Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp.

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 µm (10 µg mL-1) and IVM at 1.1 µm (1 µg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Dealing with double trouble: Combination deworming against double-drug resistant cyathostomins.

An alternative control regimen for drug-resistant parasites is combination deworming, where two drugs with different modes of action are administered simultaneously to target the same parasite. Few studies have investigated this in equine cyathostomins. We previously reported that an oxibendazole (OBZ) and pyrantel pamoate (PYR) combination was not sustainable against a cyathostomin population with high levels of OBZ and PYR resistance. This study consisted of a field study and two computer simulations to evaluate the efficacy of a moxidectin-oxibendazole (MOX-OBZ) combination against the same cyathostomin population. In the field study, anthelmintic treatments occurred when ten horses exceeded 100 eggs per gram. Fecal egg counts and efficacy evaluations were performed every two weeks. The two simulations utilized weather data as well as equine and parasite population parameters from the field study. The first simulation repeated the treatment schedule used in the field study over a 40 year period. The second evaluated efficacies of combination treatments using selective therapy over 40 years. In the field study, efficacies of MOX and both combination treatments were 100%. The egg reappearance period for MOX was 16 weeks, and the two combination treatments were 12 and 18 weeks. The first (46.7%) and last (40.1%) OBZ efficacies were not significantly different from each other. In the simulation study, the combination treatment delayed MOX resistance development compared to when MOX was used as a single active. This occurred despite the low efficacy of OBZ. The second set of simulations identified combination treatments used with selective therapy to be the most effective at delaying MOX resistance. Overall, this study supports the use of combination treatment against drug-resistant cyathostomins, when one of the actives exhibits high efficacy, and demonstrates benefits of this approach despite substantially lowered efficacy of the other active ingredient.

Nocturnal Gamma-Hydroxybutyrate Reduces Cortisol-Awakening Response and Morning Kynurenine Pathway Metabolites in Healthy Volunteers.

BACKGROUND: Gamma-hydroxybutyrate (GHB; or sodium oxybate) is an endogenous GHB-/gamma-aminobutyric acid B receptor agonist. It is approved for application in narcolepsy and has been proposed for the potential treatment of Alzheimer's disease, Parkinson's disease, fibromyalgia, and depression, all of which involve neuro-immunological processes. Tryptophan catabolites (TRYCATs), the cortisol-awakening response (CAR), and brain-derived neurotrophic factor (BDNF) have been suggested as peripheral biomarkers of neuropsychiatric disorders. GHB has been shown to induce a delayed reduction of T helper and natural killer cell counts and alter basal cortisol levels, but GHB's effects on TRYCATs, CAR, and BDNF are unknown. METHODS: Therefore, TRYCAT and BDNF serum levels, as well as CAR and the affective state (Positive and Negative Affect Schedule [PANAS]) were measured in the morning after a single nocturnal dose of GHB (50 mg/kg body weight) in 20 healthy male volunteers in a placebo-controlled, balanced, randomized, double-blind, cross-over design. RESULTS: In the morning after nocturnal GHB administration, the TRYCATs indolelactic acid, kynurenine, kynurenic acid, 3-hydroxykynurenine, and quinolinic acid; the 3-hydroxykynurenine to kynurenic acid ratio; and the CAR were significantly reduced (P < 0.05-0.001, Benjamini-Hochberg corrected). The quinolinic acid to kynurenic acid ratio was reduced by trend. Serotonin, tryptophan, and BDNF levels, as well as PANAS scores in the morning, remained unchanged after a nocturnal GHB challenge. CONCLUSIONS: GHB has post-acute effects on peripheral biomarkers of neuropsychiatric disorders, which might be a model to explain some of its therapeutic effects in disorders involving neuro-immunological pathologies. This study was registered at ClinicalTrials.gov as NCT02342366.

Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro.

In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms obtained from naturally infected foals at necropsy were immediately placed in culture flasks containing 200 mL of culture media. A variety of media types, nutrient supplementation and environmental conditions were examined. A motility-based scoring system was used to assess worm viability. Worms maintained in Roswell Park Memorial Institute-1640 had significantly better viability than any other media (P < 0.0001) and all media types supplemented with any of the nutrients examined (P < 0.0001). The use of a platform rocker also significantly improved viability (P = 0.0305). This is the first study to examine the requirements for maintaining Parascaris spp. intestinal stages in vitro and to evaluate their viability based on movement using an objective scoring system.

Effects of homogenizing methods on accuracy and precision of equine strongylid egg counts.

Recommendations for control of equine strongylid parasites are based on regular determination of fecal egg counts to identify high strongylid shedders and to evaluate treatment efficacy. The McMaster technique has long been used as the standard egg counting technique in equine veterinary practice in most parts of the world, but recent work has found the Mini-FLOTAC technique to perform with significantly better accuracy and precision. The Mini-FLOTAC system comes with a homogenizing device, termed the Fill-FLOTAC, and it has been hypothesized that this device might have a significant impact on accuracy and precision. The aim of the present study was to investigate the impact of the Fill-FLOTAC homogenizer in comparison with the classical McMaster approach, where samples are suspended in flotation medium by stirring with tongue depressor in a plastic cup. The study compared the McMaster and Mini-FLOTAC techniques, but also included cross-over versions where the Fill-FLOTAC was used with the McMaster chamber, and the tongue depressor and plastic cup homogenizing method was used with the Mini-FLOTAC counting disc. Fecal samples were collected from horses naturally infected with mixed strongylid species. Five samples were included from each of the following egg count levels: 0-500, 501-1000, and >1000 eggs per gram (EPG). Each sample was then analyzed with all four set-ups with three subsamples collected from the same suspension, and three repeated counts determined on each subsample. Both the Fill-FLOTAC homogenizer (p = 0.0098) and the McMaster counting chamber (p = 0.0298) were significantly associated with higher strongylid egg counts, whereas the Mini-FLOTAC chamber was associated with a lower coefficient of variation (p < 0.0001). Precision, however, was not associated with homogenization method (p = 0.9341). Taken together, this study suggests that while the homogenizing method has a positive effect on egg count accuracy, the counting chamber appears to primarily affect precision.

Evaluation of the mucosal inflammatory responses to equine cyathostomins in response to anthelmintic treatment.

Members of Cyathostominae are pervasive parasites of equids that can cause larval cyathostominosis, a potentially life-threatening disease that occurs when a multitude of encysted larvae synchronously excyst from the wall of the large intestine. Moxidectin and fenbendazole are the two current labeled drugs that target the encysted larval stages; however, there is limited knowledge of the local inflammatory response to the larvae and to the two treatments in clinically healthy horses. This study is the first to evaluate the local inflammatory response to cyathostomin larvae and to larvicidal treatment at 2 and 5 weeks post treatment. Thirty-six ponies with naturally acquired cyathostomin infections were randomly allocated into 3 groups: Group 1, fenbendazole at 10 mg/kg for 5 days, Group 2, a single dose of moxidectin at 0.4 mg/kg, and Group 3, untreated controls. Tissue samples from the cecum and dorsal and ventral colons were used for histopathological and immunohistochemical evaluation. Tissues were stained with routine hematoxylin and eosin (H&E) for light microscopy and immunohistochemically for MAC387, CD20, and CD3 for differentiation of activated macrophages, B cells, and T cells, respectively. Semiquantitative scores were assigned for all inflammatory cell types and fibrous connective tissue. Larvae observed by light microscopy were enumerated and classified by stage. Mucosal ulcerations and submucosal granulomas were also enumerated. Mean macrophage scores were higher in the moxidectin group than the fenbendazole group (p = 0.0185) and the control group had a higher activated macrophage score than both treatment groups (p = 0.0104, p = 0.0004). T lymphocyte scores were higher in the moxidectin group when compared to the control group (p = 0.0069). Goblet cell hyperplasia scores were elevated at 5 weeks post treatment compared to 2 weeks post treatment (p = 0.0047) and were elevated in the ventral colon compared to the dorsal colon (p = 0.0301). Eosinophil scores were elevated surrounding degenerative larvae when compared to intact larvae (p = 0.0001). Mucosal ulcerations were found only in the control group at 2 weeks post treatment. This study found subtle inflammatory differences between treatment groups but provided new information about goblet cells and eosinophils in relation to encysted cyathostomin larvae.

Management practices associated with strongylid parasite prevalence on horse farms in rural counties of Kentucky.

Anthelmintic resistance among cyathostomin parasites is a wide-spread problem. The parasite control guidelines written by the American Association of Equine Practitioners (AAEP) encourages the preservation of anthelmintic efficacy by reducing treatment frequency, using targeted deworming, and implementing environmental management practices. While there is knowledge regarding parasite management practices of affluent horse farms in the United States, surveys rarely explore the rural and underserved regions. The purpose of this study was to observe the management practices of horse farms in rural regions Kentucky, including working Amish farms, and determine factors associated with strongyle prevalence. A total of 160 horses among 38 owners from 28 different farms were enrolled in this study. A questionnaire survey regarding equine information, farm management, and deworming history was performed with each owner. Fecal samples were collected to determine fecal egg counts, perform coprocultures for subsequent strongyle larvae identification, and Strongylus vulgaris specific PCR. Serum samples were collected for the S. vulgaris antibody specific ELISA. The mean number of deworming treatments given in the last year was 2.1 with a 95% confidence interval of 1.9-2.3 with ivermectin being the most common active used. Statistical analysis showed horses treated within the last three months with a macrocylic lactone (ML) drug had significantly lower egg counts than horses treated with a ML 7-9 months ago (p = .0005). Despite the AAEP recommendations to reduce the overall number of treatments by using a surveillance-based approach and to no longer rotate treatments, only 17 horses reportedly had a fecal sample submitted for a fecal egg count and 65 horses were dewormed in a rotational manner. Horses whose owners utilized an informative deworming source (i.e., veterinarian, internet, magazine, local feed store) also had significantly lower counts (p = .0026). All coprocultures were negative for S. vulgaris while five horses were PCR positive. Interestingly, 95 horses tested ELISA positive for S. vulgaris. The strongyle egg counts of the working Amish horses were not significantly different from the other horses in this study and deworming practices including the use of efficacious drugs and low treatment frequencies were in accordance with the AAEP guidelines. This study was the first to summarize deworming management practices of rural regions in Kentucky, including a working Amish community. Overall, horse owners employed deworming practices recommended by the AAEP, however rotational deworming is still commonly implemented and fecal egg counts are rarely used.

Elasticity and tumorigenic characteristics of cells in a monolayer after nanosecond pulsed electric field exposure.

Nanosecond pulsed electric fields (nsPEFs) applied to cells can induce different biological effects depending on pulse duration and field strength. One known process is the induction of apoptosis whereby nsPEFs are currently investigated as a novel cancer therapy. Another and probably related change is the breakdown of the cytoskeleton. We investigated the elasticity of rat liver epithelial cells WB-F344 in a monolayer using atomic force microscopy (AFM) with respect to the potential of cells to undergo malignant transformation or to develop a potential to metastasize. We found that the elastic modulus of the cells decreased significantly within the first 8 min after treatment with 20 pulses of 100 ns and with a field strength of 20 kV/cm but was still higher than the elasticity of their tumorigenic counterpart WB-ras. AFM measurements and immunofluorescent staining showed that the cellular actin cytoskeleton became reorganized within 5 min. However, both a colony formation assay and a cell migration assay revealed no significant changes after nsPEF treatment, implying that cells seem not to adopt malignant characteristics associated with metastasis formation despite the induced transient changes to elasticity and cytoskeleton that can be observed for up to 1 h.

Ultrasonographic and radiographic results from a two-year controlled trial of immediate or one-year-delayed addition of infliximab to ongoing methotrexate therapy in patients with erosive early rheumatoid arthritis.

OBJECTIVE: To compare the impact of immediate and delayed introduction of anti-tumor necrosis factor therapy on inflammation and structural damage in methotrexate (MTX)-treated patients with early rheumatoid arthritis (RA). METHODS: Twenty-four patients with erosive early RA (duration < 3 years) who were receiving MTX were randomized to receive infliximab 5 mg/kg or placebo infusions at weeks 0, 2, and 6, and then every 8 weeks through week 46. Beginning at week 54 and thereafter, all patients received infliximab 5 mg/kg. Metacarpophalangeal joints were scanned using high-frequency ultrasonography and power Doppler imaging. Radiographs were evaluated using the modified Sharp/van der Heijde scoring system. RESULTS: From baseline to week 54, total synovial thickness was significantly improved in the infliximab + MTX group compared with the placebo + MTX group (median reduction 95.8% versus 37.5%; P = 0.005), as was the total color Doppler area (CDA; vascularity assessment) (median reduction 100% and 47.1%, respectively; P = 0.025). From week 0 to week 110, no significant between-group difference was observed in the change from baseline for total synovial thickening or the total CDA. At week 54, greater progression in the Sharp/van der Heijde score was apparent in patients receiving placebo + MTX compared with those receiving infliximab + MTX. Although radiographic progression in the placebo + MTX group was greatly reduced in the second year (after initiation of infliximab therapy), marked differences were observed between the infliximab + MTX group (median change in the Sharp/van der Heijde score 4.0) and the placebo + MTX group (median change 14.5) from baseline to week 110 (P = 0.076). CONCLUSION: The results indicate that the efficacy of 2 years of combination therapy with infliximab + MTX for inhibiting cumulative structural damage was superior to that of 1 year of treatment with MTX alone followed by the addition of infliximab.

Comparison of ultrasonographic assessment of synovitis and joint vascularity with radiographic evaluation in a randomized, placebo-controlled study of infliximab therapy in early rheumatoid arthritis.

OBJECTIVE: To investigate sensitive ultrasonographic imaging methods for detection of synovial thickness and vascularity to discriminate between patients with early rheumatoid arthritis (RA) receiving infliximab + methotrexate (MTX) versus placebo + MTX over 18 weeks, and to compare the relationship between synovial thickening and vascularity at baseline and radiologic damage to joints of the hands and feet at 54 weeks. METHODS: Patients with early RA (duration <3 years) receiving stable dosages of MTX were randomly assigned to receive blinded infusions of 5 mg/kg infliximab (n = 12) or placebo (n = 12) at weeks 0, 2, 6, and then every 8 weeks until week 46. At baseline and week 18, clinical assessments were performed, and metacarpophalangeal joints were assessed by high-frequency ultrasonography and power Doppler ultrasonography measurements. Radiographs of the hands and feet taken at baseline and at 54 weeks were evaluated using the van der Heijde modification of the Sharp method (vdH-Sharp score). RESULTS: Using changes in the total vdH-Sharp score over 54 weeks and changes in synovial thickening and joint vascularity at 18 weeks, we were able to distinguish those patients receiving infusions of infliximab + MTX from those receiving placebo + MTX. Sonographic measurements of synovial thickening and vascularity at baseline in the placebo + MTX group demonstrated clear relationships with the magnitude of radiologic joint damage at week 54. Infliximab + MTX treatment abolished these relationships. CONCLUSION: The delay or reversal of inflammatory and joint-destructive mechanisms in patients with early RA was already apparent following 18 weeks of treatment with infliximab + MTX and was reflected in radiologic changes at 54 weeks.

Osmic acid revisited: factors that predict a favourable response.

OBJECTIVE: Chemical synovectomy with osmic acid has been used for many years in the treatment of chronic synovitis that is unresponsive to systemic treatment and intra-articular corticosteroid. Our aims were to compare the safety and the efficacy of this procedure with that of alternative treatment modalities, and to identify any factors that may predict the response to treatment. METHODS: A retrospective review of 103 osmic acid injections was performed in 65 patients with chronic knee synovitis, between 1992 and 1998. After a clinical review 6 weeks after injection, the length of remission was determined by telephone survey. Thirty-six months of follow-up was available for 96 injections, with a minimum of 12 months for the remainder. Remission was defined as complete absence of pain and swelling. RESULTS: Sixty-nine (67.0%) knee joints remained completely free of pain and swelling at 6 months, falling to 52 (50.5%) at 12 months, 32 (31.1%) at 24 months, and 19 (18.4%) at 36 months. Knees with mild radiological changes experienced significantly better results compared with those with moderate or severe changes (P=0.006 and 0.046 respectively). In patients undergoing bilateral injections, there was a correlation between the duration of remission achieved for each of the two knees (r=0.83, P < 0.01). A correlation was also observed between the responses obtained after first and repeated synovectomies of the same knee (r=0.62, P < 0.05). Eighty-six per cent of injections were uncomplicated. Side-effects included pain, which followed 13 injections (12.4%), and two skin burns. CONCLUSIONS: Long-term complete remission was achieved in 18.4% of knees. This treatment may be best reserved for joints with less severe radiological changes. The response to a first osmic acid injection has a strong predictive value when considering further treatment of the same or the contralateral joint.

Thrombosis in Behçet's disease: a retrospective survey from a single UK centre.

OBJECTIVE: To contribute to knowledge of vascular occlusion in Behçet's disease (BD), prevalence and relative risk for thrombosis were ascertained retrospectively in a cohort of Caucasian patients with the disease. PATIENTS: The study included 73 BD patients (36 males, 37 females, mean age 45+/-11 yr) attending the Immunology/Rheumatology Department of Northwick Park Hospital. A group of 146 patients without BD, attending the same department, served as a control group. RESULTS: Thrombosis was more frequent in BD patients than in controls (23/73, 32% vs 7/146, 5%, P<0.001). This was accounted for by a higher prevalence of venous thrombosis in BD patients (18/73, 25% vs 4/146, 3%, P<0.001). Gender-adjusted data revealed an 11-fold risk of developing any thrombosis and a 14-fold risk of developing venous thrombosis in BD. After adjusting for differences in age at first symptoms, male BD patients showed a 6-fold higher risk of vein thrombosis. Males reported more often thrombophlebitis (13/36, 36% vs 0/0, P<0.001), folliculitis (8/36, 22% vs 1/37, 3%, P<0.01) and retinal vasculitis (13/36, 36%, vs 4/37, 11%, P=0.01) than females, in whom arthralgia prevailed (23/37, 62%, vs 12/36, 33%, P=0.01). CONCLUSION: In our population, BD confers a 14-fold risk of developing venous thrombosis. The risk is sixfold higher in male BD patients, who fare worse than females with regard to thrombophlebitis, folliculitis and retinal vasculitis.

Neurological complications in Behçet's syndrome.

The neurological complications of Behçet's syndrome have not been characterized with clarity. We present the clinical features, imaging characteristics and CSF findings of a series of 50 patients seen at the National Hospital for Neurology and Neurosurgery over the past 10 years. In this series, vascular complications had a low prevalence, whereas involvement of the brainstem was common; spinal cord lesions, hemisphere lesions and meningoencephalitis also occurred. Optic neuropathy, vestibulocochlear and peripheral nerve involvement occurred, but were rare. The prognosis for recovery was in general good, and the majority of those followed-up over a median of 3 years (range 1-19 years) had only single attacks. One-third of patients underwent further attacks, and four underwent progressive deterioration leading to disability. Factors suggesting a poor prognosis are repeated attacks, incomplete recovery, progressive disease course and a high level of CSF leucocytosis during acute attack. These data should be of help in the further definition of the clinical characteristics of this rare neurological disorder and in the planning of treatment trials.