Exploratory screening for micro-RNA biomarkers in canine multicentric lymphoma.

Lymphoma is one of the most frequent hematopoietic tumors in dogs and shares similar features with human counterparts. MicroRNAs (miRNA, small non-coding RNAs) are pivotal in gene regulation fine tuning and cancer hallmarks are influenced by their aberrant expression. Consequently, miRNA biomarkers may assist predicting therapeutic response and clinical outcome by providing less-invasive novel diagnostics tools. The aim of this study was to detect dysregulated miRNAs in lymphomatous lymph node tissues in comparison to lymph node material or PBMCs from healthy control dogs. Potential significant differences in miRNA expression profiles between four lymphoma entities were evaluated. A customized PCR array was utilized to profile 89 canine target miRNAs. Quantification was performed using qPCR, relative expression was determined by the delta-delta Ct method, and p-values were calculated with student's t-test. In the 14 diffuse large B-cell lymphoma (DLBCL) patients, 28 and 24 different miRNAs were significantly dysregulated compared to lymph node material or PBMCs. Sixteen miRNAs occurred in both control groups, with 12 miRNAs being down- and four miRNAs being upregulated. The six peripheral T-cell lymphoma (PTCL) samples showed 24 and 25 dysregulated miRNAs when compared to the healthy controls. A combined analysis of DLBCL and PTCL samples revealed seven shared and 19 differently expressed miRNAs. Potential biomarkers in T- and B-cell lymphoma could be the miRNA-17/92 cluster and miRNA-181-family together with miRNA-34a and miRNA-150. Diagnostic utility of potential biomarkers must be validated in larger, prospective cohorts of canine lymphoma cases and in higher numbers of physiological patient material.

Patients With Psoriatic Arthritis-Related Enthesitis and Persistence on Tofacitinib Under Real-World Conditions.

OBJECTIVE: Information on the persistence of tofacitinib (TOF) in psoriatic arthritis (PsA) is scarce in real-world conditions. Our objective was to analyze the persistence and safety of TOF under these conditions. METHODS: This was a single-center retrospective longitudinal observational study of all patients with PsA who received at least 1 dose of TOF. The main focus was on adverse events (AEs) and drug survival. Drug survival was analyzed by Kaplan-Meier curves and persistence explanatory factors by multivariate Cox regression models. The hazard ratio (HR) was used to measure association. RESULTS: Seventy-two patients were included, 54 women and 18 men, mean age 51.9 (SD 11.1) years, mean disease duration of 10.4 (SD 6.99) years. TOF was ≥ third line of therapy in > 70% of cases. The median survival was 13.0 (IQR 5.3-29.0) months. One-year retention rate was 52.7% (95% CI 42.4-65.6). TOF survival was not influenced by sex, disease duration, comorbidities, or line of treatment. Younger patients (HR 0.96, P = 0.01) and those with enthesitis (HR 0.37, P = 0.03) showed lower odds of drug discontinuation. The overall rate of AEs was 52.9 (95% CI 38.5-70.6)/100 person-years. Most AEs occurred during the first 6 months of exposure. CONCLUSION: In this real-world study, TOF showed a reasonably good retention rate in a PsA population that was mostly refractory to biologic and oral targeted synthetic disease-modifying antirheumatic drugs. There were no new causes for concern regarding safety. Patients with refractory PsA and enthesitis might be a specific target population for this drug.

Influence of the HLA-Cw6 Allele and IFIH1/MDA5 Gene Variants on the Cardiometabolic Risk Profile of Patients with Psoriatic Disease.

BACKGROUND: Cardiovascular comorbidity is a common companion of psoriasis and psoriatic arthritis (PsA). Recently, a significant link has been found between the HLA-Cw6 allele and a better cardiometabolic profile in these patients. We aimed to check this finding in our setting. METHODS: A cross-sectional observational study (n: 572 psoriasis patients, 30% with PsA) was conducted. Different study variables were collected in detail, as well as classic cardiometabolic risk factors. The distribution of the HLA-Cw6 allele and the IFIH1/MDA5 gene variants previously linked to disease risk were determined in the study cohort and stratified according to the cardiometabolic comorbidity. Linear and logistic regression models were constructed to analyze these associations. RESULTS: The study cohort included 309 men and 263 women, with a mean age of 46.7 years (SD 14.5) and a mean disease duration of 19.4 years (SD 14.8). We confirmed the known association between HLA-Cw6 and type I psoriasis (familial, severe, and early onset). Psoriasis severity (OR: 2.14), female sex (OR: 1.63), and the IFIH1/MDA5 rs1990760 TT genotype (OR: 1.62) were significantly related to PsA, while HLA-Cw6 was protective (OR: 0.65). HLA-Cw6 carriers showed a lower waist perimeter, lower BMI, and lower risk of both hypertension (OR: 0.52, p < 0.001) and diabetes (OR: 0.36, p < 0.001), but these findings were no longer apparent upon adjusting the regression models. No IFIH1/MDA5 gene variant was associated with any cardiometabolic risk factor. CONCLUSIONS: The influence of HLA-Cw6 on the cardiometabolic risk profile of psoriatic patients seems to be explained by other factors (age, sex, duration of the disease or arthritis) and not by this biomarker itself.

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death.

Apical-out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical-out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these 'inside-out' organoids die relatively quickly and cannot be propagated as long as their basal-out counterparts. Here, we show that apical-out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time.

Acroosteolysis and facial dysmorphia: a new case of Hajdu-Cheney syndrome.

Hajdu-Cheney syndrome or acro-dento-osteo-dysplasia syndrome is a rare disease characterized by band osteolysis of distal phalanges and facial dysmorphia, among other manifestations. We present the case of a 45-year-old male who consulted for mechanical joint pain of both hands, facial dysmorphism, cranio-facial alterations, and digital telescoping with acroosteolysis.

Are Patients with Axial Spondyloarthritis Who Were Breastfed Protected against the Development of Severe Disease?

BACKGROUND AND AIMS: Breastfeeding is recognized as one of the most influential drivers of the gut microbiome. In turn, alterations in the gut microbiome may play a role in the development and severity of spondyloarthritis (SpA). We aimed to analyze different disease outcomes in patients with axial SpA (axSpA) based on the history of breastfeeding. PATIENTS AND METHODS: A random sample was selected from a large database of axSpA patients. Patients were divided based on history of breastfeeding and several disease outcomes were compared. Both groups were also compared based on disease severity. Adjusted linear and logistic regression statistical methods were used. RESULTS: The study included 105 patients (46 women and 59 men), and the median age was 45 years (IQR: 16-72), and the mean age at diagnosis was 34.3 ± 10.9 years. Sixty-one patients (58.1%) were breastfed, with a median duration of 4 (IQR: 1-24) months. After the fully adjusted model, BASDAI [-1.13 (95%CI: -2.04, -0.23), p = 0.015] and ASDAS [-0.38 (95%CI: -0.72, -0.04), p = 0.030] scores were significantly lower in breastfed patients. Forty-two percent had severe disease. In the adjusted logistic model for age, sex, disease duration, family history, HLA-B27, biologic therapy, smoking, and obesity, breastfeeding had a protective effect against the development of severe disease (OR 0.22, 95%CI: 0.08-0.57, p = 0.003). The selected sample size was sufficient to detect this difference with a statistical power of 87% and a confidence level of 95%. CONCLUSION: Breastfeeding might exert a protective effect against severe disease in patients with axSpA. These data need further confirmation.

Treatment Retention and Safety of Ixekizumab in Psoriatic Arthritis: A Real Life Single-Center Experience.

Background and objectives: Information on the performance of ixekizumab (IXE) in patients with psoriatic arthritis (PsA) in clinical practice is scarce. We aimed to analyze the retention rate and safety of IXE in patients with PsA in routine clinical practice. Methods: A retrospective longitudinal observational single-center study of all patients with PsA who had received at least one dose of IXE. Adverse events (AEs) and drug retention rate were the main study focus. Survival was analyzed using Kaplan−Meier curves and predictive factors using multivariate Cox regression analysis. The hazard ratio (HR) was used as a measure of the association. Results: Seventy-two patients were included (52 women and 20 men). Median disease duration was 5 years (IQR 3−9). More than 90% received ≥2 biologic and/or targeted synthetic disease-modifying anti-rheumatic drugs (DMARDs) prior to IXE. Ixekizumab showed a 1-year retention rate of 65% and a 2-year retention rate of 57%. Regarding discontinuation due to AEs, 0.18 AEs per person-year were identified. The number of previous biologics did not influence drug survival but prior use of methotrexate (HR 2.31 (95% CI 1.05−5.10), p < 0.05) and depression (HR 2.40 (95% CI 1.07−5.41), p < 0.05) increased the risk of IXE discontinuation. Conclusions: Ixekizumab showed a good retention rate in a PsA population mostly refractory to biologic and targeted synthetic DMARDs. Drug survival was consistently good regardless of age, gender, metabolic comorbidities, smoking status, or prior number of biologic therapies. This information may be of interest to better position this drug in the PsA treatment algorithms.

Example for process validation in biobanking: Fit for purpose testing of a cryopreservation method without isopentane.

Background: The freezing process of tissue samples is crucial for the preservation of morphological and molecular features. Several biobanking guidelines describe freezing techniques for optimal outcomes. As the Vetbiobank standard freezing protocol does not comply with those recommendations in detail, a process validation was performed to demonstrate that samples are suitable for downstream applications. Here we give a formal example of a process validation in the biobanking setting, as required by the biobanking guideline ISO 20387 (2018). Methods: Three different freezing protocols, freezing in liquid nitrogen, freezing via isopentane precooled on dry ice and freezing via liquid nitrogen vapor, were assessed based on morphological integrity of mouse liver and muscle tissue samples. Samples were either frozen in cryotubes (without Optimal Cutting Temperature compound, OCT) or in cryomolds (with OCT). The protocol providing the best results was validated for reproducibility and robustness in terms of defined acceptance criteria for morphological evaluability, A260/A280 ratio, and RNA integrity number values (RIN). In addition, performance tests were run by gene expression analyzes of selected, tissue specific biomarkers to confirm that processed samples are fit for purpose. Results: From the three applied freezing protocols, freezing in liquid nitrogen generated best results. Reproducibility acceptance criteria were met for both, morphological integrity and RNA quality. The freezing method was robust for the tested tissue types and the application of OCT, with exception of liver tissue, where it led to a significant decrease of the RIN value. Gene expression analyzes showed good comparability of results regardless of the applied freezing method. Conclusion: Freezing of tissue samples in liquid nitrogen provides samples of adequate quality for subsequent RNA investigations. A negative impact of OCT on the RIN value of liver samples was observed, which was independent from the applied freezing protocol and showed no impact on subsequent gene expression analysis.

Potential Differences in the Cardiometabolic Risk Profile of Patients with Psoriatic Disease according to Their HLA-C∗06 Status.

The human leukocyte antigen-C∗06 (HLA-C∗06, formerly HLA-Cw6) is the main genetic biomarker in psoriatic disease. It has been related to several phenotypic traits in psoriatic disease, but its role in relation to cardiometabolic comorbidities is unknown at present. Here, we analyze the potential connections between this biomarker and the cardiometabolic profile of these patients. We carried out a cross-sectional observational study including 400 patients recruited at a single university hospital. Clinical and classical cardiometabolic factors were compared between HLA-C∗06-positive and HLA-C∗06-negative individuals (OR with 95% CI). Multivariate regression analyses were carried out to check for disease traits associated with different cardiometabolic risk factors. The study population included 215 men (53.8%) and 185 women (46.2%), mean age of 46 ± 15 years, and an average disease evolution of 17 ± 12.6 years. Ninety-three (23.3%) patients met CASPAR criteria for psoriatic arthritis. HLA-C∗06 carriers (n: 160, 40%) showed an earlier age at disease onset, psoriasis family history, and more severe skin disease (type I disease). After correcting for age, sex, and disease duration, they also showed less hypertension (13.8% vs. 24.2%, OR 0.7 (95% CI: 0.42-0.78), p = 0.025), lower waist circumference (94.4 ± 13.7 vs. 98.3 ± 13.8 cm), and lower BMI (27 ± 4.4 vs. 28.1 ± 4.8, p < 0.05). We confirmed the well-known association between HLA-C∗06 and type I psoriatic disease. As a novel finding, patients carrying HLA-C∗06 showed a better cardiometabolic profile. In any case, these findings need further confirmation.

Proliferation Activity in Canine Gastrointestinal Lymphoma.

Gastrointestinal lymphomas are uncommon in dogs and little is known about their distinct subtypes or proliferation rate. The aim of this study was to stratify 33 canine gastrointestinal lymphoma samples according to the latest World Health Organization classification and to determine the Ki67 proliferation index by manual counting, digital image analysis and visual estimation. The Ki67 index was then correlated with subtype, immunophenotype, mitotic index, grade and tumour location. The mitotic index correlated positively with the Ki67 index. A significantly higher number of Ki67-positive cells was found in enteropathy-associated T-cell lymphoma type I and in diffuse large B-cell lymphoma compared with enteropathy-associated T-cell lymphoma type II. There was also a significant difference in Ki67 immunolabelled cells between grade 1 and grade 2 lymphomas. Moderate agreement was found between the Ki67 index as obtained by manual counting and visual estimation, but there was strong agreement between manual counting and digital image analysis. The user-friendly digital imaging system used in this study could have potential for future determination of the Ki67 index in lymphoid neoplasms.

Human osteosarcoma cells respond to sorafenib chemotherapy by downregulation of the tumor progression factors S100A4, CXCR4 and the oncogene FOS.

Osteosarcoma is a rare but aggressive bone neoplasm in humans, which is commonly treated with surgery, classical chemotherapy and radiation. Sorafenib, an inhibitor of a number of kinases targeting the Raf/MEK/ERK pathway, is a promising new chemotherapeutic agent in human medicine that has been approved since 2006 for the therapy of renal cell carcinoma and since 2007 for the treatment of hepatocellular carcinoma. Here, we studied the antimetastatic potential of 4 µM of this multikinase inhibitor in a human osteosarcoma cell line. DNA microarray-based gene expression profiling detected 297 and 232 genes upregulated or downregulated at a threshold of >2-fold expression alteration (P<0.05) in the sorafenib-treated cells. Three genes (CXCR4, FOS and S100A4) that are involved in tumor progression were chosen for validation by quantitative PCR (qPCR) and protein expression analysis. The decrease in RNA expression detected by microarray profiling was confirmed by qPCR for all three genes (P<0.01). On the protein level, sorafenib-induced reduction of S100A4 was verified both by western blotting and immunohistochemistry. For CXCR4 and c-Fos, a reduced protein expression was shown by immunohistochemistry, for c-Fos also by immunoblotting. We conclude that sorafenib could serve as a potent chemotherapeutical agent by which to inhibit the metastatic progression of osteosarcomas.

Diagnostic errors in the new millennium: a follow-up autopsy study.

A systematic review of the second half of the last century suggested that diagnostic errors have decreased over time. Our previous study covering the years 1972-1992 was then the only time series showing a significant reduction of diagnostic errors from a single institution. We report here the results of a follow-up study a decade later. We analyzed discrepancies between clinical and autoptic diagnoses in 100 randomly selected medical patients who died in the wards and in the medical intensive care unit at a tertiary-care teaching hospital in Switzerland in the year 2002. Autopsy rate declined from around 90% in the years from 1972 to 1992 to 54% in the present study. Major diagnostic errors (class I and II) declined significantly from 30 to 7% (P<0.001) over the last 30 years. Class I errors decreased from 16 to 2% (P<0.001) in the year 2002. Sensitivity for cardiovascular diseases increased from 69 to 92% (P=0.006), for infectious diseases from 25 to 90% (P=0.013) and for neoplastic diseases from 89 to 100% (P=0.053). Specificity for cardiovascular diseases increased from 85 to 98% (P<0.001) but was unchanged at a high level for infectious diseases and neoplastic diseases. The number of diagnostic procedures increased from 144 to 281 (P<0.001) with an increase in the number of computer tomography investigations and of tissue sampling in the last decade. The frequency of major diagnostic errors has been further reduced at the beginning of the new millennium probably due in large part to new diagnostic tools.

BPV-1 infection is not confined to the dermis but also involves the epidermis of equine sarcoids.

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive.

Promiscuous arbuscular mycorrhizal symbiosis of yam (Dioscorea spp.), a key staple crop in West Africa.

Yam (Dioscorea spp.) is a tuberous staple food crop of major importance in the sub-Saharan savannas of West Africa. Optimal yields commonly are obtained only in the first year following slash-and-burn in the shifting cultivation systems. It appears that the yield decline in subsequent years is not merely caused by soil nutrient depletion but might be due to a loss of the beneficial soil microflora, including arbuscular mycorrhizal fungi (AMF), associated with tropical "tree-aspect" savannas and dry forests that are the natural habitats of the wild relatives of yam. Our objective was to study the AMF communities of natural savannas and adjacent yam fields in the Southern Guinea savanna of Benin. AMF were identified by morphotyping spores in the soil from the field sites and in AMF trap cultures with Sorghum bicolor and yam (Dioscorea rotundata and Dioscorea cayenensis) as bait plants. AMF species richness was higher in the savanna than in the yam-field soils (18-25 vs. 11-16 spp.), but similar for both ecosystems (29-36 spp.) according to the observations in trap cultures. Inoculation of trap cultures with soil sampled during the dry season led to high AMF root colonization, spore production, and species richness (overall 45 spp.) whereas inoculation with wet-season soil was inefficient (two spp. only). The use of D. cayenensis and D. rotundata as baits yielded 28 and 29 AMF species, respectively, and S. bicolor 37 species. AMF root colonization, however, was higher in yam than in sorghum (70-95 vs. 11-20%). After 8 months of trap culturing, the mycorrhizal yam had a higher tuber biomass than the nonmycorrhizal controls. The AMF actually colonizing D. rotundata roots in the field were also studied using a novel field sampling procedure for molecular analyses. Multiple phylotaxa were detected that corresponded with the spore morphotypes observed. It is, therefore, likely that the legacy of indigenous AMF from the natural savanna plays a crucial role for yam productivity, particularly in the low-input traditional farming systems prevailing in West Africa.