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Livestock production is a fundamental source of revenue and nutrition, wherein cattle-farming constitutes one of the major agricultural industries. Vectors and vector-borne diseases constitute one of the major factors that decrease the livelihood of all farming communities, more so in resource-poor communities and developing countries. Understanding the immunological responses during tick infestation in cattle is instrumental in the development of novel and improved tick control strategies, such as vaccines. In this study, gene expression patterns were compared within the lymph nodes of three cattle breeds at different life stages of the cattle tick, Rhipicephalus microplus. For Bonsmara (5/8Bos taurus indicus × 3/8B. t. taurus) cattle specifically, some 183 genes were found to be differentially expressed within the lymph nodes during larval and adult tick feeding, relative to uninfested cattle. Overall, the data provides evidence for a transcriptional regulatory network that is activated during immature tick infestation, but is down-regulated towards basal transcriptional levels when adult ticks are feeding. Specific processes in the lymph nodes of Bonsmara cattle were found to be differentially regulated on a transcriptional level. These include: (1) Leukocyte recruitment to the lymph node via chemokines and chemotaxis, (2) Trans-endothelial and intranodal movement on the reticular network, (3) Active regulation of cellular transcription and translation in the lymph node (including leukocyte associated cellular regulatory networks) and (4) Chemokine receptors regulating the movement of cells out of the lymph node. This work provides a first transcriptome analysis of bovine lymph node responses in tick-infested cattle. Findings show a dynamic immune response to tick infestation for the Bonsmara cattle breed, and that suppression of the maturation of the cattle hosts' immunity is especially evident during the larval feeding stages.

Light microscopic manual count is the current gold standard for parasite quantification. The ability to determine parasite density in whole blood is crucial to understanding disease pathogenesis and finding a suitable automated method of Babesia rossi parasite quantification would facilitate higher throughput and provide results that are more objective. This study investigated both peripheral capillary and central venous whole blood to estimate the correlations between light microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qPCR). Peripheral capillary and central venous blood were sampled from 40 naturally B. rossi-infected dogs and 10 healthy control dogs. Samples were analysed by reverse line blot hybridization assay to confirm a mono-B. rossi infection. Capillary blood parasite density was detected using light microscopic manual counting and venous blood parasitaemia detected by manual counts, flow cytometry and qPCR. A significant correlation was found between the venous manual counts and flow cytometry (rs = 0.465; P

BackgroundBabesia rossiis a leading cause of morbidity and mortality among the canine population of sub-Saharan Africa, but pathogenesis remains poorly understood. Previous studies ofB. rossiinfection were derived from clinical cases, in which neither the onset of infection nor the infectious inoculum was known. Here, we performed controlledB. rossiinoculations in canines and evaluated disease progression through clinical tests and whole blood transcriptomic profiling.ResultsTwo subjects were administered a low inoculum (104parasites) while three received a high (108parasites). Subjects were monitored for 8 consecutive days; anti-parasite treatment with diminazene aceturate was administered on day 4. Blood was drawn prior to inoculation as well as every experimental day for assessment of clinical parameters and transcriptomic profiles. The model recapitulated natural disease manifestations including anemia, acidosis, inflammation and behavioral changes. Rate of disease onset and clinical severity were proportional to the inoculum. To analyze the temporal dynamics of the transcriptomic host response, we sequenced mRNA extracted from whole blood drawn on days 0, 1, 3, 4, 6, and 8. Differential gene expression, hierarchical clustering, and pathway enrichment analyses identified genes and pathways involved in response to hemolysis, metabolic changes, and several arms of the immune response including innate immunity, adaptive immunity, and response to viral infection.ConclusionsThis work comprehensively characterizes the clinical and transcriptomic progression ofB. rossiinfection in canines, thus establishing a large mammalian model of severe hemoprotozoal disease to facilitate the study of host-parasite biology and in which to test novel anti-disease therapeutics. The knowledge gained from the study ofB. rossiin canines will not only improve our understanding of this emerging infectious disease threat in domestic dogs, but also provide insight into the pathobiology of human diseases caused byBabesiaandPlasmodiumspecies.

To provide useful information based on the macropathology, histopathology and immunohistochemical investigation in the spleens of dogs with Babesia rossi infection. Control spleens were collected from four healthy dogs euthanized for welfare reasons. Nine dogs that died naturally because of a mono-infection with Babesia rossi were selected for the diseased group. One haematoxylin-and-eosin-stained section of splenic tissue from each of the infected and control dogs was examined under the light microscope. Immunohistochemical markers were applied to characterize different immunocyte populations. The application of analytic software enabled semi-quantitative comparison of leucocyte subpopulations. Routine splenic histopathology revealed diffuse intermingling of white and red pulp from infected dogs with a clear loss of distinction between these zones. Immunohistochemistry revealed an increase in the proportion of tissue resident and bone marrow origin macrophages in the infected spleens. Apart from a few remnant lymphocytes within the peri-arteriolar lymphatic sheaths and follicles, the majority of the immunocytes redistributed to the red pulp, supporting the observation of white and red pulp intermingling. The majority of our findings are in agreement with histomorphological descriptions of the spleen in a variety of noncanid mammalian hosts with lethal malaria or babesiosis.

Although varying natural resistance to ticks between highly resistant Brahman (Bos taurus indicus), resistant Bonsmara (5/8 B. t. indicus x 3/8 B. t. taurus) and susceptible Holstein-Friesian (B. t. taurus) breeds is documented in skin and blood, little information is available describing draining lymph nodes. To elucidate the cellular dynamics during Rhipicephalus microplus induced immune responses, this study analysed immune factors from these cattle breeds using histology, immunohistochemistry and flow cytometry. Following the collection of skin and lymph node samples before artificial tick infestation, cattle were infested with R. microplus larvae. Subsequent sampling coincided with the tick larvae and adult developmental stages. A significant influx of CD20+ B-lymphocytes in the dermis all cattle breeds was observed while CD3+ T-lymphocytes were significantly increased for more tick resistant breeds. Eosinophil infiltration in germinal centres of lymph nodes was significant for all cattle breeds while tingible body macrophages were significantly increased for adult infested Brahman animals. A negligible fluctuation in CD20+ and CD79α+ B-lymphocyte numbers was present in the lymph node of more resistant cattle breeds, while susceptible animals showed a decrease in B-lymphocytes after infestation, followed by an increase between larvae to adult infested time points. Increased variability of γd T-lymphocyte populations in lymph nodes was correlated with tick susceptibility. In addition, a more stable T helper lymphocyte population was identified in the lymph nodes for the Brahman cattle breed. Results suggest the association of tick susceptibility with differential B-lymphocyte regulation in lymph node tissues, increased variability of WC1+ γδ T-lymphocyte populations in the lymph node as well as a decrease in T helper lymphocytes in the lymph node.

Dogs with babesiosis can present with multiple complications, including acute kidney injury (AKI). The objective of this study was to characterize AKI in dogs with babesiosis caused by Babesia rossi at presentation and after treatment. Thirty-five client-owned dogs with B. rossi infection and 10 control dogs were included in this prospective observational study. Blood and urine were collected in Babesia-infected dogs at presentation (T-0, n = 35), after 24 h (T-24h, n = 11), and after 1 month (T-1m, n = 9). The following urinary kidney injury biomarkers were assessed: urinary protein to creatinine ratio (UPC), urinary glomerular injury biomarkers (immunoglobulin G (uIgG) and C-reactive protein (uCRP)), and urinary tubular injury biomarkers (retinol-binding protein (uRBP) and neutrophil gelatinase-associated lipocalin (uNGAL)). Serum functional renal biomarkers were creatinine (sCr) and symmetric dimethylarginine (sSDMA). Post-mortem kidney biopsies were analyzed by light and transmission electron microscopy. At T-0, all kidney injury biomarkers were significantly higher in Babesia-infected dogs compared to healthy controls (P < 0.001), while functional renal biomarkers were not significantly different (P > 0.05). At T-24h, all urinary tubular injury biomarkers and UPC decreased significantly (P < 0.01), while glomerular injury biomarkers did not (P = 0.084). At T-1m, all urinary kidney injury biomarkers decreased to values not significantly different from healthy controls (P > 0.5). Significant changes in functional renal biomarkers were not seen after treatment (P > 0.05). Dogs with complicated babesiosis had significantly higher glomerular injury biomarkers, UPC, and sSDMA compared to uncomplicated cases (P < 0.05), while all tubular injury biomarkers and sCr were not significantly different (P > 0.1). Dogs with babesiosis caused by B. rossi showed transient kidney injury, which was detected by all kidney injury biomarkers, but remained undetected by functional biomarkers. All infected dogs, irrespective of disease severity, suffered comparable kidney injury based on tubular injury biomarker concentrations, while loss of function was seen more often in dogs with complicated babesiosis based on sSDMA results.

Babesia rossi is an important, tick-borne intraerythrocytic protozoan parasite; however, its natural history and epidemiology is poorly understood. Babesia rossi is the most virulent Babesia sp. in domestic dogs and is generally considered to cause severe babesiosis, which is fatal if left untreated. However, subclinical infections and mild disease from B. rossi have been reported, although the clinical progression of these cases was not reported. Therefore, to better understand B. rossi under field conditions, we evaluated its clinical progression and seroprevalence in an owned, free-roaming dog population in Zenzele, South Africa, where the parasite is endemic and prevention is not routine. The entire dog population in Zenzele was monitored intensively at the individual level from March 2008 until April 2014, primarily for a longitudinal study on rabies control. Subsequent evaluation of B. rossi comprised analyses of clinical and laboratory data collected from the Zenzele dog population during the 6 year study period. A substantial proportion (31% (n = 34)) of 109 dogs (randomly selected from every available dog in February/March 2010 older than ~6–8 weeks (n = 246)) tested by Indirect Fluorescent Antibody Test had seroconverted strongly to B. rossi. All 34 dogs were generally consistently healthy adults, determined from regular clinical examinations between March 2008 and April 2014. Blood smear examinations at multiple time points between July 2009 and February 2011 were also undertaken for almost all of these (34) seropositive dogs and all those tested were consistently negative for Babesia spp. Subclinical infections and mild disease were also the main findings for a separate group of 18 dogs positive for Babesia spp. on blood smear examination and confirmed to be infected with B. rossi by Polymerase Chain Reaction – Reverse Line Blot. Almost all of these dogs were positive at only one time point from repeat blood smear examinations between July 2009 and February 2011. We suggest that these observations are consistent with immunity acquired from repeated, low-level exposure to the parasite, generating transient subclinical infections or mild disease. Should this be the case, the use of tick control, particularly in adult dogs in free-roaming populations in B. rossi endemic regions, should be carefully considered.

// Benjamin Garcia-Bloj 1, 2, 4 , Colette Moses 1, 2 , Agustin Sgro 1 , Janice Plani-Lam 1 , Mahira Arooj 1, 5 , Ciara Duffy 1, 2 , Shreyas Thiruvengadam 1 , Anabel Sorolla 1 , Rabab Rashwan 1 , Ricardo L. Mancera 5 , Andrea Leisewitz 4 , Theresa Swift-Scanlan 6 , Alejandro H. Corvalan 3, 4 , Pilar Blancafort 1, 2 1 Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia 2 School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia 3 Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 4 Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 5 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia 6 School of Nursing, Virginia Commonwealth University, Richmond, Virginia, 23298, USA Correspondence to: Pilar Blancafort, email: pilar.blancafort@uwa.edu.au Keywords: CRISPR/dCas9, ZF, TALE, tumor suppressor genes, gene reactivation Received: June 01, 2016 Accepted: July 19, 2016 Published: August 09, 2016 ABSTRACT The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO , in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Aims Babesia rossi causes severe disease in dogs. Here, we describe the association between serum cytokine concentrations and disease severity. Methods Seventeen controls and 55 PCR confirmed B rossi-infected dogs were included. Diseased dogs were subdivided into 23 critically ill and 32 relatively well cases. Serum concentrations of 11 cytokines and biochemical markers of disease severity were determined. Results Significant differences were detected for IL-6, IL-8, IL-10, MCP-1 and TNF-α between the groups. Generally, the more complicated the disease, the more pro-inflammatory the cytokine milieu. IL-8 showed a reverse trend and was negatively correlated with disease severity. IL-6, MCP-1 and TNF-α were also significantly higher in the dogs that died (n = 9) compared to the dogs that survived (n = 46). IL-8 showed the opposite. MCP-1 and TNF-α were negatively correlated with biochemical markers of severity. Glucose was negatively correlated with IL-6. Cortisol, peripheral parasite density and band neutrophil count were positively correlated, whilst thyroid hormone was negatively correlated with IL-6, MCP-1 and TNF-α. Conclusions As in malaria and sepsis, B rossi infection induces a pro-inflammatory cytokine storm that correlates with disease severity and adverse outcome. The multiplicity of cytokines involved argues for redundancy in the system once the disease is established.

Babesia rossi causes the most severe clinical disease in dogs of all the babesia parasites. We included 320 naturally-infected dogs that presented for care at the Onderstepoort Veterinary Academic Hospital between 2006 and 2016. All dogs had mono-infections confirmed by multiplex PCR. The data allowed more accurate clinical classification of the disease and identified parameters that were associated with disease severity and death. Odds ratios for dying were significant (P

Canine babesiosis is caused by tick-transmitted intraerythrocytic protozoan parasites occurring worldwide. In southern Africa, babesiosis is caused by Babesia rossi and B. vogeli and is one of the most common and important infectious diseases affecting dogs. There is no reliable, rapid and sensitive method for the detection of these parasites, especially when parasitaemia is low. The aim of this study was to develop a sensitive and specific multiplex TaqMan® MGB PCR assay for the diagnosis of canine babesiosis infections occurring in southern Africa, and to discriminate between Babesia rossi and B. vogeli. The fitness of purpose of the assay was to confirm diagnosis of suspect or clinical cases, and estimate prevalence of infection for research purposes. A total of 648 published sequences were used to design the assay. A set of group-specific canine Babesia spp. primers were designed to amplify a 117 nucleotide region of the 18S rRNA gene of all canine Babesia spp. Species-specific TaqMan® MGB probes were developed for B. rossi, B. vogeli, B. canis and B. gibsoni, but analytical validation was only performed for B. rossi and B. vogeli as a multiplex assay. The assay had a broad dynamic range and amplified B. rossi and B. vogeli efficiently (98.6% and 94.7% respectively). The assay was sensitive, with a 95% LOD of 10

The occurrence of acute kidney injury in canine babesiosis is not well documented. Furthermore, interpretation of urine specific gravity (USG) to assess renal concentrating ability is hampered by the frequent presence of hemoglobinuria in this disease. This cross-sectional study aimed to test the hypothesis that renal azotemia (RA) is underdiagnosed according to current canine babesiosis literature by determining its occurrence at presentation, using urine osmolality instead of USG to measure urinary concentration. The second objective was to examine potential associations between the presence of RA and selected clinical and laboratory variables at presentation. Medical records available from 3 previously performed prospective data collections were reviewed retrospectively. Client-owned dogs that were diagnosed with babesiosis caused by Babesia rossi, were included if a complete blood count, biochemistry profile, and urinalysis was performed at admission. Urine osmolality was measured to identify dogs with RA. Differences between dogs with RA and dogs without RA were assessed by nonparametric statistics. One hundred and fifty-two dogs were included, of which 26 (17%) were azotemic at admission. The occurrence of RA was 14% (21/152), hence 81% (21/26) of all azotemic dogs were diagnosed with RA. In contrast, when diagnosis of RA was based on an admission USG 1.030, only 23% (6/26) of the azotemic dogs would have been considered to have RA. Several signalment and clinicopathological findings were found to be associated with the presence of RA, including older age, and the presence of collapse, hypoglycemia, hyperphosphatemia, cerebral babesiosis, and acute respiratory distress syndrome. Lastly, survival at discharge was significantly lower in dogs diagnosed with RA at presentation. Our results clarified that RA is more common than previously reported in B. rossi. This study also demonstrated that USG determination is not a reliable method to evaluate renal concentrating ability in azotemic dogs with babesiosis. Thus, if available, urine osmolality should be part of the diagnostic work-up of dogs infected with B. rossi to avoid misclassification of dogs with RA as having prerenal azotemia. If urine osmolality cannot be measured, clinicians should realize that most azotemic dogs with B. rossi infection have RA.

Using flow cytometry, platelet–leukocyte aggregate (PLA) formation has previously been documented in dogs with a variety of systemic inflammatory disorders and immune-mediated haemolytic anaemia. Platelet activation and subsequent interaction between platelets and leukocytes are important for regulating innate immunity and systemic inflammation. The objective of this study was to investigate PLA formation in canine babesiosis and to determine whether it was associated with outcome. Blood was collected from 36 client-owned dogs diagnosed with Babesia rossi infection and 15 healthy controls using EDTA as anticoagulant. Activated platelets and PLA formation were detected by measuring surface expression of P-selectin (CD62P) on platelets, monocytes and neutrophils. Of the Babesia -infected dogs, 29 survived and seven died. The percentage of CD62P-positive monocytes was significantly higher ( P = 0.036) in the Babesia -infected dogs (54%) than in healthy control dogs (35.3%). However, there were no significant differences between the Babesia -infected and control groups for CD62P-positive platelets (4.9% and 1.2%, respectively) and CD62P-positive neutrophils (28.3% and 17.9%, respectively). The percentage of CD62P-positive monocytes was significantly higher ( P = 0.019) in the survivors (58.9%) than in healthy control dogs; however, there were no significant differences between the non-survivors (39.2%) and the controls or between survivors and non-survivors. There were no significant differences between groups for the percentage of CD62P-positive platelets (survivors 4.8%; non-survivors 5.3%; controls 1.2%) or CD62P-positive neutrophils (survivors 31.6%; non-survivors 5.6%; controls 17.9%). In conclusion, Babesia -infected dogs, specifically dogs that survived, had a significantly increased percentage of platelet–monocyte aggregates compared to healthy control dogs.

Immunity to Babesia infection requires both innate and acquired responses, including cell mediated- and humoral responses. The aims of this study were to investigate the variation in selected peripheral blood lymphocyte phenotypes in dogs with virulent babesiosis at presentation and over time after treatment, and to determine whether these were correlated with the severity of clinical signs. Forty-four dogs naturally infected with B. rossi were studied and 5 healthy dogs were included as controls. Blood samples were collected from the jugular vein at admission, prior to any treatment, and at 24 hours and 48–72 hours. Leukocytes were incubated with canine specific, fluorochrome conjugated anti-CD3, anti-CD4, anti-CD8, and anti-B cell markers. Babesia-infected dogs were divided into complicated or uncomplicated groups on clinical grounds and in-house laboratory assays. The percentage CD3+ lymphocytes in the complicated group was lower compared to the controls (P = 0.014) and uncomplicated group (P = 0.007). The percentage CD4+ T lymphocytes in the complicated group was lower compared to the controls (P = 0.027) and uncomplicated group (P = 0.014). Both the complicated as well as the uncomplicated groups expressed a lower percentage CD8+ T lymphocytes compared to the control group (P < 0.001 and P = 0.005, respectively). The percentage B lymphocytes was higher in the complicated group at 48–72 hours. These findings could indicate the presence of a functional immune suppression secondary to increased apoptosis or redistribution of effector lymphocytes and/or a combination of other immune modulatory mechanisms induced by B. rossi infection.

The acid–base disturbances in canine parvoviral (CPV) enteritis are not well described. In addition, the mechanisms causing these perturbations have not been fully elucidated. The purpose of the present study was to assess acid–base changes in puppies suffering from CPV enteritis, using a modified strong ion model (SIM). The hypothesis of the study was that severe acid–base disturbances would be present and that the SIM would provide insights into pathological mechanisms, which have not been fully appreciated by the Henderson–Hasselbalch model. The study analysed retrospective data, obtained from 42 puppies with confirmed CPV enteritis and 10 healthy control dogs. The CPV-enteritis group had been allocated a clinical score, to allow classification of the data according to clinical severity. The effects of changes in free water, chloride, l-lactate, albumin and phosphate were calculated, using a modification of the base excess algorithm. When the data were summated for each patient, and correlated to each individual component, the most important contributor to the metabolic acid–base changes, according to the SIM, was chloride (P

Canine parvovirus (CPV) enteritis has, since its emergence in 1978, remained a common and important cause of morbidity and mortality in young dogs. The continued incidence of parvoviral enteritis is partly due to the virus' capability to evolve into more virulent and resistant variants with significant local gastrointestinal and systemic inflammatory sequelae. This paper reviews current knowledge on historical-, signalment-, and clinical factors as well as several haematological-, biochemical- and endocrine parameters that can be used as diagnostic and prognostic biomarkers in CPV enteritis. These factors include season of presentation, purebred nature, bodyweight, vomiting, leukopaenia, lymphopaenia, thrombocytopaenia, hypercoagulability, hypercortisolaemia, hypothyroxinaemia, hypoalbuminaemia, elevated C-reactive protein and tumour necrosis factor, hypocholesterolaemia and hypocitrullinaemia. Factors contributing to the manifestations of CPV infection are multiple with elements of host, pathogen, secondary infections, underlying stressors and environment affecting severity and outcome. The availability of several prognosticators has made identification of patients at high risk of death and their subsequent targeted management more rewarding.

Acute myeloid leukemia (AML) has a high mortality rate despite chemotherapy and transplantation. Both CXCR4/SDF-1 and VLA-4/VCAM1 axes are involved in leukemia protection but little is known about the role of CCL2/CCR2 in AML biology and protection against chemotherapy. We measured CCR2 expression in AML cell lines and primary AML cells by flow cytometry (FCM), real time PCR (RT-PCR) and western blot (WB). CCL2 production was quantified by solid phase ELISA in peripheral blood (PB) and bone marrow (BM) serum. We measured chemotaxis in a transwell system with different concentrations of CCL2/CCR2 blockers; cell cycle with BrDU and propidium iodide and proliferation with yellow tetrazolium MTT. We determined synergy in in vitro cell apoptosis combining chemotherapy and CCL2/CCR2 blockade. Finally, we performed chemoprotection studies in an in vivo mouse model. Of 35 patients, 23 (65%) expressed CCR2 by FCM in PB. Two cell lines expressed high levels of CCR2 (THP-1 and murine AML). RT-PCR and WB confirmed CCR2 production. CCL2 solid phase ELISA showed significantly lower levels of CCL2 in PB and BM compared to normal controls. Chemotaxis experiments confirmed a dose-dependent migration in AML primary cells expressing CCR2 and THP-1 cells. A significant inhibition of transmigration was seen after CCL2/CCR2 blockade. Proliferation of CCR2+ AML cell lines was slightly increased (1.4-fold) after axis stimulation. We observed a non-significant increase in phase S THP-1 cells exposed to CCL2 and a concomitant decrease of cells in G1. The chemotherapy studies did not show a protective effect of CCL2 on cytarabine-induced apoptosis or synergy with chemotherapy after CCL2/CCR2 blockade both in vitro and in vivo. In conclusion, CCL2/CCR2 axis is expressed in the majority of monocytoid AML blasts. The axis is involved in cell trafficking and proliferation but no in vitro and in vivo chemotherapy protective effect was seen.

Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.

Envenomation of domestic animals by snakes occurs frequently in certain geographic areas. However, reports describing clinical signs, clin- icopathologic abnormalities, therapeutic approaches, and outcomes are sparse. This review summarizes various snake families, venom types asso- ciated with harmful snakes, and the significant hematologic, hemostatic, and biochemical abnormalities associated with envenomation. Hematolog- ic abnormalities include RBC membrane abnormalities, hemolysis, hemo- concentration, leukogram changes, and platelet abnormalities, specifically thrombocytopenia. Coagulopathies associated with snake envenomation are well described in human medicine, and many studies have demon- strated properties of venoms that lead to both procoagulation and antico- agulation. As expected, similar abnormalities have been described in domestic animals. Biochemical abnormalities are associated with the effects of venom on tissues such as liver, skeletal and cardiac muscle, vas- cular endothelium, and kidney as well as effects on protein components and cholesterol. This comprehensive review of clinicopathologic abnor- malities associated with envenomation and their relationships to charac- terized venom constituents should be useful both in the diagnosis and management of envenomation and should serve as a foundation for future research in this field.

Equilibrative Nucleoside Transporters (ENTs) are facilitative transporters broadly selective for pyrimidine and purine nucleosides and are essential for the modulation of nucleoside concentration and nucleoside analog availability. Resistance to nucleoside-derived drugs strongly correlates with a deficiency of ENT1 expression in several tumor cells. Thus, it is crucial to understand the mechanisms by which this transporter is modulated. Using a mouse myeloid leukemic cell line as a model, we investigated whether stress-activated kinases regulate ENT1 expression and function. JNK activation, but not p38 MAPK results in rapid loss of mENT1 function, mRNA expression and promoter activity. c-Jun but not the mutant c-Jun Ser63/73Ala, decreased mENT1 promoter activity. Moreover cJun bound to an AP-1 site identified at −1196 of the promoter, suggesting a specific role for this transcription factor in mENT1 regulation. We propose that activation of JNK-cJun pathway negatively regulates mENT1 and suggest that this mechanism might contribute to the development of nucleoside analog-derived drug resistance.

Babesia rossi , an intraerythrocytic protozoan, causes a severe, often life-threatening disease of domestic dogs. Dogs treated early for B. rossi infection usually recover from the disease, but dogs left untreated or treated at a later stage of infection seldom survive. Dogs infected with B. rossi have varied clinical manifestations that can be categorized as uncomplicated (with a good prognosis) or complicated (with a poor prognosis). One hundred twenty-one blood samples were collected from dogs presented to the Onderstepoort Veterinary Academic Hospital and diagnosed with babesiosis by the use of a thin blood smear. An additional 20 samples were obtained from Babesia -infected dogs from private clinics around the Onderstepoort, Johannesburg, Durban, White River, and Cape Town areas. The samples were screened by PCR targeting the Babesia rossi erythrocyte membrane antigen gene (Br EMA1 ) and by sequencing of the polymorphic region (i.e., region with a variable number of hexapeptide repeats). Analysis of PCR products revealed 11 different gene profiles, visualized by gel electrophoresis. Twelve distinct Br EMA1 genotypes were identified by sequencing, but the numbers of hexapeptide repeats varied from 6 to 31 (classified as genotype6 to genotype31). The genotypes were retrospectively compared to the clinical case data. The most frequently encountered B. rossi parasites were those attributed to genotype19 (36.2%), genotype28 and genotype29 (20.6% each), and genotype11 (12.7%). These genotypes were also the ones associated with the poorest prognosis. This preliminary finding suggests clinically important differences between the various B. rossi genotypes identified.

The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.

Blood specimens from wild dogs (n = 301) were obtained from De Wildt Cheetah and Wildlife Centre (Pretoria) and five game reserves (4 in the North-West Province and 1 in Limpopo Province), South Africa. Specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. Positive results were obtained in 18 (6%) wild dogs. Sixteen specimens were found positive for Babesia rossi and two dogs were Hepatozoon sp. positive. It appears that these tick-borne pathogens are not widely distributed in wild dog populations.

A Theileria species was detected by PCR in blood samples collected from dogs in the Pietermaritzburg area and was also found in dogs presented at the Outpatients Clinic of the Onderstepoort Veterinary Academic Hospital (OVAH), in the Pretoria area, South Africa. In the Pietermaritzburg area, 79 of the 192 samples were positive, while 3 out of 1137 of the Onderstepoort samples were positive. Three positive samples from Pietermaritzburg were co-infected with Ehrlichia canis. PCR positive samples were further analysed by the Reverse Line Blot (RLB) and sequence analysis. Phylogenetic analysis of the 18S rRNA full-length gene sequences of one sample (VT12) from Pietermaritzburg and two samples from OVAH (BC281 and BC295) revealed a close relationship with sequences of Theileria species (sable). Clinical signs of the dogs that were examined at Pietermaritzburg and OVAH included an immune-mediated condition with severe thrombocytopenia. These findings identify a Theileria sp. in dogs for the first time in South Africa and add yet another microorganism to the growing list of haemoprotozoan parasites infecting dogs worldwide. The clinical significance of this infection in dogs is poorly resolved.

A total of 1,138 blood specimens were collected over a 6-year period (2000-2006) from domestic dogs in South Africa. Specimens from domestic dogs were obtained from the Onderstepoort Veterinary Academic Hospital (OVAH) in Pretoria, the Society for the Prevention of Cruelty to Animals (Johannesburg, Durban, East London and Bloemfontein) and private practices from four provinces (Gauteng, Mpumalanga, KwaZulu-Natal and Western Cape). All specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. On RLB, 560/1,137 domestic dog-specimens were positive for one or more parasites. Of the positive domestic dog-specimens, 420 (75%) were infected with Babesia rossi; 82 (15%) dogs were infected with Theileria sp. (dog); 18 (3%) dogs were infected with Babesia vogeli; 14 (3%) specimens were infected with Ehrlichia canis. Mixed infections were also found: B. rossi and E. canis were detected in 12 (2%) specimens; B. vogeli and E. canis occurred in 7 (1%) specimens; Theileria sp. (dog) and E. canis in 3 (0.5%) specimens; B. rossi and B. vogeli in one specimen. B. rossi, B. vogeli and E. canis occurred simultaneously in one dog. There was also one incidental finding of a dog positive for Trypanosoma congolense. The results indicate that a wide range of tick-borne pathogens are circulating in the canine populations in South Africa.

Babesia rossi infection causes a severe inflammatory response in the dog, which is the result of the balance between pro- and anti-inflammatory cytokine secretion. The aim of this study was to determine whether changes in cytokine concentrations were present in dogs with babesiosis and whether it was associated with disease outcome. Ninety-seven dogs naturally infected with B. rossi were studied and fifteen healthy dogs were included as controls. Diagnosis of babesiosis was confirmed by polymerase chain reaction and reverse line blot. Blood samples were collected from the jugular vein at admission, prior to any treatment. Cytokine concentrations were assessed using a canine-specific multiplex assay on an automated analyser. Serum concentrations of interleukin (IL)-2, IL-6, IL-8, IL-10, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF) and monocyte chemotactic protein-1 (MCP-1) were measured. Twelve of the Babesia-infected dogs died (12%) and 85 survived (88%). Babesia-infected dogs were also divided into those that presented within 48 hours from displaying clinical signs, and those that presented more than 48 hours after displaying clinical signs. Cytokine concentrations were compared between the different groups using the Mann-Whitney U test. IL-10 and MCP-1 concentrations were significantly elevated for the Babesia-infected dogs compared to the healthy controls. In contrast, the IL-8 concentration was significantly decreased in the Babesia-infected dogs compared to the controls. Concentrations of IL-6 and MCP-1 were significantly increased in the non-survivors compared to the survivors. Concentrations for IL-2, IL-6, IL-18 and GM-CSF were significantly higher in those cases that presented during the more acute stage of the disease. These findings suggest that a mixed cytokine response is present in dogs with babesiosis caused by B. rossi, and that an excessive pro-inflammatory response may result in a poor outcome.

SUMMARY Bacillus Calmette-Guerin (BCG) infection of the spleen is a potent modifier of splenic function. Prior to malaria infection, we infected two mouse strains of differing susceptibility to Plasmodium chabaudi AS (C57BL/6 and A/J) with this mycobacterium. We then evaluated aspects of spleen cell composition, architecture and cytokine expression, and correlated these with the outcome. BCG preinfection resulted in protection of the A/J mice but paradoxically resulted in mortality of the C57BL/6 mice. The latter developed higher parasitaemias that peaked earlier than the A/J mice rendered resistant by BCG. BCG infection induced remarkable changes to splenic histology examined by H&E staining, but there were no consistent differences between mouse strains. C57BL/6 mice had higher absolute numbers of all immune cell phenotypes than did A/J mice, and higher macrophage and dendritic cell proportions. BCG-induced resistance in A/J mice was associated with an increased CD4 + expression of IFN- γ whilst induced death in C57BL/6 mice was associated with excessive IFN- γ expression. A moderate TH1 response in the A/J model may have been responsible for the improved survival, and an excessive TH1 response in the C57BL/6 model may have contributed to their death.

Dendritic cells, particularly those residing in the spleen, are thought to orchestrate acquired immunity to malaria, but it is not known how the splenic dendritic cell population responds to malaria infection and how this response compares with the responses of other antigen-presenting cells. We investigated this question for Plasmodium chabaudi AS infection in C57BL/6 mice. We found that dendritic cells, defined here by the CD11c marker, migrated from the marginal zone of the spleen into the CD4 + T-cell area within 5 days after parasites entered the bloodstream. This contrasted with the results observed for the macrophage and B-cell populations, which expanded greatly but did not show any comparable migration. Over the same time period dendritic cells showed upregulation of CD40, CD54, and CD86 costimulatory molecules that are required for successful T-cell activation. In dendritic cells, the peak intracellular gamma interferon expression (as shown by fluorescence-activated cell sorting) was on day 5, 2 days earlier than the peak expression in B-cells or macrophages. These findings show that splenic dendritic cells are actively engaged in the earliest phase of malarial infection in vivo and are likely to be critical in shaping the subsequent immune response.

Cases of snakebite envenomation are frequently presented to veterinary practitioners in southernAfrica. Despite this, no published guidelines exist onhowthis medical emergency should be managed. Southern African snake venoms can be classified into 3 main types based on the main mechanism of venom action and clinical presentation. A polyvalent antivenom is manufactured in South Africa and contains antibodies against the most important southern African snake venoms. The cytotoxic venoms are represented mainly by the puff-adder (Bitis arietans), Mozambique spitting cobra (Naja mossabica), black-necked spitting cobra (Naja nigricollis) (in the Western Cape and Namibia) and the stiletto snake (Atractaspis bibronii). These venoms may cause dramatic local swelling, high morbidity and low mortality and infrequently require the use of antivenom for survival (the only cytotoxic venoms used to prepare the antivenom are the puff-adder and Mozambique spitting cobra). The neurotoxic venoms (represented chiefly by the non-spitting cobras and mambas) cause high mortality due to rapid onset of paresis and require antivenom and mechanical ventilatory support which is life-saving. The boomslang (Dispholidus typus) and the vine snake (coagulopathic venom) rarely bite humans but dogs may be bitten more frequently. These venoms cause a consumption coagulopathy and successful treatment of boomslang bites requires the use of snake species-specific monovalent antivenom. There is no antivenom available for treating vine snake (Thelotornis capensis), berg adder (Bitis atropos), night adder (Causus spp.), stiletto snake and other lesser adder bites. There are some important differences between the way snakebites are managed in humans and dogs.

Peroxisome proliferator-activated receptors (PPARs) are key transcription factors in the control of lipid homeostasis and cell differentiation, but little is known about their function in oligodendrocytes, the major lipid-synthesizing cells in the central nervous system (CNS). Using the B12 oligodendrocyte-like cell line and rat spinal cord-derived oligodendrocytes, we evaluated the importance of PPARgamma in the maturation process of these cells. B12 cells express all PPAR isoforms (alpha, beta/delta, and gamma), as assessed by RT-PCR, Western-blot, and transactivation assays. B12 cells respond specifically to PPARgamma agonists by arresting cell proliferation and extending cell processes, events that are blocked by the PPARgamma antagonist GW9662. In addition, alkyl-dihydroxyacetone phosphate synthase (ADAPS), a key peroxisomal enzyme involved in the synthesis of myelin-rich lipid plasmalogens, is increased in PPARgamma agonist-treated B12 cells. In contrast with B12 cells, both immature and mature isolated spinal cord oligodendrocytes presented a high and similar expression level of ADAPS, as assessed by immunocytochemistry. However, as in B12 cells, isolated spinal cord oligodendrocytes were also found to respond specifically to PPARgamma agonists with a four-fold increase in the number of mature cells. Our data suggest a relevant role for PPARgamma in oligodendrocyte lipid metabolism and differentiation.

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors that control important genes involved in lipid metabolism. Their role in nerve cells is uncertain, although anomalous myelination of the corpus callosum has been described in the PPARbeta-null mouse, and abnormalities of this tissue have been documented in fetal alcohol syndrome in humans. We report here that ethanol treatment of B12 oligodendrocyte-like cells induces a concentration- and time-dependent decrease in the mRNA and protein levels of PPARbeta, with no effect on PPARalpha or PPARgamma. The effect on PPARbeta is seen as an increase in mRNA degradation, as assessed by run-off assays, due to a significant decrease in PPARbeta mRNA half-life, with no observed changes in intracellular localization. Our results suggest a possible link between PPARbeta function and ethanol-induced abnormal myelination in oligodendrocytes.

Background The favoured animal model of cerebral malaria is an artificial host-parasite combination caused by Plasmodium berghei in inbred mouse strains [1]. Canine babesiosis caused by natural infection in dogs with Babesia rossi causes cerebral disease in some cases [2]. This disease demonstrates both similarities and differences to human malarial and comparisons may be of value in elucidating the pathogenesis of this serious complication in both hosts [3].

Canine distemper virus is a member of the genus Morbillivirus of the family Paramyxoviridaethat causes severe disease in dogs and a range of wild mammals. The clinical signs relate essentially to the respiratory, gastrointestinal and central nervous systems. In South Africa, infection with Ehrlichia canis and canine parvovirus may present similarly. Many dogs will initially present with a wide range of central nervous system signs without any history of systemic disease. A recent South African study evaluating ante mortem diagnosis highlighted the importance of recognising clinical signs, cerebrospinal fluid IgG titres, serum IgM titres and immunocytochemistry of epithelial tissue. A 2-year retrospective evaluation of cerebrospinal fluid samples collected from dogs presented to the Onderstepoort Veterinary Academic Hospital indicates that distemper infection is common, and this disease should routinely be suspected in cases of diverse neurological disease in dogs. The South African dog population is specifically at high risk for the disease because of the large pool of unvaccinated, reproductively-active dogs that expose the wildlife resources of the country to risk of fatal disease. Outbreaks of disease in dogs continue to occur in developed and developing communities in both vaccinated and unvaccinated dogs worldwide, and have also been described in a wide range of free-ranging wildlife, including seals, dolphins and lions, and in endangered zoo animals. Modified live virus vaccines have contributed markedly to disease control in the dog population but have caused mortality in some wild carnivores. New recombinant vaccines are being developed that will be safe in wild animals. The pathogenesis of CNS demyelination has been compared to various important demyelinating diseases in humans and, amongst other things, relates to down-regulation of the oligodendrocyte gene coding for myelin synthesis and non-immunocyte CNS cell expression of type II major histocompatibility receptors. Early CNS lesions are characterised by demyelination and later lesions by perivascular round cell cuffing. Treatment is supportive.

Previous studies have shown that palmitoyl-carnitine is an anti-proliferative agent and a protein kinase C inhibitor. Two new palmitoyl-carnitine analogs were synthesized by replacing the ester bond with a metabolically more stable ether bond. An LD50 value in the nm range was found in anti-proliferative assays using HL-60 cells and was dependent on the alkyl-chain length. The inhibitory action of these water-soluble compounds on protein kinase C in vitro was greatly increased with respect to palmitoyl-carnitine and was dependent on the length of the alkyl chain. Its effect was mediated by an increase in the enzyme’s requirement for phosphatidylserine. Inhibition of the in situ phosphorylation of a physiological platelet protein kinase C substrate and of phorbol ester-induced differentiation of HL-60 cells was also observed. Finally, to test for isoenzyme selectivity, several human recombinant protein kinase C isoforms were used. Only the Ca2+-dependent classic protein kinase Cs (α, βΙ, βΙΙ and γ) were inhibited by these compounds, yet the activities of casein kinase I, Ca2+/calmodulin-dependent kinase and cAMP-dependent protein kinase were unaffected. Thus, these novel inhibitors appear to be both protein kinase C and isozyme selective. They may be useful in assessing the individual roles of protein kinase C isoforms in cell proliferation and tumor development and may be rational candidates for anti-neoplasic drug design.

The regulation of CNS lipid metabolism by nuclear receptors and their relation to cell differentiation remains undetermined. Since myelinating oligodendrocytes are the major lipid-synthesizing cells in the CNS, we characterized the effect of PPAR activation in a CNS derived cell line that expresses oligodendrocyte markers and compared these effects with oligodendrocyte primary cultures (90–95% pure). The rat glioma derived B12 cell line express the three major PPAR isoforms (PPAR α, β and γ) and present a large number of peroxisomes, indicating an important lipid metabolism. Treatment with ciprofibrate, a general PPAR activator and clinical hypolipidemic, induces proliferation arrest, process extension and a moderate rise in the expression of acyl-CoA oxidase, a specific marker of peroxisomal proliferation. Cell growth arrest by ciprofibrate is enhanced 100-fold by low concentrations of retinoic acid (0.01 μm), suggesting the involvement of the PPAR-retinoid acid receptor heterodimers. Since ciprofibrate possibly acts by modifying the concentration of endogenous PPAR ligands, we traced its effects to PPARγ by using isoform specific ligands: Troglitazone and 15-deoxy-prostaglandin J2, both of which induce growth arrest and process extension in B12 cells. These effects were corroborated on rat spinal cord derived oligodendrocytes primary cultures, where a significant rise in the number of mature oligodendrocytes is observed in response to PPARγ activators. These results show that PPARγ, a master gene in the differentiation of adipose tissue could be involved in the lipid metabolism of maturing oligodendrocytes.

Human equilibrative nucleoside transporter 1 (hENT1) is an important determinant for nucleoside analog based chemotherapy success. Preliminary data suggest hENT1 regulation by PPARs. Using A2780 cells, we investigated the role of PPARs on hENT1 expression and activity. PPARα and PPARγ agonists, Wy14,643 and RGZ, increased hENT1 expression, but only PPARα activation or overexpression resulted in higher hENT1 transport activity. On the other hand, promoter analysis showed two putative PPRE in hENT1 promoter and luciferase-coupled promoter constructs were generated and analyzed. Our results suggest that PPARα-but not PPARγ-mediated expression regulation of hENT1 is PPRE-dependent. In conclusion, PPARα and PPARγ activation modulate hENT1 expression.

International audience; In order to treat osteoporosis and other bone mass disorders it is necessary to understand the regulatory processes that control the cell fate decisions responsible for going from bone precursor cells to bone tissue. Many processes interact to regulate cell division, differentiation and apoptosis. There are models for these basic processes, but not for their interactions. In this work we use the theory of switched systems, reuse and composition of validated models to describe the cell fate decisions leading to bone and fat formation. We describe the differentiation of osteo-adipo progenitor cells by composing its model with differentiation stimuli. We use the activation of the Wnt pathway as stimulus to osteoblast lineage, including regulation of cell division and apoptosis. This model is our first step to simulate physiological responses in silico to treatments for bone mass disorders.

Recent evidence indicates that transcription factor Runx1 modulates the expression of several phenotypic markers in dorsal root ganglia (DRGs) neurons, including the pain-related P2X3 receptor. In several cell lineages C/EBP transcription factors interact with the Runx factor family members to jointly bind and activate transcription of target genes. Here, we examine whether these two transcription factors directly regulate P2X3 gene expression. Through in silico analyses of the first 2 kb of the P2X3 gene promoter we identified putative consensus-binding sites for both Runx1 and C/EBPβ transcription factors. Transient over-expression in PC12 cells of either Runx1 or C/EBPβ increases P2X3 gene promoter activity and co-expression of both factors results in an additive stimulatory effect on the promoter function. Accordingly, chromatin immunoprecipitation assays demonstrate that both Runx1 and C/EBPβ bind to the P2X3 promoter in PC12 cells expressing this gene. Site-directed mutagenesis of the proximal Runx1 and C/EBPβ consensus elements in the P2X3 promoter decrease Runx1- and C/EBPβ-mediated transcriptional activity. Moreover, C/EBPβ-mediated enhancement of the P2X3 promoter requires a functional Runx1 binding site. Altogether our results support a functional and coordinated role for Runx1 and C/EBPβ transcription factors during activation of P2X3 gene transcription.

The extent of trauma in a patient can be difficult for a clinician to quantify. A prospective study was performed on 2 groups of dogs undergoing either ovariohysterectomy or hemilaminectomy. The serum activity of creatine kinase and serum concentration of C-reactive protein were evaluated preoperatively and then at 4, 6, 8, 12, 24 and 48 hours postoperatively in both groups. The results were compared statistically both within and between the 2 groups. A wide range of results was found at each time point for both analytes although there were no significant differences for either analyte between the 2 surgical groups preoperatively. Thereafter there were significant differences in creatine kinase activity levels between the 2 groups. C-reactive protein concentration results were very similar in the 2 groups with no statistical difference at any time point. The results of this study suggest that the evaluation of CK and CRP at any one time point in a traumatised animal is of limited value. However, the evaluation of the trend of these 2 analytes, even over a relatively short time period, may allow for useful prognostication in clinical cases.

Peroxisome proliferator activated receptors (PPARs, alpha, beta/delta, gamma) control lipid homeostasis and differentiation in various tissues and tumor cells. PPARbeta and PPARgamma increase oligodendrocyte maturation in glial mixed populations and spinal cord oligodendrocytes, respectively, and PPARbeta is known to modulate the activity of other PPARs. To assess a possible interaction between PPARs in glial cell differentiation we used the undifferentiated C6 glioma cell line as model. These cells express all three PPARs, but only PPARgamma shows transcriptional activity in agonist-based reporter gene assay. Agonist-activated PPARgamma up-regulates oligodendrocyte markers, down-regulates an astrocyte marker, and increases alkyl-dihydroxyacetone phosphate synthase, enzyme involved in the synthesis of myelin-rich plasmalogens. Similar effects are induced in PPARgamma overexpressing cells, which in addition show PPARbeta up-regulation. PPARbeta or PPARalpha agonists show no effect. Nevertheless, PPARbeta overexpression up-regulates PPARgamma and commits C6 cells to oligodendrocytes; effect that is abrogated by a PPARgamma antagonist or PPARgamma interference RNA. Moreover, PPARbeta overexpression also induces PPARalpha and its target genes, including acyl-CoA oxidase, enzyme involved in very long chain fatty acid recycling, and in the synthesis of myelin components such as docosahexaenoic acid. These results indicate for the first time, that PPARs concertedly cooperate in C6 glioma cell differentiation to oligodendrocytes. Further, they suggest that active PPARbeta might be essential for increasing oligodendrocyte distinctive markers and enzymes required for myelin synthesis in C6 glioma cells through up-regulation of PPARgamma and PPARalpha.

Philadelphia chromosome-positive chronic myelogenus leukemia (CML) is widely treated with imatinib mesylate (imatinib), a potent inhibitor of the Bcr-Abl tyrosine kinase. However, resistance to this compound remains a concern. Current treatment approaches include combinations of imatinib with nucleoside analogs such as gemcitabine, which requires equilibrative nucleoside transporters (ENTs) for uptake, to overcome this resistance. Here we report that imatinib treatment decreased ENT1-dependent activity and mRNA expression. Although, imatinib-resistant cells showed decreased levels of both ENT1 and ENT2 activity and expression, these cells remained sensitive to gemcitabine, suggesting that nucleoside analogs can be used as adjunctive therapy.

This observational study of 100 dogs naturally infected with Babesia canis rossi determined whether severity of parasitaemia was associated with outcome of infection and documented the relative distribution of parasitised red blood cells (pRBC) in capillary and venous circulation. The association between increased parasitaemias and outcome with a clinically compromised circulation was also investigated. Outcome was defined as either hospitalisation with death, or hospitalisation with eventual recovery or treatment as an outpatient. Dogs were enrolled if large babesias were found on stained thin capillary blood smears made from an ear prick. Thin venous smears were prepared from jugular or cephalic blood. Parasitaemias were manually counted and expressed as the percent pRBC. Ten dogs died, 50 recovered after hospitalisation and 40 were treated as outpatients. Venous sampling site did not affect venous parasitaemia (P=0.6). Both capillary and venous parasitaemias of dogs that died were significantly higher than those of dogs that recovered after hospitalisation (P=0.002) and dogs that were treated as outpatients (P

BACKGROUND: Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. METHODS: Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of (3)H-adenosine. RESULTS: Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. CONCLUSION: The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.

Introduction: Gastric cancer (GC) is the second cause of cancer-related death worldwide and the leading one in Latin America. It has been proposed that the silencing of Reprimo (RPRM), by epigenetic mechanisms, is a putative biomarker of GC. RPRM is a p53 dependent G2 arrest mediator and is considered to be tumor suppressor gene involved in the pathogenesis of numerous cancers such as gastric, colon, pituitary, among others. We propose that the reactivation of RPRM expression, by means of an activator Artificial Transcription Factor (ATF), modifies the proliferation rate of GC cell lines in vitro. Methods: An activator ATF for RPRM was designed using the method proposed by zincfingertools.org. Thus, an ATF with a unique domain of six zinc-finger capable of binding specifically to 18 consecutive base pairs, located in the promoter region of RPRM, was engineered. This ATF was then inserted into a modified pcDNA3.1+ vector coupled with a potent transcription activator domain (VP64). A luciferase reporter assay was performed using a pGL3 vector (Promega), linked to a 634 base pairs region of the promoter region of RPRM, in order to evaluate the efficiency and specificity of the synthetized ATF. Finally, the ATF was transiently transfected with lipofectamine2000 (Invitrogen). Subsequently, RPRM mRNA expression was evaluated by a RT-qPCR and proliferation rate was evaluated by a MTS assay. The experiments were conducted on AGS and MKN28 GC cell lines. Results: The ATF designed was able to significantly increase the luciferase activity vs controls, both in MKN28 and AGS cell lines. Accordingly, RPRM mRNA was upregulated in the presence of the ATF. These findings were associated with a lower proliferation rate in both cell lines. Since the AGS cell line is highly methylated in the promoter region of RPRM, it might explain a significantly lower response to the presence of ATF, in comparison with that of MKN28. Discussion: Our work suggests that a RPRM specific ATF could be used as a novel tool to reactivate this epigenetically silenced tumor suppressor gene. Moreover, the upregulation of RPRM modifies one of the hallmarks of cancer pathways by reducing the proliferation rate of cancerous cell lines in vitro. Grant Support: Fondecyt #1111014 and Fondef #D09I1137 from the Government of Chile to AHC.

This article reviews Pneumocystis carinii and presents four cases in the miniature dachshund. The cases presented with hyperpnoea, tachypnoea and exercise intolerance. There were also clinical signs suggestive of immune incompetence in all the dogs. P carinii pneumonia was diagnosed in all four cases on transtracheal aspirate cytology. Immunological studies showed low globulin levels on serum electrophoresis, decreased lymphoblast transformation response (in the two cases that were tested) and a deficiency of immunoglobulins A, G and M. Light and electron microscopy as well as anti-canine immunoglobulin G immunoperoxidase staining studies were performed on one case which had died because of the disease. From these four cases, it appears that P carinii pneumonia in the miniature dachshund may be the result of an immunodeficiency. It does not, however, appear to be a classic primary severe combined immunodeficiency syndrome as the dogs appeared to respond to treatment, did not show growth failure and did not manifest overwhelming commensurate bacterial infections.

Uric acid was used as a test for liver disease before the advent of enzymology. Three old studies criticised uric acid as a test of liver function. Uric acid, as an end-product of purine metabolism in the liver, deserved re-evaluation as a liver function test. Serumtotal bile acids are widely accepted as the most reliable liver function test. This study compared the ability of serumuric acid concentration to assess liver function with that of serumpre-prandial bile acids in dogs. In addition, due to the renal excretion of uric acid the 2 assays were also compared in a renal disease group. Using a control group of healthy dogs, a group of dogs with congenital vascular liver disease, a group of dogs with non-vascular parenchymal liver diseases and a renal disease group, the ability of uric acid and pre-prandial bile acids was compared to detect reduced functional hepatic mass overall and in the vascular or parenchymal liver disease groups separately. Sensitivities, specificities and predictive value parameters were calculated for each test. The medians of uric acid concentration did not differ significantly between any of the groups, whereas pre-prandial bile acids medians were significantly higher in the liver disease groups compared with the normal and renal disease group of dogs. The sensitivity of uric acid in detecting liver disease overall was 65% while the specificity of uric acid in detecting liver disease overall was 59 %. The sensitivity and specificity of uric acid in detecting congenital vascular liver disease was 68%and 59 %, respectively. The sensitivity and specificity of uric acid in detecting parenchymal liver disease was 63%and 60 %, respectively. The overall positive and negative predictive values for uric acid in detecting liver disease were poor and the data in this study indicated uric acid to be an unreliable test of liver function. In dogs suffering from renal compromise serum uric acid concentrations may increase into the abnormal range due to its renal route of excretion.

Carboxyhaemoglobin fractions were found to be significantly higher (p0,05) in dogs (n=5) with severe babesiosis than in control subjects (n=5). The enzymatic conversion of haem to biliverdin by haem oxygenase is the only known source of endogenous carbon monoxide. We propose that the increased production of endogenous carbon monoxide following the haemolysis associated with babesiosis, results in the carboxyhaemoglobinaeroia observed in this study. The superimposition of carboxyhaemoglobinaemia on severe anaemia results in further compromise of the oxygen status of dogs with severe babesiosis, and probably plays a role in the pathogenesis of the hypoxic tissue damage associated with this condition.