Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9. Methods: Flow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively. Results: Cell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9. Conclusions: Patients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

Publisher's version (útgefin grein)Background: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.[Figure not available: see fulltext.]This work was supported by the Swedish research council FORMAS (grant no. 942-2015-508).Peer Reviewe

Cytochrome P450 3A and ABC-transport proteins in horse

In this thesis cytochrome P450 3A (CYP3A) and the ABC-transport proteins P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance protein 1 (MRP1) and multidrug resistance protein 2 (MRP2) have been studied in horse. In the first part of the thesis the gene and protein expression, cellular localization and metabolic activity of CYP3A were investigated in the intestines, liver and upper respiratory airways. High levels of CYP3A gene and protein expression and metabolic activity were found in the small intestines. The CYP3A-related activity in the upper respiratory airways was high in spite of rather low levels of CYP3A gene and protein expression. A possible explanation in this observation is that there is an efficient electron transport from NADPH P450 reductase and cytochrome b5 to CYP3A in these tissues. In the second part of the thesis gene and protein expression and cellular localization of P-gp, BCRP, MRP1 and MRP2 were studied in the intestines, liver and kidney. The expression and localization of the transporters in the tissues of horse showed both similarities and differences compared to other species. Collectively the results of the present thesis indicate that CYP3A and the examined ABC-transport proteins have important roles for bioavalability and elimination of substrate compounds in horse

Parasites in Horses Kept in A 2.5 Year-Round Grazing System in Nordic Conditions without Supplementary Feeding

Horse grazing can be favorable from a biological diversity perspective. This study documented the occurrence of endo- and ectoparasites and sought to reduce parasite egg excretion with the anthelmintic drug pyrantel in 12 Gotlandsruss stallions maintained in a year-round grazing system for 2.5 years. Feces samples were collected monthly and all horses were treated with pyrantel, the anthelmintic drug of choice in biological diversity preservation, at study population mean cyathostomin eggs per gram (EPG) of &gt;200. The relationship between cyathostomin EPG and body condition was studied, as was horse behavioral response to Bovicola equi (chewing louse) infestation. Eggs of cyathostomins (small strongyles), Parascaris spp. (roundworm), Oxyuris equi (pinworm), Anoplocephala perfoliata (tapeworm), and Gasterophilus spp. (botfly) were detected at least once during the trial. Excretion of cyathostomin eggs was highest during summer&ndash;autumn and increased year-on-year. No relationship was found between cyathostomin EPG and body condition. Infestation with B. equi did not affect the number of scratching sessions compared with unaffected horses. Therefore, in this year-round grazing system, pyrantel treatment had to be complemented with moxidectin to reduce excretion of cyathostomin eggs, thus compromising biological diversity

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

Background: Parascaris univalensis a pathogenic parasite of foals and yearlings worldwide. In recent years,Parascarisspp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adultP. univalensworms afterin vitroexposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways.Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10(-9) M, 10(-11) M, 10(-13) M), pyrantel citrate (10(-6) M, 10(-8) M, 10(-10) M), thiabendazole (10(-5) M, 10(-7) M, 10(-9) M) or without anthelmintics (control) at 37 degrees C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system.Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj &lt; 0.05 and log2 fold change >= 1 or &lt;= - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunitlgc-37, which was upregulated only in worms exposed to 10(-9) M of ivermectin.Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described inP. univalensand future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance

The separation and identification of parasite eggs from horse feces

Freely grazing horses are at risk of infection by parasites such as Parascaris equorum (roundworm), Strongylus spp. (large bloodworms), Cyathostomes (small bloodworms), and Anoplochephala perfoliata (tapeworms). Mixed infections are common and diagnosis is based on demonstrations of eggs in feces followed by identification of larvae after fecal culture. Drug resistance is a growing problem, not least because treatments tend to be cheaper than diagnosis and “just in case” treatments common. There is a need for improved methods that are easy to use, rapid and cheap. Furthermore, a successful approach may find use with other livestock such as ruminants and pigs

Evaluation of Strategies to Reduce Equine Strongyle Infective Larvae on Pasture and Study of Larval Migration and Overwintering in a Nordic Climate

Horses, as grazing animals, are inadvertently exposed to intestinal parasites that, if not controlled, may cause disease. However, the indiscriminate use of anthelmintic drugs has led to drug resistance, highlighting the need for pasture-management practices to reduce the level of parasitic exposure and lessen reliance on drugs. The efficacy of such methods depends both on the epidemiology of the parasites and the prevailing weather conditions. The aim of the study was to investigate the effect of faecal removal and harrowing on reducing the number of parasite larvae in herbage. Moreover, the migratory and survival ability of strongyle larvae in a Nordic climate was studied. Faeces from horses naturally infected with strongyle nematodes were used to contaminate pastures and grass samples were collected to harvest larvae. Twice-weekly faecal removal significantly reduced larval yields, whereas harrowing on a single occasion under dry weather conditions in the summer did not. Strongyle larvae were able to migrate 150 cm from the faecal pats, but most larvae were found within 50 cm. Both Cyathostominae and S. vulgaris survived the winter months with larvae harvested up to 17&ndash;18 months after faecal placement. Resting of pastures for one year greatly reduced the parasite level, but two years of rest were required for parasite-free pasture

Demonstration of reduced efficacy against cyathostomins without change in species composition after pyrantel embonate treatment in Swedish equine establishments

Consisting of approximately 50 different species, the cyathostomin parasites are ubiquitous in grazing horses. Co-infection with several species is common, and large burdens can cause the fatal disease of larval cyathostominosis. Due to intense anthelmintic drug use, cyathostomin resistance has developed to all available anthelmintic drug groups. Resistance to the anthelmintic drug pyrantel (PYR) has been documented in over 90% of studies published over the past two decades. In Sweden, a study performed in the early 2000s only confirmed resistance in 4.5% of farms. Further, prescription-only administration of equine anthelmintic drugs was enforced in Sweden in 2007. However, it is unknown if this conservative drug use has maintained PYR efficacy in cyathostomins. The aim of the present study was to investigate the effect of PYR on cyathostomin infection in Sweden using fecal egg count reduction tests (FECRTs). Further, the effect of PYR treatment on cyathostomin species composition was studied using metabarcoding.Sixteen farms with at least six horses excreting a minimum of 100 eggs per gram feces were included. Using the current World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines, PYR resistance was demonstrated in nine of farms, with seven farms showing full susceptibility. Farms with low biosecurity measures had significantly lower efficacy of PYR treatment. The most common cyathostomin species were Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cys. calicatus, Cys. goldi, Cys. minutus, Coronocyclus coronatus and Cya. pateratum, accounting for 97% of all sequence reads prior to treatment. Of these, Cyc. nassatus and Cya. catinatum had the highest occurrence, accounting for 68% of all sequence reads prior to PYR treatment. Treatment did not significantly affect the species composition. The results highlight the importance of drug efficacy testing when using PYR to treat cyathostomin infection, even when selective anthelmintic treatment and thus low treatment intensity, is used on the farm

Transcriptomics of ivermectin response in Caenorhabditis elegans: Integrating abamectin quantitative trait loci and comparison to the Ivermectin-exposed DA1316 strain.

Parasitic nematodes pose a significant threat to human and animal health, as well as cause economic losses in the agricultural sector. The use of anthelmintic drugs, such as Ivermectin (IVM), to control these parasites has led to widespread drug resistance. Identifying genetic markers of resistance in parasitic nematodes can be challenging, but the free-living nematode Caenorhabditis elegans provides a suitable model. In this study, we aimed to analyze the transcriptomes of adult C. elegans worms of the N2 strain exposed to the anthelmintic drug Ivermectin (IVM), and compare them to those of the resistant strain DA1316 and the recently identified Abamectin Quantitative Trait Loci (QTL) on chromosome V. We exposed pools of 300 adult N2 worms to IVM (10-7 and 10-8 M) for 4 hours at 20°C, extracted total RNA and sequenced it on the Illumina NovaSeq6000 platform. Differentially expressed genes (DEGs) were determined using an in-house pipeline. The DEGs were compared to genes from a previous microarray study on IVM-resistant C. elegans and Abamectin-QTL. Our results revealed 615 DEGs (183 up-regulated and 432 down-regulated genes) from diverse gene families in the N2 C. elegans strain. Of these DEGs, 31 overlapped with genes from IVM-exposed adult worms of the DA1316 strain. We identified 19 genes, including the folate transporter (folt-2) and the transmembrane transporter (T22F3.11), which exhibited an opposite expression in N2 and the DA1316 strain and were deemed potential candidates. Additionally, we compiled a list of potential candidates for further research including T-type calcium channel (cca-1), potassium chloride cotransporter (kcc-2), as well as other genes such as glutamate-gated channel (glc-1) that mapped to the Abamectin-QTL