and lymph nodes. Based on histologic findings, the morphologic diagnoses were disseminated myeloid neoplasia, chronic regionally extensive tooth root abscess, and membranous glomerulonephritis. The neoplastic cells were CD172a-positive on flow cytometry, chloroacetate esterase-positive by cytochemistry, and myeloperoxidase-positive by immunohistochemistry, confirming myeloid origin. To our knowledge, this is the first case of AML with multilineage dysplasia in an alpaca, with only one other case of myelodysplasia described previously in this species.

Characterization of the canine urinary proteome

BACKGROUND Urine is an attractive biofluid for biomarker discovery as it is easy and minimally invasive to obtain. While numerous studies have focused on the characterization of human urine, much less research has focused on canine urine. OBJECTIVES The objectives of this study were to characterize the universal canine urinary proteome (both soluble and exosomal), to determine the overlap between the canine proteome and a representative human urinary proteome study, to generate a resource for future canine studies, and to determine the suitability of the dog as a large animal model for human diseases. METHODS The soluble and exosomal fractions of normal canine urine were characterized using liquid chromatography tandem mass spectrometry (LC-MS/MS). Biological Networks Gene Ontology (BiNGO) software was utilized to assign the canine urinary proteome to respective Gene Ontology categories, such as Cellular Component, Molecular Function, and Biological Process. RESULTS Over 500 proteins were confidently identified in normal canine urine. Gene Ontology analysis revealed that exosomal proteins were largely derived from an intracellular location, while soluble proteins included both extracellular and membrane proteins. Exosome proteins were assigned to metabolic processes and localization, while soluble proteins were primarily annotated to specific localization processes. Several proteins identified in normal canine urine have previously been identified in human urine where these proteins are related to various extrarenal and renal diseases. CONCLUSIONS The results of this study illustrate the potential of the dog as an animal model for human disease states and provide the framework for future studies of canine renal diseases.

Comparison of methods for depletion of albumin and IgG from equine serum

BACKGROUND Disease-specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. OBJECTIVE The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. METHODS One-dimensional gel electrophoresis followed by densitometry was used to determine percent of albumin, IgG, and both in pooled serum from 3 horses before and after application of 7 depletion methods. Repeatability was determined by applying the 2 best methods to serum samples from 6 grade horses. RESULTS For pooled serum, depletion rates varied from 35-90% for albumin and 0-94% for IgG. In the repeatability study, the ProteoExtract method combined with protein G Sepharose beads to remove additional IgG provided the best overall performance with 66% albumin depletion and 100% IgG depletion. A protocol using protein G Sepharose beads to remove IgG followed by ethanol precipitation of nonalbumin proteins with albumin remaining in the supernatant was the second most effective, with 85% albumin depletion and 55% IgG depletion. Although a multiprotein immunodepletion column effectively removed 90% of the albumin, the method was ineffective at removing IgG. CONCLUSION Albumin and IgG removal kits optimized for human use have variable efficacy for equine serum. Combined use of the ProteoExtract kit and manual incubation with protein G Sepharose beads provided the most effective depletion.