Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease

Background: Leukocyte cytosolic proteins (e.g., calprotectin) are emerging biomarkers for inflammatory bowel disease. Leukocyte aryl esterase activity has been commonly used for sensitive detection of leukocytes in human body fluids such as urine. Urine test strip results are generally reported in categories. As automated strip readers allow quantitative data to be reported, sensitive quantitative detection of leukocytes in body fluids has become possible. Here, we explored the use of leukocyte esterase as a potential alternative faecal biomarker for inflammatory bowel disease. Methods: We evaluated leukocyte esterase activity in faecal extracts and compared Cobas u 411 (Roche) quantitative reflectance data with calprotectin concentration for 107 routine samples. Stability of leukocyte esterase for trypsin digestion was carried out by adding trypsin to the extract. Incubation occurred at 37 ° C for 24 h or 48 h. Results: Reproducibility of the reflectance signal was good (within-run imprecision: 6.1%; between-run imprecision: 6.2%). Results were linear in the range 10 3 – 10 6 WBC/100 mg faeces. The lower limit of detection was 4 WBC/ μ L and the lower limit of quantification was 5 WBC/ μ L. Stability of LE activity in stool and faecal matrix was good. An adequate correlation was obtained between leukocyte esterase activity and the faecal calprotectin concentration: log(y)  =  4.28 + 0.29log(x). In vitro experiments monitored the digestion of leukocyte esterase and faecal calprotectin. Leukocyte esterase activity was significantly less affected by trypsin activity than calprotectin immunoreactivity. Conclusions: Quantitative leukocyte esterase activity of faecal extracts provides information about the leukocyte count in the gut lumen. Leukocyte esterase is a promising and affordable alternative biomarker for monitoring inflammatory bowel disease

Accuracy of transcranial magnetic stimulation and a Bayesian latent class model for diagnosis of spinal cord dysfunction in horses

Background: Spinal cord dysfunction/compression and ataxia are common in horses. Presumptive diagnosis is most commonly based on neurological examination and cervical radiography, but the interest into the diagnostic value of transcranial magnetic stimulation (TMS) with recording of magnetic motor evoked potentials has increased. The problem for the evaluation of diagnostic tests for spinal cord dysfunction is the absence of a gold standard in the living animal. Objectives: To compare diagnostic accuracy of TMS, cervical radiography, and neurological examination. Animals: One hundred seventy-four horses admitted at the clinic for neurological examination. Methods: Retrospective comparison of neurological examination, cervical radiography, and different TMS criteria, using Bayesian latent class modeling to account for the absence of a gold standard. Results: The Bayesian estimate of the prevalence (95% CI) of spinal cord dysfunction was 58.1 (48.3%-68.3%). Sensitivity and specificity of neurological examination were 97.6 (91.4%-99.9%) and 74.7 (61.0%-96.3%), for radiography they were 43.0 (32.3%-54.6%) and 77.3 (67.1%-86.1%), respectively. Transcranial magnetic stimulation reached a sensitivity and specificity of 87.5 (68.2%-99.2%) and 97.4 (90.4%-99.9%). For TMS, the highest accuracy was obtained using the minimum latency time for the pelvic limbs (Youden's index = 0.85). In all evaluated models, cervical radiography performed poorest. Clinical Relevance: Transcranial magnetic stimulation-magnetic motor evoked potential (TMS-MMEP) was the best test to diagnose spinal cord disease, the neurological examination was the second best, but the accuracy of cervical radiography was low. Selecting animals based on neurological examination (highest sensitivity) and confirming disease by TMS-MMEP (highest specificity) would currently be the optimal diagnostic strategy

Single domain antibody multimers confer protection against rabies infection

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection

Proteolysis is a confounding factor in the interpretation of faecal calprotectin

Background: Calprotectin is a 36 kDa calcium and zinc binding protein. An increased level of calprotectin points towards inflammatory bowel disease. However, the biomarker calprotectin shows 14 potential cleavages sites for trypsin. Next to trypsin, also the presence of its inhibitor α1-antitrypsin after a gastrointestinal bleeding may affect calprotectin testing. In this study, effects of trypsin and α1-antitrypsin as potential confounders for faecal calprotectin testing are investigated. Methods: An in vitro model was created. As calprotectin source, leukocytes were isolated and subsequently lysed (1% Triton X-100) and diluted in faecal matrix. Trypsin digestion was carried out by adding trypsin. Incubation occurred for 24 h or 48 h (37 °C). To study the influence of α1-antitrypsin on trypsin, the same experiment was repeated after adding serum containing α1-antitrypsin. Results: In vitro experiments enabled monitoring of the faecal calprotectin digestion, leading to loss of immunoreactivity. Trypsin activity was a potential confounder in the interpretation of calprotectin, in particular for proximal lesions, where exposure of calprotectin to trypsin is prolonged. Relative calprotectin loss was proportional to the amount of trypsin. Decrease of calprotectin was more pronounced after 48 h of incubation in comparison to 24 h of incubation. Analogue experiments also showed stable calprotectin values after adding α1-antitrypsin. Conclusions: Transit time, trypsin activity and addition of blood as a source of α1-antitrypsin may be regarded as potential confounders in the interpretation of calprotectin results. Age-related cut-off values depending on the anatomical localisation of the lesions could improve the diagnostic efficiency of calprotectin testing

The benefits of computed tomography imaging in the diagnosis, prognosis and treatment planning of equine orthopedic patients illustrated by six cases = De voordelen van computertomografie in de diagnose, prognose en behandeling van orthopedische aandoeningen bij het paard : zes casussen

Radiography and/or ultrasonography are the first imaging modalities for diagnosing ortho-pedic pathology in equine patients. However, in some cases, cross-sectional imaging is necessary to reach a more accurate diagnosis. Six cases were retrospectively selected from the imaging database of the Faculty of Veterinary Medicine (Ghent University) to illustrate the benefits of computed tomography (CT) in orthopedic patients. In two cases, CT demonstrated osteomyelitis lesions in two young foals, which could not be detected with radiography and ultrasonography. In three cases, CT was performed for surgical planning of fracture repair, and in one case CT demonstrated multiple lesions at the soft tissues and ligamentous insertions in the stifle. In all cases, CT revealed additional findings, which were important for the treatment and prognosis of the patient