Association of Vitamin D with Severity and Outcome of COVID-19: Clinical and Experimental Evidence.

INTRODUCTION: The role of vitamin in COVID-19 remains controversial. We investigated the association between endogenous vitamin D and the severity of COVID-19 as well as the mechanisms of action of vitamin D supplementation. METHODS: 25(OH)D3 in serum was associated with disease severity and outcome in 190 COVID-19 patients. In a COVID-19 animal model using intravenous injection of plasma from patients with COVID-19 acute respiratory distress syndrome into C57/BL6 mice, mice were treated with 0.25 µg human 1,25(OH)D3 or vehicle. Mice were sacrificed on day 4. Cytokines and myeloperoxidase (MPO) in tissues were measured. Changes in gene expression after vitamin D supplementation were measured. RESULTS: Vitamin D deficiency and insufficiency were associated with increased severity and unfavorable outcome after 28 days. Vitamin D levels were negatively associated with biomarkers of COVID-19 severity. Vitamin D supplementation after challenge of mice with COVID-19 plasma led to reduced levels of TNFα, IL-6, IFNγ, and MPO in the lung, as well as down-regulation of pro-inflammatory pathways. CONCLUSION: Normal levels of endogenous vitamin D are associated with reduced severity and risk of unfavorable outcome in COVID-19, possibly through attenuation of tissue-specific hyperinflammation.

Deficiency of hydrogen sulfide production and pregnancy rate in an experimental model: Association with preterm delivery.

PROBLEM: Pro-inflammatory phenomena drive preterm delivery (PTD). Hydrogen sulfide is a gasotransmitter with anti-inflammatory properties produced through the activity of the enzyme cystathionine-γ-lyase (CSE), and its impact was studied in models of normal delivery and PTD in mice. METHOD OF STUDY: Female CSE+/+ and CSE-/- mice were mated with male CSE+/+ mice; mating was done with drinking water unsupplemented and supplemented with cysteine. The pregnancy rate was monitored. PTD was induced by the intraperitoneal injection of bacterial lipopolysaccharide (LPS) on day 14.5 of pregnancy. Mice were sacrificed for tissue collection and splenocyte isolation after 6 and 12 h. Isolated splenocytes were stimulated for the production of tumor necrosis factor-alpha (TNFα), interleukin (IL)-10 and interferon-gamma (IFNγ); TNFα and vascular endothelial growth factor (VEGF) were measured in the fetuses and the placenta. RESULTS: The successful pregnancy rate was lower in CSE-/- mice and it was restored with cysteine supplementation. CSE deficiency was associated with higher tissue concentrations of TNFα in the fetuses, attenuated IL-10 responses and higher IFNγ production from splenocytes. CSE deficiency was not associated with PTD. Following PTD induction, CSE-/- mice did not show attenuated IL-10 responses but the production of TNFα and IFNγ was lowered over-time; placental VEGF was also increased over-time. CONCLUSIONS: CSE deficiency has an unfavorable impact on pregnancy. H2 S deficiency through CSE does not drive PTD but mediates pro-inflammatory phenomena in fetuses.

Specificity of a pancreatic lipase point-of-care test and agreement with pancreatic lipase immunoreactivity in cats without clinical evidence of pancreatitis.

OBJECTIVES: The aim of this study was to determine the specificity of a rapid point-of-care test for the estimation of feline pancreatic lipase (SNAP fPL) in healthy and sick cats without clinical evidence of pancreatitis. A second objective was to evaluate the agreement between SNAP fPL and serum pancreatic lipase immunoreactivity (fPLI), as measured by Spec fPL. METHODS: A total of 150 cats were prospectively enrolled into this study. Of them, 82 cats were healthy while 68 cats had various diseases but no clinical signs (eg, anorexia, depression, vomiting) raising a suspicion of pancreatitis. RESULTS: SNAP fPL was normal in 133/150 cats (specificity 89%) without obvious clinical pancreatitis. SNAP fPL was normal in 74/82 healthy cats (specificity 90%) and in 59/68 cats that were sick but without typical signs of pancreatitis (specificity 87%). The agreement between SNAP fPL and Spec fPL was substantial (k = 0.64) in healthy cats and almost perfect (k = 0.93) in sick cats. The overall agreement between SNAP fPL and Spec fPL was almost perfect (k = 0.81). CONCLUSIONS AND RELEVANCE: The specificity of SNAP fPL in this group of cats was high. There was a substantial and almost perfect agreement between the SNAP fPL and Spec fPL in healthy cats and sick cats without suspected pancreatitis, respectively. In the small percentage of cats with abnormal SNAP fPL and/or Spec fPL results, the possibility of subclinical pancreatitis cannot be excluded.

Dimethyl itaconate induces long-term innate immune responses and confers protection against infection.

Itaconate is an immunomodulatory metabolite produced by immune cells under microbial stimulation and certain pro-inflammatory conditions and triggers antioxidant and anti-inflammatory responses. We show that dimethyl itaconate, a derivative of itaconate previously linked to suppression of inflammation and widely employed as an alternative to the endogenous metabolite, can induce long-term transcriptional, epigenomic, and metabolic changes, characteristic of trained immunity. Dimethyl itaconate alters glycolytic and mitochondrial energetic metabolism, ultimately leading to increased responsiveness to microbial ligand stimulation. Subsequently, mice treated with dimethyl itaconate present increased survival to infection with Staphylococcus aureus. Additionally, itaconate levels in human plasma correlate with enhanced ex vivo pro-inflammatory cytokine production. Collectively, these findings demonstrate that dimethyl itaconate displays short-term anti-inflammatory characteristics and the capacity to induce long-term trained immunity. This pro-and anti-inflammatory dichotomy of dimethyl itaconate is likely to induce complex immune responses and should be contemplated when considering itaconate derivatives in a therapeutic context.

IL-1 Mediates Tissue-Specific Inflammation and Severe Respiratory Failure in COVID-19.

Acute respiratory distress syndrome (ARDS) in COVID-19 has been associated with catastrophic inflammation. We present measurements in humans and a new animal model implicating a role in danger-associated molecular patterns. Calprotectin (S100A8/A9) and high-mobility group box 1 (HMGB1) were measured in patients without/with ARDS, and admission calprotectin was associated with soluble urokinase plasminogen activator receptor (suPAR). An animal model was developed by intravenous injection of plasma from healthy or patients with COVID-19 ARDS into C57/BL6 mice once daily for 3 consecutive days. Mice were treated with one anti-S100A8/A9 antibody, the IL-1 receptor antagonist anakinra or vehicle, and Flo1-2a anti-murine anti-IL-1α monoclonal antibody or the specific antihuman IL-1α antibody XB2001 or isotype controls. Cytokines and myeloperoxidase (MPO) were measured in tissues. Calprotectin, but not HMGB1, was elevated in ARDS. Higher suPAR indicated higher calprotectin. Animal challenge with COVID-19 plasma led to inflammatory reactions in murine lung and intestines as evidenced by increased levels of TNFα, IL-6, IFNγ, and MPO. Lung inflammation was attenuated with anti-S100A8/A9 pre-treatment. Anakinra treatment restored these levels. Similar decrease was found in mice treated with Flo1-2a but not with XB2001. Circulating alarmins, specifically calprotectin, of critically ill COVID-19 patients induces tissue-specific inflammatory responses through an IL-1-mediated mechanism. This could be attenuated through inhibition of IL-1 receptor or of IL-1α.

Evaluation of Heller's reaction and the urine dipstick method alone or in combination to detect proteinuria in sheep.

BACKGROUND: Urine dipstick and Heller's reaction are easy first-line screening tests for the detection of proteinuria; however, the performance of these methods in alkaline ovine urine is largely unknown. OBJECTIVE: The aim of this study was to evaluate the diagnostic performance of Heller's reaction alone or in combination with dipstick for the detection of proteinuria in sheep, using the urine protein to creatinine ratio (UP/C) with two cut-off values as the reference method. METHODS: Ninety-eight urine samples were collected from sheep using the transient apnea method. Heller's reaction, the dipstick method, and the UP/C ratio were used to assess proteinuria. The results were statistically analyzed twice, based on two different UP/C cut-off values of 0.2 and 0.5. Cohen's kappa value was used to determine the agreement between the UP/C ratios and Heller's reaction, the dipstick method, or the combination of methods. Sensitivity, specificity, and positive and negative likelihood ratios were calculated. ROC curves were also generated, and the areas under the curve (AUC) were evaluated to determine the optimal threshold for the numerical values of the two methods. RESULTS: Heller's reaction is more specific (96.67% and 96.00% when the cut-off value is 0.2 and 0.5, respectively) than the dipstick method, while the dipstick method was more sensitive (91.18% and 91.30%, when the cut-off value was 0.2 and 0.5, respectively) than Heller's reaction for the detection of proteinuria. Both tests were accurate when any grade >0 was considered positive. CONCLUSIONS: Proteinuria can almost be excluded in ovine urine samples with negative Heller's reaction and dipstick test.

Effect of storage time on the urine protein: creatinine ratio in alkaline ovine urine.

The urine protein:creatinine (UPC) ratio is considered the reference method to assess proteinuria. Its diagnostic value in ovine medicine needs further elucidation. In population monitoring and/or for research purposes, it is convenient to collect many samples simultaneously and store them for later analysis. However, analyte stability data are required to ensure reliable results. We used 15 of 90 urine samples collected from sheep to assess the effect of storage time on the UPC ratio. After centrifugation, the supernatant of each sample was divided into 6 aliquots. Urine protein and creatinine concentrations were determined immediately in one aliquot using the pyrogallol red and a modified Jaffè method, respectively. The other aliquots were stored at -18°C. Based on the absence of active sediment, alkaline urine pH, and UPC ratio ≥0.2, we included 15 samples in our study. The UPC ratio was determined in the stored aliquots 2, 7, 14, 21, and 60 d after collection. The data were analyzed with univariate ANOVA. No significant difference was observed in the urinary concentrations of protein, creatinine, and the UPC ratio (0.8 ± 0.84 in conventional units and 0.09 ± 0.095 in SI units) among different times (p > 0.05). The UPC ratio remained stable for 2 mo in ovine urine samples stored at -18°C.

Neospora caninum and/or Toxoplasma gondii Seroprevalence: Vaccination against PCV2 and Muscle Enzyme Activity in Seropositive and Seronegative Pigs.

Neospora caninum and Toxoplasma gondii affect both humans and animals worldwide. To investigate their seroprevalence and differences in seropositivity between pigs vaccinated and unvaccinated against porcine circovirus 2 (PCV2), as well as differences in muscle enzyme activity between seropositive and seronegative pigs, blood samples were collected from 380 sows. Antibodies against T. gondii and N. caninum were detected by an indirect immunofluorescence antibody (IFA) assay, while the activities of creatine kinase (CK) and aspartate aminotransferase (AST) were biochemically assessed. Out of the 364 sows finally included in the study, 4.4%, 3.5%, and 0.5% were seropositive to T. gondii, N. caninum, or both. A significantly higher percentage of seropositivity against T. gondii and/or N. caninum in PCV2 unvaccinated pigs compared with vaccinated pigs was observed. Increased serum activities of CK and AST were detected in 71.43% and 100% of only against T. gondii (T+) and 63.64% and 90.91% of only against N. caninum (N+) seropositive sows, respectively, and were significantly higher compared to seronegative animals. T. gondii and N. caninum seropositivity, especially in presumed immunocompromised pigs, and the evidence of muscle damage highlight their importance as a zoonotic pathogen and animal model of human infection, respectively.

Hematological Features in Sheep with IgG and IgM Antibodies against Borrelia burgdorferi sensu lato.

Exposure of sheep to Borreliaburgdorferi sensulato (s.I.) complex, the causative agent of Lyme borreliosis (LB), has been reported in tick-abundant areas worldwide, while no data have been reported in Greece. The aim of the study was to identify the hematological alterations in sheep with seropositivity against Borrelia burgdorferi (s.I.). Blood samples were obtained from 318 tick infested sheep for blood analysis and serological determination of IgG and IgM antibodies against B. burgdorferi by indirect immunofluorescence antibody (IFA) assay after exclusion of endo-ectoparasites and other tick-borne infections. A total number of 162 sheep met the inclusion criteria, allocated in four groups based on the presence or absence of IgG and/or IgM; sheep found negative for IgM and IgG (Group A), positive for IgM (Group B), positive for both IgM and IgG (Group C) and positive for IgG (Group D). Anemia, thrombocytopenia and normal or decreased leukocyte count, mainly due to lymphopenia were the main hematological features observed in seropositive sheep. The presence of these features raises the suspicion of Borrelia infection in tick infested sheep. The seropositivity of 23.58% in sheep raises concerns of Borrelia circulation, especially in rural areas and potential risk of transmission to humans.

Potential Acute Renal Injury in Sheep with Bluetongue Serotype 4.

Bluetongue is a vector-borne disease with epidemic potential. Recently, outbreaks of Bluetongue were reported across Greece, caused by the Bluetongue virus (BTV) serotype 4. Regarding its pathogenesis, BTV infection involves various target organs with limited data referring to the kidneys. The objective of this study was to identify the possible impact of BTV infection on kidneys using common renal biomarkers. Urine and blood samples collected from 30 sheep with clinical signs of bluetongue (BTV sheep) and 30 clinically healthy sheep (normal sheep) from the same farms were finally selected and included in the study from an initial population of 47 sheep per group, based on the absence of active urine sediment. Complete urinalysis was performed and urine protein to creatinine ratio (UPC) and urine gamma-glutamyl transferase to creatinine (UGGTC) ratio were determined. Blood urea nitrogen (BUN), creatinine, total proteins, albumin (ALB), and inorganic phosphate (P) were determined in serum samples. UPC and UGGTC were significantly higher (p < 0.05) in BTV sheep compared to normal, whereas urine specific gravity (USG) was significantly lower (p < 0.05). Cylindruria was also detected in BTV sheep, and absence of azotemia in BTV and normal sheep. All these findings are indicative of renal tubular injury and/or dysfunction and suggestive of an association between BTV infection and acute damage of renal tissue.