High dimensional profiling identifies specific immune types along the recovery trajectories of critically ill COVID19 patients.

The COVID-19 pandemic poses a major burden on healthcare and economic systems across the globe. Even though a majority of the population develops only minor symptoms upon SARS-CoV-2 infection, a significant number are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are rapidly expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed whole blood samples serially collected from 40 surviving COVID-19 patients throughout their recovery in ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil-to-lymphocyte ratio (NLR), we defined four sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We identified classical monocytes as the first immune cell type to recover by restoration of HLA-DR-positivity and the reduction of immunosuppressive CD163 + monocytes, followed by the recovery of CD8 + and CD4 + T cell and non-classical monocyte populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19.

Nonsyndromic hearing impairment is associated with a mutation in DFNA5.

Nonsyndromic hearing impairment is one of the most heterogeneous hereditary conditions, with more than 40 loci mapped on the human genome, however, only a limited number of genes implicated in hearing loss have been identified. We previously reported linkage to chromosome 7p15 for autosomal dominant hearing impairment segregating in an extended Dutch family (DFNA5). Here, we report a further refinement of the DFNA5 candidate region and the isolation of a gene from this region that is expressed in the cochlea. In intron 7 of this gene, we identified an insertion/deletion mutation that does not affect intron-exon boundaries, but deletes five G-triplets at the 3' end of the intron. The mutation co-segregated with deafness in the family and causes skipping of exon 8, resulting in premature termination of the open reading frame. As no physiological function could be assigned, the gene was designated DFNA5.

On-farm prevalence of and potential risk factors for boar taint.

Boar taint is an unpleasant taste and odor that can occur in entire male pigs and is caused by androstenone, skatole, and to a lesser extent indole accumulating in fat tissue. In the present observational study, we evaluated an extensive list of such potential risk factors which influence boar taint: social hierarchy and puberty attainment, housing, health, preslaughter conditions, season, feed, carcass composition, slaughter weight or age, and breed. Details on these factors were collected by interviews with the participating farmers, observations on each farm by trained observers and farmers, as well as slaughterhouse data. Twenty-two farms (in West- and East-Flanders, ranging from 160 to 600 sows, selected on suitability) raising entire male pigs were included in the study to evaluate the link between boar taint and potential risk factors related to the farm and slaughter batch (114 slaughter batches and 16 791 entire male pigs in total). Average olfactory boar taint prevalence was 1.8 ±â€¯0.8%. Boar taint prevalence varied also within farms up to a maximum range between slaughter batches of 9.1% which suggests an effect of factors varying between slaughter batches such as season or other variables varying between slaughter batches. Less aggressive behavior at the end of fattening as well as lower skin lesion scores at fattening as well as at slaughter could be associated with less boar taint. The same might be said for sexual behavior, though less convincingly from this study. Measures that reduce aggression and stress have therefore have the potential to lower boar taint prevalence. The same might be said for sexual behavior, though less convincingly from this study. Furthermore, boar taint prevalence was generally higher in winter than in summer, which is relevant from a planning perspective for the slaughterhouses to seek alternative markets. Finally, increased CP gave significantly lower boar taint prevalences. This may to some extent be explained by the negative association between boar taint and lean meat percentage, as increased dietary CP levels promote the carcass lean meat percentages which can then be associated with lower boar taint levels.

Early lung ultrasound assessment for the prognosis of patients hospitalized for COVID-19 pneumonia. A pilot study.

OBJECTIVE: SARS CoV-2 is an epidemic viral infection that can cause mild to severe lung involvement. Newly apprehended knowledge on thoracic imaging abnormalities and the growing clinical experience on the evolution of this disease make the radiographic follow-up of hospitalized patients relevant. The value of consecutive bedside lung ultrasonography in the follow-up of hospitalized patients with SARS CoV-2 pneumonia and its correlation with other clinical and laboratory markers needs to be evaluated. METHODS: We assessed 39 patients [age: 64 y(60.1-68.7)] with confirmed SARS CoV-2 pneumonia. A total of 24 patients were hospitalized until the follow-up test, 9 were discharged early and 6 required a transfer to critical care unit. Two ultrasound scans of the lung were performed on day 1 and 4 of patients' hospitalization. Primary endpoint was the magnitude of association between a global lung ultrasound score (LUS) and clinical and laboratory markers. Secondary endpoint was the association between the evolution of LUS with the corresponded changes in clinical and laboratory outcomes during hospitalization period. RESULTS: LUS score on admission was higher among the deteriorating patients and significantly (P=0.038-0.0001) correlated (Spearman's rho) with the levels of C-reactive protein (0.58), lymphocytes (-0.33), SpO2 (-0.48) and oxygen supplementation (0.48) upon admission. The increase in LUS score between the two scans was significantly correlated (0.544, P=0.006) with longer hospital stay. CONCLUSION: Lung ultrasound assessment can be a useful as an imaging modality for SARS CoV-2 patients. Larger studies are needed to further investigate the predictive role of LUS in the duration and the outcome of the hospitalization of these patients.

A randomized, open-label, adaptive, proof-of-concept clinical trial of modulation of host thromboinflammatory response in patients with COVID-19: the DAWn-Antico study.

BACKGROUND: The peak of the global COVID-19 pandemic has not yet been reached, and many countries face the prospect of a second wave of infections before effective vaccinations will be available. After an initial phase of viral replication, some patients develop a second illness phase in which the host thrombotic and inflammatory responses seem to drive complications. Severe COVID-19 disease is linked to high mortality, hyperinflammation, and a remarkably high incidence of thrombotic events. We hypothesize a crucial pathophysiological role for the contact pathway of coagulation and the kallikrein-bradykinin pathway. Therefore, drugs that modulate this excessive thromboinflammatory response should be investigated in severe COVID-19. METHODS: In this adaptive, open-label multicenter randomized clinical trial, we compare low molecular weight heparins at 50 IU anti-Xa/kg twice daily-or 75 IU anti-Xa twice daily for intensive care (ICU) patients-in combination with aprotinin to standard thromboprophylaxis in hospitalized COVID-19 patients. In the case of hyperinflammation, the interleukin-1 receptor antagonist anakinra will be added on top of the drugs in the interventional arm. In a pilot phase, the effect of the intervention on thrombotic markers (D-dimer) will be assessed. In the full trial, the primary outcome is defined as the effect of the interventional drugs on clinical status as defined by the WHO ordinal scale for clinical improvement. DISCUSSION: In this trial, we target the thromboinflammatory response at multiple levels. We intensify the dose of low molecular weight heparins to reduce thrombotic complications. Aprotinin is a potent kallikrein pathway inhibitor that reduces fibrinolysis, activation of the contact pathway of coagulation, and local inflammatory response. Additionally, aprotinin has shown in vitro inhibitory effects on SARS-CoV-2 cellular entry. Because the excessive thromboinflammatory response is one of the most adverse prognostic factors in COVID-19, we will add anakinra, a recombinant interleukin-1 receptor antagonist, to the regimen in case of severely increased inflammatory parameters. This way, we hope to modulate the systemic response to SARS-CoV-2 and avoid disease progressions with a potentially fatal outcome. TRIAL REGISTRATION: The EU Clinical Trials Register 2020-001739-28 . Registered on April 10, 2020.

Impact of supplemental parenteral nutrition early during critical illness on invasive fungal infections: a secondary analysis of the EPaNIC randomized controlled trial.

OBJECTIVE: In the EPaNIC RCT (N=4640), postponing the administration of parenteral nutrition (PN) to beyond 1 week in the intensive care unit (ICU) (late-PN) reduced the number of ICU-acquired infections and the costs for antimicrobial drugs compared with initiation of PN within 24-48 hours of admission (early-PN). In a secondary analysis, we hypothesize that late-PN reduces the odds to acquire an invasive fungal infection (IFI) in the ICU. METHODS: The impact of late-PN (N=2328) versus early-PN (N=2312) on acquired IFI and on the likelihood to acquire an IFI over time was assessed in univariable and multivariable analyses. Subsequently, we performed multivariable analyses to assess the effect of the mean total daily administered calories from admission until day 3, day 5, and day 7 on the likelihood over time of acquiring an IFI. RESULTS: Fewer late-PN patients acquired an IFI compared with early-PN patients (77/2328 versus 112/2312) (p 0.008). After adjusting for risk factors, the odds to acquire an IFI and the likelihood of acquiring an IFI at any time were lower in late-PN (adjusted odds ratio 0.66, 95% CI 0.48-0.90, p 0.009; adjusted hazard ratio (HRadj) 0.70, 95% CI 0.52-0.93, p 0.02). Larger caloric amounts from admission until day 7 were associated with a higher likelihood to acquire an IFI over time (HRadj 1.09, 95% CI 1.02-1.16, p 0.009). CONCLUSION: Postponing PN to beyond 1 week and smaller caloric amounts until day 7 in the ICU reduced ICU-acquired IFIs and the likelihood to develop an IFI over time.

Monitoring of behavior, sex hormones and boar taint compounds during the vaccination program for immunocastration in three sire lines.

Immunocastration (vaccination against boar taint) is an alternative method to prevent boar taint without the need for surgical castration. This study investigates the evolution of boar taint compounds in serum and fat, serum steroid compounds as well as behavior in immunocastrated pigs from 3 sire lines: 15 stress positive Belgian Piétrain (BP), 20 stress negative French Piétrain (FP), and 20 stress negative Canadian Duroc (CD). Hormone and boar taint compounds in serum were determined at 4 time points; boar taint compounds in fat were determined at 3 time points. Behavior, skin lesions, animal and pen fouling were also recorded before the first vaccination (V2). Aggressiveness, eating and drinking and general activity behavior declined from  V2 for all sire lines. Pigs from BP were cleaner than FP and CD pigs. Even though immunocastration was effective in general (reduced testosterone, estradiol as well as androstenone in serum) for all sire lines, some individual pigs showed either androstenone or skatole levels in fat above cutoff values. While the immunocastration mechanism works as intended for androstenone, and also for skatole for the three sire lines, the risk of carcasses with boar taint compounds above cutoff levels (respectively 1.9 and 3.7%) still remains to some extent.

On farm intervention studies on reduction of boar taint prevalence: Feeding strategies, presence of gilts and time in lairage.

One of the challenges in the production of entire male pigs is the occurrence of boar taint. We separately tested the effect of 3 management strategies to reduce boar taint on respectively 2, 3, and 6 Flemish pig farms: 1) adapted feeding strategies, 2) presence of gilts in the compartment, and 3) varying lairage duration at the slaughterhouse. A commercialized feed concept resulted in a significant reduction of olfactory boar taint prevalence when fed for 2 weeks (T2W) compared to control (T-CON) (P = 0.030). For T2W and when fed for 3 weeks (T3W), androstenone (AND) (P = 0.002 for T2W, P = 0.029 for T3W) and skatole (SKA) (P < 0.001 for T2W and T3W) were significantly reduced compared to T-CON. Olfactory boar taint prevalence was significantly reduced when feeding 5% dried chicory roots (FI5%) (P = 0.032), but not for 3% dried chicory roots (FI3%) (P = 0.958). SKA concentration was significantly lower when feeding FI5% (P < 0.001) and when feeding FI3% (P = 0.034). Rearing entire male pigs separately from gilts and increasing lairage duration from <1 h to >3 h did not significantly affect boar taint.

Discrimination between Synthetically Administered and Endogenous Thiouracil Based on Monitoring of Urine, Muscle, and Thyroid Tissue: An in Vivo Study in Young and Adult Bovines.

Thiouracil (TU), synthesized for its thyroid-regulating capacities and alternatively misused in livestock for its weight-gaining effects, is acknowledged to have an endogenous origin. Discrimination between low-level abuse and endogenous occurrence is challenging and unexplored in an experimental setting. Therefore, cows (n = 16) and calves (n = 18) were subjected to a rapeseed-supplemented diet or treated with synthetic TU. Significant higher urinary TU levels were recorded after TU administration (

Olfactory evaluation of boar taint: effect of factors measured at slaughter and link with boar taint compounds.

There is a commitment by the European pig sector to ban surgical castration of male piglets in the European Union in 2018. One alternative to castration is to raise entire male pigs, with an increased risk of boar taint. A field study was performed to: (1) evaluate inter- and intra-farm variation in boar taint prevalence, (2) investigate factors measured at slaughter influencing boar taint and (3) evaluate the relationship between sensorial scoring by a trained panel and the concentration of boar taint components. From 34 farms, neck fat samples were collected from all entire male pigs in at least two slaughter batches per farm (78 batches; 9167 animals). In addition to olfactory boar taint analysis, data were also collected on fresh skin lesions (score 0 to 3) at the slaughter line, slaughter weight, lean meat percentage, duration of transport, time spent in lairage, total delivery duration, day length, shortening of days and outdoor mean temperature. Using the hot iron method, neck fat samples were scored (eight-point scale) for boar taint. Average boar taint prevalence (score ≥3) was 5.6±2.5% and the mean difference between the maximum and minimum prevalence per farm was 4.3±3.2%. Androstenone (AND), skatole (SKA) and indole concentrations were measured for a subset (n=254) of the samples. According to binomial univariate mixed models, entire male pigs with a higher skin lesion score had higher odds of having boar taint (P=0.031), as did fatter entire male pigs (P<0.001). In the binomial multivariate mixed model lean meat percentage (P<0.001) and outdoor mean temperature (P=0.005) remained as only significant factors. Based on our results, we can conclude that these statistically significant at least partially influence the prevalence of boar taint. According to the binomial univariate mixed models SKA concentration in liquid fat seems a better predictor for boar taint than AND. There were no significant synergetic effects between boar taint compounds.

Fingerprinting and validation of a LC-DAD method for the analysis of biflavanones in Garcinia kola-based antimalarial improved traditional medicines.

African populations use traditional medicines in their initial attempt to treat a range of diseases. Nevertheless, accurate knowledge of the composition of these drugs remains a challenge in terms of ensuring the health of population and in order to advance towards improved traditional medicines (ITMs). In this paper chromatographic methods were developed for qualitative and quantitative analyses of a per os antimalarial ITM containing Garcinia kola. The identified analytical markers were used to establish TLC and HPLC fingerprints. G. kola seeds were analysed by HPLC to confirm the identity of the extract used by the Congolese manufacturer in the ITM. The main compounds (GB1, GB2, GB-1a and Kolaflavanone) were isolated by preparative TLC and identified by UPLC-MS and NMR. For the quantification of the major compound GB1, a simple and rapid experimental design was applied to develop an LC method, and then its validation was demonstrated using the total error strategy with the accuracy profile as a decision tool. The accurate results were observed within 0.14-0.45mg/mL range of GB1 expressed as naringenin. The extracts used in several batches of the analysed oral solutions contained GB1 (expressed as naringenin) within 2.04-2.43%. Both the fingerprints and the validated LC-DAD were found suitable for the quality control of G. kola-based raw material and finished products, respectively.

An intervention study demonstrates effects of MC4R genotype on boar taint and performances of growing-finishing pigs.

The Asp298Asn polymorphism of the melanocortin-4 receptor (MC4R) in pigs is known to affect economically important traits such as growth rate and backfat thickness. We have assessed the possible use of this polymorphism as a molecular marker to perform genetic selection toward lower boar taint levels without compromising growth performance and carcass and meat quality in commercial boars and gilts. Homozygous boars and gilts of the AA genotype and GG genotype were compared in an intervention study with a 2 × 2 design to assess main effects and possible interactions between sex and genotype. The concentrations of the 3 boar taint compounds androstenone ( = 0.044), skatole ( = 0.049), and indole ( = 0.006) were significantly higher in fat of AA boars compared to GG boars. However, no effect on the sensory analysis of the fat samples could be observed. Between 20 and 115 kg BW, AA pigs showed higher ADFI than GG pigs ( < 0.001). An interaction between genotype and sex was observed for ADG ( = 0.044): AA boars had a significantly higher ADG than GG boars but there was no significant difference between the gilts. Daily lean meat gain tended to be higher in boars compared to gilts ( = 0.051), independent of genotype. Similarly, boars showed higher G:F compared to gilts ( < 0.001), without effect of genotype. Genotype and sex affected several carcass quality parameters but there was no interaction. Pigs of the AA genotype displayed a lower dressing percentage ( = 0.005), lower ham width ( = 0.024), lower muscle thickness ( = 0.011), and higher fat thickness ( < 0.001), resulting in a lower lean meat percentage ( < 0.001) in comparison with GG pigs. Gilts had a significantly higher dressing percentage ( < 0.001), higher muscle thickness ( < 0.001), higher ham width ( < 0.001), and lower ham angle ( < 0.001) compared to boars. Other than the boar taint compounds, meat quality was not affected by genotype. Pork of gilts was darker ( = 0.014) and less exudative during cooking ( < 0.001) and contained more intramuscular fat ( = 0.013). These results indicate that genetic selection against boar taint is possible using this marker. This will also result in lower feed intake and ADG and, consequently, better carcass quality.

Development of a quantitative method for the simultaneous analysis of the boar taint compounds androstenone, skatole and indole in porcine serum and plasma by means of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry.

Boar taint is an off-odour occurring while heating meat or fat from boars. A method detecting the three compounds (androstenone, skatole and indole) simultaneously in blood would offer substantial advantages since it would allow monitoring the impact of rearing strategies. Therefore, a UHPLC-HR-Orbitrap-MS analysis method is optimized and validated for the quantification of these compounds in plasma or serum. Sample pre-treatment involved an extraction with diethylether followed by a centrifugal filtration (30 kDa). Limits of detection and quantification varied between 0.5 and 1 µg L(-1) and 2 and 3 µg L(-1) for the three compounds, respectively. Besides, an excellent repeatability (RSD < 7.6%), within-laboratory reproducibility (RSD<10.5%), recovery (87-97%) and linearity (R(2)>0.99) were recorded. Correlations between serum/plasma and fat levels of the boar taint compounds were positive for skatole (r(serum) = 0.39 and r(plasma) = 0.84) and androstenone (r(serum) = 0.73-0.78 and r(plasma) = 0.32-0.80).

The effect of the MC4R gene on boar taint compounds, sexual maturity and behaviour in growing-finishing boars and gilts.

Societal pressure to ban surgical castration of male piglets is rising due to animal welfare concerns, thus other methods to prevent boar taint need to be explored. Genetic selection against boar taint appears to be a long-term sustainable alternative. However, as boar taint is linked to reproductive hormones, it is important to consider possible negative side effects such as delayed sexual maturity or changes in behaviour. We reported earlier that the melanocortin-4 receptor (MC4R) marker can be used to reduce boar taint levels in fat of boars. The objective of this study was to evaluate whether MC4R marker-assisted selection for lower boar taint prevalence affects plasma levels of boar taint compounds and testosterone; sexual maturity; behaviour; skin lesions; and lameness in boars and gilts. Using an intervention study with a 2×2 design, 264 boars and gilts differing on position 893 of the MC4R gene (AA v. GG) were compared. The MC4R polymorphism did not affect the plasma concentration of either androstenone or testosterone at different time points, whereas the concentration of skatole was significantly lower (P=0.003) and the concentration of indole tended to be lower (P=0.074) in GG compared with AA boars. A higher percentage of gilts of the GG genotype were in puberty at slaughter age compared with AA gilts (P<0.001). The age of the boars at sexual maturity (as indicated by the first positive preputial smear test) did not differ between AA and GG boars. In contrast, weight of GG boars at sexual maturity tended to be lower (P=0.065). During the period from 6 weeks of age to slaughter, boars and gilts of the GG genotype showed more playing behaviour (P=0.015) and less passive and feeding behaviour (P=0.003). They showed more skin lesions on their back and caudal area (P=0.022), and tended to show more skin lesions on their head and anterior area (P=0.093) compared with AA animals. In conclusion, the polymorphism in the MC4R gene can be used as a marker without negative effects on reproduction characteristics in boars and gilts. Genetic selection towards a lower prevalence of boar taint will lead to more active pigs with more skin lesions. Management strategies may therefore be necessary to reduce skin lesions in the selected animals.

Transmission of Pneumocystis carinii from AIDS patients to other immunosuppressed patients: a cluster of Pneumocystis carinii pneumonia in renal transplant recipients.

Five renal transplant recipients developed Pneumocystis carinii pneumonia (PCP) over a 22-month period, while no cases had been observed over a 5-year period in 114 transplanted patients treated with the same immunosuppressive protocol. All patients were HIV-negative, and no modification in diagnostic techniques for P. carinii could account for this observation. All five patients developed PCP within 2 months of an acute graft rejection episode. All of them attended the same outpatient facility as AIDS patients attending the hospital, where they shared the waiting and treatment rooms. Comparison of cases with matched controls was possible in three instances and revealed that the cases had had more outpatient clinic encounters with AIDS patients who had presented, or subsequently developed, PCP. This observation suggests that AIDS patients developing PCP may transmit the infection to other immunosuppressed patients.

Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes.

The human extracellular matrix protein 1 (Ecm1) gene is located at chromosome band 1q21 close to the epidermal differentiation complex and is transcribed in two discrete mRNAs: a full length Ecm1a and a shorter, alternatively spliced, Ecm1b transcript, the expression of which is restricted to tonsils and skin. The chromosomal localization and the Ecm1b expression in skin prompted us to investigate the role of Ecm1 in keratinocyte differentiation. In this study, we provide evidence for the existence of a relationship between keratinocyte differentiation and expression of the Ecm1b transcript. Cultures of subconfluent undifferentiated normal human keratinocytes express only Ecm1a. Upon reaching confluence, the cells start to differentiate, as measured by keratin K10 mRNA expression. Concomitantly Ecm1b mRNA expression is induced, although expression of Ecm1a mRNA remains unchanged. In addition, treatment of undifferentiated normal human keratinocyte cells with 12-O-tetradecanoyl-phorbol-13-acetate strongly induces the expression of Ecm1b mRNA. Expression of Ecm1b can also be induced by coculturing normal human keratinocytes with lethally irradiated feeder cells and by a diffusible factor secreted by stromal cells. In adult human skin, Ecm1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers, whereas expression of Ecm1b mRNA is predominantly found in spinous and granular cell layers. Immunohistochemically, Ecm1a expression is almost completely restricted to the basal cell layer, whereas Ecm1b is detected in the suprabasal layers. These results are strongly suggestive of a role for Ecm1b in terminal keratinocyte differentiation, which is also supported by the localization of the Ecm1 gene at 1q21. Refinement of its genomic localization, however, placed Ecm1 centromeric of the epidermal differentiation complex.