Prospective Application of the Erythrocyte Sedimentation Rate (ESR) as a Possible Inflammatory Marker in Feline Patients.

The application of the erythrocyte sedimentation rate (ESR) in feline medicine is currently unavailable, while in canine medicine it has been rediscovered due to the introduction of an automated ESR device. Our aims were to (1) define the reference interval (RI) of the ESR in healthy cats, (2) evaluate the ESR values between healthy and ill cats, (3) evaluate relationships between the ESR and some inflammatory markers, and (4) assess ESR changes in different durations of illness (acute, chronic, or acute-on-chronic). A prospective multicentric cohort study on 200 client-owned cats: 57 healthy cats and 143 ill cats for the other aims. Healthy cats were blood donors, or young cats underwent desexing procedures. Ill cats with full clinical medical records, hematobiochemical profiles, and diagnostic procedures to reach a final diagnosis were included. The ESR was performed with MINI-PET using the same K3-EDTA tubes used for CBC, with no additional sample required. The total leukocyte count (WBC), neutrophil-to-lymphocyte ratio (NLR), fibrinogen, serum amyloid A, and albumin/globulin ratio (A/G) were concurrently measured. Based on the clinical presentation and the final diagnosis, cats were classified as having the following: acute, chronic, and acute-on-chronic conditions. The RI of the ESR ranged between 1 and 23 mm/h. Ill cats showed a significantly higher ESR (median 29 mm/h; range 12-46 mm/h) than healthy cats (median 10 mm/h; range 1-12 mm/h; p < 0.0001). The ESR was positively correlated only with fibrinogen (p < 0.001; r = 0.43). Cats with acute-on-chronic diseases had the highest ESR (median 47 mm/h; range 35-56 mm/h) compared with acute (median 16 mm/h; range 14-42 mm/h; p=0.003) and chronic cats (median 14 mm/h; range 10-31 mm/h; p < 0.0001). Although further studies are needed, the ESR could be a useful ancillary inflammatory marker in cats, specifically in cats with acute diseases, with or without an underlying chronic condition.

Increased Erythrocyte Sedimentation Rate in Dogs: Frequency in Routine Clinical Practice and Association with Hematological Changes.

(1) Background: the erythrocyte sedimentation rate (ESR) has been reported to increase in some infectious or inflammatory diseases in dogs, but no information on the frequency of increases in a routine clinical setting exists. The aim of this study was to assess the frequency of an increased ESR in dogs and to investigate its possible association with hematologic changes; (2) Methods: A total of 295 EDTA blood samples were randomly selected from the routine caseload of the Veterinary Teaching Hospital. Samples were grouped in controls and in pathologic groups based on the clinical presentation. A routine hemogram was performed, then the ESR was measured using the instrument MINI-PET; (3) Results: compared with controls, the ESR was significantly higher in all the pathologic groups, except for the hematological disorders group. The highest ESR was found in samples from dogs with chronic kidney disease or inflammation, followed by those from dogs with mild chronic disorders, severe/acute diseases, tumors and urinary disorders. The ESR negatively correlated with hematocrit and positively with neutrophil counts. (4) Conclusions: The ESR increases more frequently in dogs with clinically evident inflammation or CKD, but also in several other conditions, likely as a consequence of anemia and acute phase response.

Evaluation of Stability and Accuracy Compared to the Westergren Method of ESR Samples Analyzed at VES-MATIC 5.

The Erythrocyte Sedimentation Rate (ESR) is a diagnostic estimator of systemic inflammation as a reflection of acute phase proteins circulating in the blood. The purpose of this manuscript is to evaluate the blood stability at room temperature (RT) and at 4 °C to avoid ESR diagnostic errors, as well as the accuracy of the VES-MATIC 5 analyzer. The ESR stability evaluation at RT for 24 h (4 h "T1", 6 h "T2", 8 h "T3", 10 h "T4", 24 h "T5") and at 4 °C (24 h, 36 h, 48 h) was carried out using 635 total samples, starting with T0 (2 h of venipuncture). For method comparison, 164 patients were analyzed using VES-MATIC 5 and then the Westergren reference method. The sample at RT is established by a significant gradual decrease in correlation R = 0.99 (T0 vs. T1), R = 0.97 (T0 vs. T2), R = 0.92 (T0 vs. T3), R = 0.87 (T0 vs. T4), and R = 0.40 (T0 vs. T5). The stability at 4 °C after 24 h, 36 h, and 48 h showed a regression of R = 0.99, R = 0.97, and R = 0.95, respectively. Therefore, ESR measurements on RT samples beyond 6 h after collection cannot be carried out, but the ESR can be measured until 36 h for samples stored at 4 °C. Moreover, the VES-MATIC 5 accuracy performance compared to the Westergren method (R = 0.96) is confirmed.

Validation of the New MINI-CUBE for Clinic Determination of Erythrocyte Sedimentation Rate.

Background: Erythrocyte sedimentation rate (ESR) indirectly measures blood fibrinogen, and it is altered by all those pathological conditions that modify the aggregation of red blood cells. The international guidelines by the International Council for Standardization in Hematology (ICSH) define the Westergren method as the gold standard for ESR, although it is completely operator-dependent, time-consuming, and requires a patient's blood consumption. Therefore, the validation of new ESR analyzers is needed. The aim of this study is the validation of a new automated ESR analyzer, MINI-CUBE (DIESSE, Diagnostica Senese, Italy). Methods: The samples (n = 270) were collected at the University Hospital of the University of Rome Tor Vergata. A comparison between the automated instrument and the gold standard was performed. Statistical analyses were processed by MedCalc software. Results: The comparison analysis performed on the overall samples reported a good agreement, showing a Spearman rank correlation coefficient of 0.94 (P < 0.001), compared to the Westergren test. The Bland-Altman analysis showed a mean bias of 1.5 (maximum (max.):19.6; minimum (min.): -16.6). Inter-run (level 1 coefficient of variation (CV): 4.9%; level 2 CV: 0.8%), intra-run (level 1 CV: 21.1%; level 2 CV: 3.2%), and inter-instrument (level 1 CV: 27.1%; level 2 CV: 5.6%) precision were also assessed. The hematocrit value did not interfere with the analysis: Spearman rank correlation coefficient of 0.929 (P < 0.001); mean bias of 1.3 (max.:18.3; min.: -15.6). Conclusions: Overall results from MINI-CUBE asserted a good correlation rate with the gold standard, and it could be considered an accurate, and objective alternative for the Westergren test.

Analytical Validation of MINI-PET as Point-of-Care for Erythrocyte Sedimentation Rate Measure in Horses.

The erythrocyte sedimentation rate (ESR) is a widely used diagnostic assay in human medicine but nowadays poorly applied in veterinary medicine. This test measures the speed (millimeters per hour) at which red blood cells settle in a whole anticoagulated blood tube. In human medicine, high ESR values are associated with various disorders, including infections, rheumatoid arthritis, oncologic diseases, and other inflammatory conditions. The ESR can also be influenced by some factors such as age and gender. In veterinary medicine, the ESR with the Westergren manual method was almost forgotten over the years due to blood consumption and long turn-around time. The instrument MINI-PET, using a modified Westergren method, does not require blood consumption or release waste product and recently has been applied in canine medicine. The aims of the study in the horse were as follows: to establish the appropriate time to read the ESR with the Westergren reference method; to compare the MINI-PET ESR results with the reference technique; to assess the ESR reference intervals with MINI-PET; and to establish the ESR stability from collection at different time points by MINI-PET. Using 150 horses, we established 60 minutes as the appropriate time for ESR reading with the Westergren method. Moreover, ESR results obtained in 8 minutes with MINI-PET showed a good correlation with the Westergren ESR. Reference intervals (RIs) with MINI-PET were established in mm/h for the healthy horses (geldings 18.6-100.1; stallions, 13.8-55.7; and mares 1-73.7) according to the American Society of Veterinary Clinical Pathology guidelines. In addition, the ESR stability from the blood collection time was evaluated in the MINI-PET on 15 horses: at room temperature, ESR is stable up to 8 hours and at 4°C up to 24 hours. In conclusion, MINI-PET represents a rapid and reliable tool for measuring ESR in horses, offering a valid option to replace the traditional manual technique.

Effect of time and storage temperature on canine and feline erythrocyte sedimentation rate.

We aimed to test the influence of storage temperature and time for canine and feline ESR. Forty dogs and 12 cats were included and randomly allocated in "room temperature" and "refrigerated" groups. Both groups had the T0 ESR measures few minutes after complete blood count. Afterwards, room temperature group had ESR measured at 2, 4, 6 and 8h after T0, whereas the "refrigerated" group had the blood sample stored at 4-6°C for 24 and then T24h ESR was measured. In each ESR measurement, [1] blood samples were put on a tube rocker waiting for ESR analysis; [2] before inserting the blood tube in the MINI-PET ESR instrument, samples were gently mixed again by complete inversion 10 times; (3) each mixed blood tube was inserted in the one of the four MINI-PET tubes position; (4) on the machine display, patient's species has to be chosen and the 14 minutes countdown started; (5) after the 14 minutes optical reading, the ESR result (mm/h) is displayed on the machine. ESR of canine samples at room temperature were significantly stable until T6, while feline samples remained stable at T8. After 24h at refrigerated temperature, both canine and feline samples were stable. • MINI-PET is an ESR automatic continuous-loading instrument that can analyze up to four EDTA blood samples simultaneously using an optical system that measures the erythrocytes sedimentation level • We aimed to test influence of storage temperature and time for canine and feline ESR • At room temperature, dogs' samples were stable within 6 hours from collection, and cats' samples were stable until 8h. At refrigerated temperature, there was no difference in T0-T24 ESR in both canine and feline samples.

Magnetic resonance imaging of infarct-induced canonical wingless/integrated (Wnt)/ß-catenin/T-cell factor pathway activation, in vivo.

AIMS: Combined magnetic resonance imaging (MRI) of molecular and morpho-functional changes might prove highly valuable for the elucidation of pathological processes involved in the development of cardiac diseases. Our aim was to test a novel MRI reporter gene for in vivo assessment of the canonical Wnt/ß-catenin/TCF pathway activation, an important regulator of post-ischaemic cardiac remodelling. METHODS AND RESULTS: We designed and developed a chimeric construct encoding for both of iron-binding human ferritin heavy chain (hFTH) controlled by the ß-catenin-responsive TCF/lymphoid-enhancer binding factor (Lef) promoter and constitutively expressed green fluorescent protein (GFP). It was carried by adeno-associated virus serotype 9 (rAAV9) vectors and delivered to the peri-infarct myocardium of rats subjected to coronary ligation (n = 11). By 1.5 T MRI and a multiecho T2* gradient echo sequence, we detected iron accumulation only in the border zone of the transduced infarcted hearts. In the same cardiac area, post-mortem histological analysis confirmed the co-existence of iron accumulation and GFP. The iron signal was absent when rats (n = 6) were chronically treated with SEN195 (10 mg/kg/day), a small-molecular inhibitor of ß-catenin/TCF-dependent gene transcription. Canonical Wnt pathway inhibition attenuated the post-ischaemic remodelling process, as demonstrated by the significant preservation of cardiac function, the 42 ± 1% increase of peri-infarct arteriolar density and 43 ± 3% reduction in infarct scar size compared with untreated animals. CONCLUSIONS: The TCF/Lef promoter-hFTH construct is a novel and reliable MRI reporter gene for in vivo detection of the canonical Wnt/ß-catenin/TCF activation state in response to cardiac injury and therapeutic interventions.

Multimodal molecular imaging system for pathway-specific reporter gene expression.

Preclinical imaging modalities represent an essential tool to develop a modern and translational biomedical research. To date, Optical Imaging (OI) and Magnetic Resonance Imaging (MRI) are used principally in separate studies for molecular imaging studies. We decided to combine OI and MRI together through the development of a lentiviral vector to monitor the Wnt pathway response to Lithium Chloride (LiCl) treatment. The construct was stably infected in glioblastoma cells and, after intracranial transplantation in mice, serial MRI and OI imaging sessions were performed to detect human ferritin heavy chain protein (hFTH) and firefly luciferase enzyme (FLuc) respectively. The system allowed also ex vivo analysis using a constitutive fluorescence protein expression. In mice, LiCl administration has shown significantly increment of luminescence signal and a lower signal of T2 values (P<0.05), recorded noninvasively with OI and a 7 Tesla MRI scanner. This study indicates that OI and MRI can be performed in a single in vivo experiment, providing an in vivo proof-of-concept for drug discovery projects in preclinical phase.

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

An exploratory double-blind, randomized clinical trial with selisistat, a SirT1 inhibitor, in patients with Huntington's disease.

AIMS: Selisistat, a selective SirT1 inhibitor is being developed as a potentially disease-modifying therapeutic for Huntington's disease (HD). This was the first study of selisistat in HD patients and was primarily aimed at development of pharmacodynamic biomarkers. METHODS: This was a randomized, double-blind, placebo-controlled, multicentre exploratory study. Fifty-five male and female patients in early stage HD were randomized to receive 10 mg or 100 mg of selisistat or placebo once daily for 14 days. Blood sampling, clinical and safety assessments were conducted throughout the study. Candidate pharmacodynamic markers included circulating soluble huntingtin and innate immune markers. RESULTS: Selisistat was found to be safe and well tolerated, and systemic exposure parameters showed that the average steady-state plasma concentration achieved at the 10 mg dose level (125 nm) was comparable with the IC50 for SirT1 inhibition. No adverse effects on motor, cognitive or functional readouts were recorded. While circulating levels of soluble huntingtin were not affected by selisistat in this study, the biological samples collected have allowed development of assay technology for use in future studies. No effects on innate immune markers were seen. CONCLUSIONS: Selisistat was found to be safe and well tolerated in early stage HD patients at plasma concentrations within the anticipated therapeutic concentration range.

Safety, pharmacokinetics, pharmacogenomics and QT concentration-effect modelling of the SirT1 inhibitor selisistat in healthy volunteers.

AIM: Selisistat (SEN0014196), a first-in-class SirT1 inhibitor, is being developed as a disease-modifying therapy for Huntington's disease. This first-in-human study investigated the safety, pharmacokinetics and pharmacogenomics of single and multiple doses of selisistat in healthy male and female subjects. METHOD: In this double-blind, randomized, placebo-controlled study, seven cohorts of eight subjects received a single dose of selisistat at dose levels of 5, 25, 75, 150, 300 and 600 mg and four cohorts of eight subjects were administered 100, 200 and 300 mg once daily for 7 days. Blood sampling and safety assessments were conducted throughout the study. RESULTS: Selisistat was rapidly absorbed and systemic exposure increased in proportion to dose in the 5-300 mg range. Steady-state plasma concentrations were achieved within 4 days of repeated dosing. The incidence of drug related adverse events showed no correlation with dose level or number of doses received and was comparable with the placebo group. No serious adverse events were reported and no subjects were withdrawn due to adverse events. There were no trends in clinical laboratory parameters or vital signs. No trends in heart rate or ECG parameters, including the QTc interval and T-wave morphology, were observed. There were no findings in physical or neurological examinations or postural control. Transcriptional alteration was observed in peripheral blood. CONCLUSION: Selisistat was safe and well tolerated by healthy male and female subjects after single doses up to 600 mg and multiple doses up to 300 mg day(-1).

Whole gene expression profile in blood reveals multiple pathways deregulation in R6/2 mouse model.

BACKGROUND: Huntington Disease (HD) is a progressive neurological disorder, with pathological manifestations in brain areas and in periphery caused by the ubiquitous expression of mutant Huntingtin protein. Transcriptional dysregulation is considered a key molecular mechanism responsible of HD pathogenesis but, although numerous studies investigated mRNA alterations in HD, so far none evaluated a whole gene expression profile in blood of R6/2 mouse model. FINDINGS: To discover novel pathogenic mechanisms and potential peripheral biomarkers useful to monitor disease progression or drug efficacy, a microarray study was performed in blood of R6/2 at manifest stage and wild type littermate mice. This approach allowed to propose new peripheral molecular processes involved in HD and to suggest different panels of candidate biomarkers. Among the discovered deregulated processes, we focused on specific ones: complement and coagulation cascades, PPAR signaling, cardiac muscle contraction, and dilated cardiomyopathy pathways. Selected genes derived from these pathways were additionally investigated in other accessible tissues to validate these matrices as source of biomarkers, and in brain, to link central and peripheral disease manifestations. CONCLUSIONS: Our findings validated the skeletal muscle as suitable source to investigate peripheral transcriptional alterations in HD and supported the hypothesis that immunological alteration may contribute to neurological degeneration. Moreover, the identification of altered signaling in mouse blood enforce R6/2 transgenic mouse as a powerful HD model while suggesting novel disease biomarkers for pre-clinical investigation.

MREdictor: a two-step dynamic interaction model that accounts for mRNA accessibility and Pumilio binding accurately predicts microRNA targets.

The prediction of pairing between microRNAs (miRNAs) and the miRNA recognition elements (MREs) on mRNAs is expected to be an important tool for understanding gene regulation. Here, we show that mRNAs that contain Pumilio recognition elements (PRE) in the proximity of predicted miRNA-binding sites are more likely to form stable secondary structures within their 3'-UTR, and we demonstrated using a PUM1 and PUM2 double knockdown that Pumilio proteins are general regulators of miRNA accessibility. On the basis of these findings, we developed a computational method for predicting miRNA targets that accounts for the presence of PRE in the proximity of seed-match sequences within poorly accessible structures. Moreover, we implement the miRNA-MRE duplex pairing as a two-step model, which better fits the available structural data. This algorithm, called MREdictor, allows for the identification of miRNA targets in poorly accessible regions and is not restricted to a perfect seed-match; these features are not present in other computational prediction methods.

A phenotypic screening assay for modulators of huntingtin-induced transcriptional dysregulation.

Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD.

Reference genes selection for transcriptional profiling in blood of HD patients and R6/2 mice.

BACKGROUND: Huntington's disease is a neurodegenerative disorder characterized by transcriptional alterations both in central and peripheral tissues. Therefore, the identification of a transcriptional signature in an accessible tissue can meaningfully complement current efforts in clinical biomarker development. Gene expression normalization represents an essential step in transcriptional signatures identification, and since many reference genes show altered expressions in several pathologies, the definition of stable genes in the desired tissue is required to allow correct result interpretations. OBJECTIVE: The present work aimed at identifying a set of suitable reference genes for expression normalization in blood of HD patients and R6/2 mice. METHODS: By crossing literature investigation and analysis of microarrays performed on blood of HD patients and healthy subjects, a set of genes was selected and tested by RT-qPCR. Employment of statistical algorithms allowed the identification of the most stable genes in human samples that were than confirmed in R6/2. RESULTS: PPIB, PGK1, ACTB and YWHAZ represent the best possible genes combination, useful to normalize blood transcriptional analysis. To link clinical and preclinical studies, the identified genes were investigated also in blood of R6/2 and wild type mice, confirming that Ppib, Actb and Ywhaz were appropriate for expression normalization. Selected references were subsequently applied to evaluate expression of genes known to be involved in Huntington's pathological progression. CONCLUSIONS: This work highlights the importance for correct data normalization to avoid misinterpretation of results, while providing a suitable method to support quantitative gene expression analysis in preclinical and clinical investigations.

Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction.

Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation.

Identification of a candidate alternative promoter region of the human Bcl2L11 (Bim) gene.

BACKGROUND: Despite the importance of the BCL2L11 (BIM) protein in various apoptotic processes in development and disease, little is known of the promoter structure of the human BCL2L11 locus and of the cis-acting elements regulating expression of the human gene. RESULTS: In the search for novel promoter sequences in the human BCL2L11 locus, we have identified previously unrecognized genomic sequences displaying promoter activity and E2F responsiveness, and driving the expression of BCL2L11 coding transcripts. In man, transcripts originating from this novel putative promoter contribute significantly to total BCL2L11 mRNA expression in testis, heart and liver. In HEK293 cells, this novel candidate promoter originates BCL2L11 transcripts whose expression can be modulated by a known modulator of BCL2L11 expression (Trichostatin A) and by E2F, a characterized transcriptional regulator of BCL2L11 expression. CONCLUSION: The identification of a novel putative human BCL2L11 promoter provides new insights into the structure and regulation of the BCL2L11 locus.

Increased expression of the oligopeptidase THOP1 is a neuroprotective response to Abeta toxicity.

In a comprehensive proteomics study aiming at the identification of proteins associated with amyloid-beta (Abeta)-mediated toxicity in cultured cortical neurons, we have identified Thimet oligopeptidase (THOP1). Functional modulation of THOP1 levels in primary cortical neurons demonstrated that its overexpression was neuroprotective against Abeta toxicity, while RNAi knockdown made neurons more vulnerable to amyloid peptide. In the TgCRND8 transgenic mouse model of amyloid plaque deposition, an age-dependent increase of THOP1 expression was found in brain tissue, where it co-localized with Abeta plaques. In accordance with these findings, THOP1 expression was significantly increased in human AD brain tissue as compared to non-demented controls. These results provide compelling evidence for a neuroprotective role of THOP1 against toxic effects of Abeta in the early stages of AD pathology, and suggest that the observed increase in THOP1 expression might be part of a compensatory defense mechanism of the brain against an increased Abeta load.