Transcriptomics reveals immune-metabolism disorder in acute-on-chronic liver failure in rats

Acute-on-chronic liver failure (ACLF) is clinical syndrome with high mortality rate. This study aimed to perform detailed transcriptomic analysis in liver cirrhosis–based ACLF rats to elucidate ACLF pathogenesis. ACLF was induced by combined porcine serum with D-galactosamine and lipopolysaccharide. Gene expression profile of liver tissues from ACLF rats was generated by transcriptome sequencing to reveal the molecular mechanism. ACLF rats successfully developed with typical characteristics. Total of 2,354/3,576 differentially expressed genes were identified when ACLF was compared to liver cirrhosis and normal control, separately. The functional synergy analysis revealed prominent immune dysregulation at ACLF stage, whereas metabolic disruption was significantly down-regulated. Relative proportions of innate immune–related cells showed significant elevation of monocytes and macrophages, whereas adaptive immune–related cells were reduced. The seven differentially expressed genes underlying the ACLF molecular mechanisms were externally validated, among them THBS1, IL-10, and NR4A3 expressions were confirmed in rats, patient transcriptomics, and liver biopsies, verifying their potential value in the ACLF pathogenesis. This study indicates immune-metabolism disorder in ACLF rats, which may provide clinicians new targets for improving intervention strategies.

Association between Equol Production Status and Nonalcoholic Steatohepatitis

Equol is a metabolite of daidzein, a major soybean isoflavone with estrogenic and antioxidant activities. As the production of equol depends on the presence of certain members of the intestinal microflora, not all individuals can produce equol. We examined the relationship between NASH histological features and equol production. In an animal model, obese OLETF rats were intraperitoneally injected with a porcine serum to augment liver fibrogenesis. Equol-rich soy product, SE5-OH was orally administered during the experimental period. Treatment with SE5-OH markedly attenuated the development of liver fibrosis and expression of alpha-smooth muscle actin. In clinical research, 38 NAFLD patients (13 men and 25 women) were included. The degree of fibrosis and ballooning in equol-nonproducers was significantly higher than in equol-producers in women. The percentage of nonproducers with NAFLD activity score (NAS) ≥5 was significantly higher than that of producers. None of the histological features were significantly different between nonproducers and producers in men. Decision tree analysis identified predictors for NAS ≥5 in women. The status of equol production was the strongest predictor, followed by fasting glucose. Since equol can be noninvasively detected in urine, it can be applied as a screening tool for the progression of NASH in women.

Prevalence of Linda Virus Neutralizing Antibodies in the Austrian Pig Population

A novel pestivirus species, termed Lateral-shaking Inducing Neuro-Degenerative Agent virus (LindaV), was discovered in a piglet-producing farm in Austria in 2015 related to severe congenital tremor cases. Since the initial outbreak LindaV has not been found anywhere else. In this study, we determined the seroprevalence of LindaV infections in the domestic pig population of Austria. A fluorophore labeled infectious cDNA clone of LindaV (mCherry-LindaV) was generated and used in serum virus neutralization (SVN) assays for the detection of LindaV specific neutralizing antibodies in porcine serum samples. In total, 637 sera from sows and gilts from five federal states of Austria, collected between the years 2015 and 2020, were analyzed. We identified a single serum showing a high neutralizing antibody titer, that originated from a farm (Farm S2) in the proximity of the initially affected farm. The analysis of 57 additional sera from Farm S2 revealed a wider spread of LindaV in this pig herd. Furthermore, a second LindaV strain originating from this farm could be isolated in cell culture and was further characterized at the genetic level. Possible transmission routes and virus reservoir hosts of this emerging porcine virus need to be addressed in future studies.

Development and Validation of an Immunoenzymatic Assay for Pig IgG Quantification in Serum Samples

This research describes the development and validation of a sandwich-type Enzyme-Linked-Immunosorbent Assay (ELISA) assay based on a sheep polyclonal antibody developed to determine porcine immunoglobulin G (IgG) concentration in serum samples. The coating and blocking conditions were established. The immunoassay demonstrated the ability to specifically quantify pig IgG in serum samples without reacts with rabbit or mouse IgG, evaluated in serum and ascites, respectively. Given its precision, specificity and accuracy, this ELISA is a tool for the measurement of IgG in porcine serum samples, to evaluate the humoral immune status of pigs.

Active Human and Porcine Serum Induce Competence for Genetic Transformation in the Emerging Zoonotic Pathogen Streptococcus suis

The acquisition of novel genetic traits through natural competence is a strategy used by bacteria in microbe-rich environments where microbial competition, antibiotics, and host immune defenses threaten their survival. Here, we show that virulent strains of Streptococcus suis, an important zoonotic agent and porcine pathogen, become competent for genetic transformation with plasmid or linear DNA when cultured in active porcine and human serum. Competence was not induced in active fetal bovine serum, which contains less complement factors and immunoglobulins than adult serum and was strongly reduced in heat-treated or low-molecular weight fractions of active porcine serum. Late competence genes, encoding the uptake machinery for environmental DNA, were upregulated in the active serum. Competence development was independent of the early competence regulatory switch involving XIP and ComR, as well as sigma factor ComX, suggesting the presence of an alternative stress-induced pathway for regulation of the late competence genes required for DNA uptake.

A nanobody‐horseradish peroxidase fusion protein‐based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2

Background The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)‑horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. Results Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15‑HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti‑PCV2 antibodies. The cut‑off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. Conclusions In brief, the newly developed cELISA based PCV2-Nb15‑HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

Nutritional Intervention in Chronic Fatigue Syndrome and Fibromyalgia (CFS/FMS) A Unique Porcine Serum Polypeptide Nutritional Supplement

Background: Clinical experience suggested that a unique porcine serum polypeptide extract, used in hospitals for people with severe malnutrition, serendipitously resulted in a dramatic improvement in many fibromyalgia cases. Aims: The study aims to determine the effectiveness of a unique polypeptide serum extract in improving the symptoms of CFS and fibromyalgia (CFS/FMS). Methods: An open-label prospective study of 43 people with CFS or Fibromyalgia recruited worldwide. Interventions: Four 500 mg tablets twice daily for five weeks. Outcome Measures: Assessed baseline at five weeks of treatment using a VAS(1-10 points) rating energy, sleep, cognitive function, pain, overall well-being, anxiety, and digestive health, as well as the FIQR. The primary outcome measure was the pre- and post-treatment VAS composite score for the first five symptoms. Results: 43 subjects completed the three-week treatment trial. 60.5% of subjects rated themselves as improved, with 18.6% rating themselves as much better. In the 60.5% of subjects that rated themselves as improved, the significant average improvement was seen in all categories: 1. 69.4% increase in energy(p<.001) 2. 69.2% increase in overall well-being(<.001) 3. 53.8% improvement in sleep(<.001) 4. 60.5% improvement in mental clarity(<.001) 5. 37.9% decrease in pain(<.013) 6. 34.8% decrease in anxiety(<.001) 7. 54.6% improvement in digestive symptoms(<.001) 8. FIQR 59.2 to 39.3(<.001) In six individuals who also had pre- and post IgG antibody levels, total IgG increased by 13.8% on average, with similar improvements seen in the IgG 1-4 subsets. Conclusion: Recovery Factors® resulted in markedly improved energy, sleep, cognition, pain relief, calming, digestion and overall well-being in those with CFS/FMS. Clinical Trial Registration Number: NCT04381793.

Bioactive peptides originating from gastrointestinal endogenous proteins in the growing pig: In vivo identification

Background: Recent in silico and in vitro studies have shown that gastrointestinal endogenous proteins (GEP) are a source of bioactive peptides. To date, however, the presence of such peptides in the lumen of the digestive tract has not been demonstrated. Objective: We investigated the generation of GEP-derived bioactive peptides in the growing pig fed a protein-free diet. Methods: Stomach chyme (SC) and jejunal digesta (JD) fractions from 6 growing pigs (two sampling times) were assessed for their angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1) inhibition, and antioxidant activity using the 2,2- diphenyl-1-picrylhydrazyl (DPPH) inhibition, ferric reducing antioxidant power (FRAP) and microsomal lipid peroxidation (MLP) inhibition assays. Results: Two of the fractions prepared from JD samples inhibited ACE-I and DPPH by 81 (± 2.80)% and 94 (±0.66)%. SC fractions were found to inhibit MLP between 15-39 (±3.52-1.40)%. The study identified over 180 novel peptide sequences that were related to the determined bioactivities, including a porcine serum albumin-derived peptide (FAKTCVADE SAENCDKS), corresponding to f(7-23) of the human serum albumin peptide LVNEVTEFAKTCVADESAEN CDKSLHTLF that was previously identified from the digests of the latter GEP. Conclusions: This study provides the first in vivo evidence for GEP as a source of bioactive peptides. These new findings help advance our knowledge of the latent bioactive role of GEP-derived peptides in mammalian nutrition and health, and their potential pharmaceutical applications.

A Nanobody-horseradish Peroxidase Fusion Protein-based Competitive ELISA for Rapid Detection of Antibodies Against Porcine Circovirus Type 2

Background: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)‑horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum.Results: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15‑HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti‑PCV2 antibodies. The cut‑off value of the cELISA was 20.72%, 360 porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68% and 95.92%, respectively. The coincidence rate of the two methods was 99.17%. When detecting 620 clinical porcine serum samples, a good consistent (kappa value=0.954) was found between the result of the cELISA and that of commercial kits.Conclusions: In brief, the newly developed cELISA based PCV2-Nb15‑HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccines efficacy and indirect diagnosis of PCV2 infection.