Serum Amyloid A Proteins Induce Pathogenic Th17 Cells and Promote Inflammatory Disease.

Lymphoid cells that produce interleukin (IL)-17 cytokines protect barrier tissues from pathogenic microbes but are also prominent effectors of inflammation and autoimmune disease. T helper 17 (Th17) cells, defined by RORγt-dependent production of IL-17A and IL-17F, exert homeostatic functions in the gut upon microbiota-directed differentiation from naive CD4+ T cells. In the non-pathogenic setting, their cytokine production is regulated by serum amyloid A proteins (SAA1 and SAA2) secreted by adjacent intestinal epithelial cells. However, Th17 cell behaviors vary markedly according to their environment. Here, we show that SAAs additionally direct a pathogenic pro-inflammatory Th17 cell differentiation program, acting directly on T cells in collaboration with STAT3-activating cytokines. Using loss- and gain-of-function mouse models, we show that SAA1, SAA2, and SAA3 have distinct systemic and local functions in promoting Th17-mediated inflammatory diseases. These studies suggest that T cell signaling pathways modulated by the SAAs may be attractive targets for anti-inflammatory therapies.

Multiplex Assay to Determine Acute Phase Proteins in Modified Live PRRSV Vaccinated Pigs.

Acute phase protein (APP) response to vaccine challenges is an attractive alternative to natural infection for identifying pigs with increased disease resilience and monitoring the productive performance. Currently, the methods used for APP quantification are diverse and often based on techniques that use antibodies that are not necessarily pig specific. The objective of this work is the development of a method based on a UPLC-SRM/MS system for simultaneous determination of haptoglobin, apolipoprotein A1, C-reactive protein, pig-major acute protein, and serum amyloid A and its application in pigs to monitor the effect of a vaccine administered against porcine reproductive and respiratory syndrome virus (PRRSV). With the aim of tracing the complete analytical process for each proteotypic peptide, a synthetic QconCat polypeptide construct was designed. It was possible to develop an SRM method including haptoglobin, apolipoprotein A1, pig-MAP, and serum amyloid A1. The PRRSV vaccine only affected haptoglobin. The pigs with positive viremia tended to show higher values than negative pigs, reaching significant differences in the three haptoglobin SRM-detected peptides but not with the data acquired by immunoenzymatic and spectrophotometric assays. These results open the door to the use of SRM to accurately monitor APP changes in experimental pigs.

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant.

Using proteomics and complexome profiling, we evaluated in a year-long study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2-glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14. As negative APPs, being downregulated upon inflammation, we identified the well-documented Serotransferrin and Transthyretin, but added Kallistatin, Heparin cofactor 2, and interalpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For the patient with the most severe APR, we performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observed that several plasma proteins displaying alike concentration patterns coelute and form macromolecular complexes. By complexome profiling, we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, replacing largely Apolipoprotein (APO)A1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on correlated variations in the abundance of several plasma proteins upon inflammatory events.

The Molecular Signature Related to Local Inflammatory and Immune Response in Canine Cutaneous Hypersensitivity Reactions: A Preliminary Study.

Cutaneous hypersensitivity reactions (CHRs) are complex inflammatory skin disorders that affect humans and dogs. This study examined the inflammatory and immune responses leading to skin damage, inflammation, and irritation by investigating gene expression through quantitative PCR (qPCR) and protein localization through the immunohistochemistry (IHC) of specific receptors and molecules involved in CHRs. Formalin-fixed paraffin-embedded (FFPE) samples from canine CHR skin (n = 20) and healthy dog skin (n = 3) were analyzed for expression levels of eight genes, including members of the pattern recognition receptor (PRR) family, CD209 and CLEC4G, the Regakine-1-like chemokine, and acute phase proteins (APPs), LBP-like and Hp-like genes. Additionally, we examined the local involvement of IL-6, Janus Kinase 1 (JAK1), and the signal transducer activator of transcription 3 (STAT3) in the CHR cases. The study demonstrated statistically significant increases in the expression levels of CD209, Hp-like (p < 0.01), LBP-like, Regakine-1-like, and CLEC4G (p < 0.05) genes in CHRs compared to healthy controls. Conversely, IL-6, JAK1, and STAT3 showed no significant difference between the two groups (p > 0.05). Protein analysis revealed JAK1 and STAT3 expression in CHR hyperplastic epithelial cells, dermal fibroblasts, and endothelial cells of small capillaries, indicating a possible involvement in the JAK/STAT pathway in local inflammatory response regulation. Our findings suggest that the skin plays a role in the development of CHRs.

Dysregulated proteasome activity and steroid hormone biosynthesis are associated with mortality among patients with acute COVID-19.

The persistence of coronavirus disease 2019 (COVID-19)-related hospitalization severely threatens medical systems worldwide and has increased the need for reliable detection of acute status and prediction of mortality. We applied a systems biology approach to discover acute-stage biomarkers that could predict mortality. A total 247 plasma samples were collected from 103 COVID-19 (52 surviving COVID-19 patients and 51 COVID-19 patients with mortality), 51 patients with other infectious diseases (IDCs) and 41 healthy controls (HCs). Paired plasma samples were obtained from survival COVID-19 patients within 1 day after hospital admission and 1-3 days before discharge. There were clear differences between COVID-19 patients and controls, as well as substantial differences between the acute and recovery phases of COVID-19. Samples from patients in the acute phase showed suppressed immunity and decreased steroid hormone biosynthesis, as well as elevated inflammation and proteasome activation. These findings were validated by enzyme-linked immunosorbent assays and metabolomic analyses in a larger cohort. Moreover, excessive proteasome activity was a prominent signature in the acute phase among patients with mortality, indicating that it may be a key cause of poor prognosis. Based on these features, we constructed a machine learning panel, including four proteins [C-reactive protein (CRP), proteasome subunit alpha type (PSMA)1, PSMA7, and proteasome subunit beta type (PSMB)1)] and one metabolite (urocortisone), to predict mortality among COVID-19 patients (area under the receiver operating characteristic curve: 0.976) on the first day of hospitalization. Our systematic analysis provides a novel method for the early prediction of mortality in hospitalized COVID-19 patients.

In silico analysis of intestinal microbial instability and symptomatic markers in mice during the acute phase of severe burns.

BACKGROUND: Severe burns may alter the stability of the intestinal flora and affect the patient's recovery process. Understanding the characteristics of the gut microbiota in the acute phase of burns and their association with phenotype can help to accurately assess the progression of the disease and identify potential microbiota markers. METHODS: We established mouse models of partial thickness deep III degree burns and collected faecal samples for 16 S rRNA amplification and high throughput sequencing at two time points in the acute phase for independent bioinformatic analysis. RESULTS: We analysed the sequencing results using alpha diversity, beta diversity and machine learning methods. At both time points, 4 and 6 h after burning, the Firmicutes phylum content decreased and the content of the Bacteroidetes phylum content increased, showing a significant decrease in the Firmicutes/Bacteroidetes ratio compared to the control group. Nine bacterial genera changed significantly during the acute phase and occupied the top six positions in the Random Forest significance ranking. Clustering results also clearly showed that there was a clear boundary between the communities of burned and control mice. Functional analyses showed that during the acute phase of burn, gut bacteria increased lipoic acid metabolism, seleno-compound metabolism, TCA cycling, and carbon fixation, while decreasing galactose metabolism and triglyceride metabolism. Based on the abundance characteristics of the six significantly different bacterial genera, both the XGboost and Random Forest models were able to discriminate between the burn and control groups with 100% accuracy, while both the Random Forest and Support Vector Machine models were able to classify samples from the 4-hour and 6-hour burn groups with 86.7% accuracy. CONCLUSIONS: Our study shows an increase in gut microbiota diversity in the acute phase of deep burn injury, rather than a decrease as is commonly believed. Severe burns result in a severe imbalance of the gut flora, with a decrease in probiotics and an increase in microorganisms that trigger inflammation and cognitive deficits, and multiple pathways of metabolism and substance synthesis are affected. Simple machine learning model testing suggests several bacterial genera as potential biomarkers of severe burn phenotypes.

Mapping dynamic molecular changes in hippocampal subregions after traumatic brain injury through spatial proteomics.

BACKGROUND: Traumatic brain injury (TBI) often results in diverse molecular responses, challenging traditional proteomic studies that measure average changes at tissue levels and fail to capture the complexity and heterogeneity of the affected tissues. Spatial proteomics offers a solution by providing insights into sub-region-specific alterations within tissues. This study focuses on the hippocampal sub-regions, analyzing proteomic expression profiles in mice at the acute (1 day) and subacute (7 days) phases of post-TBI to understand subregion-specific vulnerabilities and long-term consequences. METHODS: Three mice brains were collected from each group, including Sham, 1-day post-TBI and 7-day post-TBI. Hippocampal subregions were extracted using Laser Microdissection (LMD) and subsequently analyzed by label-free quantitative proteomics. RESULTS: The spatial analysis reveals region-specific protein abundance changes, highlighting the elevation of FN1, LGALS3BP, HP, and MUG-1 in the stratum moleculare (SM), suggesting potential immune cell enrichment post-TBI. Notably, established markers of chronic traumatic encephalopathy, IGHM and B2M, exhibit specific upregulation in the dentate gyrus bottom (DG2) independent of direct mechanical injury. Metabolic pathway analysis identifies disturbances in glucose and lipid metabolism, coupled with activated cholesterol synthesis pathways enriched in SM at 7-Day post-TBI and subsequently in deeper DG1 and DG2 suggesting a role in neurogenesis and the onset of recovery. Coordinated activation of neuroglia and microtubule dynamics in DG2 suggest recovery mechanisms in less affected regions. Cluster analysis revealed spatial variations post-TBI, indicative of dysregulated neuronal plasticity and neurogenesis and further predisposition to neurological disorders. TBI-induced protein upregulation (MUG-1, PZP, GFAP, TJP, STAT-1, and CD44) across hippocampal sub-regions indicates shared molecular responses and links to neurological disorders. Spatial variations were demonstrated by proteins dysregulated in both or either of the time-points exclusively in each subregion (ELAVL2, CLIC1 in PL, CD44 and MUG-1 in SM, and SHOC2, LGALS3 in DG). CONCLUSIONS: Utilizing advanced spatial proteomics techniques, the study unveils the dynamic molecular responses in distinct hippocampal subregions post-TBI. It uncovers region-specific vulnerabilities and dysregulated neuronal processes, and potential recovery-related pathways that contribute to our understanding of TBI's neurological consequences and provides valuable insights for biomarker discovery and therapeutic targets.

Deletion of the P2Y2 receptor aggravates internal elastic lamina calcification in chronic kidney disease mice through upregulation of alkaline phosphatase and lipocalin-2.

Calcification of the medial layer, inducing arterial stiffness, contributes significantly to cardiovascular mortality in patients with chronic kidney disease (CKD). Extracellular nucleotides block the mineralization of arteries by binding to purinergic receptors including the P2Y2 receptor. This study investigates whether deletion of the P2Y2 receptor influences the development of arterial media calcification in CKD mice. Animals were divided into: (i) wild type mice with normal renal function (control diet) (n = 8), (ii) P2Y2 R-/- mice with normal renal function (n = 8), (iii) wild type mice with CKD (n = 27), and (iv) P2Y2 R-/- mice with CKD (n = 22). To induce CKD, animals received an alternating (0.2-0.3%) adenine diet for 7 weeks. All CKD groups developed a similar degree of chronic renal failure as reflected by high serum creatinine and phosphorus levels. Also, the presence of CKD induced calcification in the heart and medial layer of the aortic wall. However, deletion of the P2Y2 receptor makes CKD mice more susceptible to the development of calcification in the heart and aorta (aortic calcium scores (median ± IQR), CKD-wild type: 0.34 ± 4.3 mg calcium/g wet tissue and CKD-P2Y2 R-/- : 4.0 ± 13.2 mg calcium/g wet tissue). As indicated by serum and aortic mRNA markers, this P2Y2 R-/- mediated increase in CKD-related arterial media calcification was associated with an elevation of calcification stimulators, including alkaline phosphatase and inflammatory molecules interleukin-6 and lipocalin 2. The P2Y2 receptor should be considered as an interesting therapeutic target for tackling CKD-related arterial media calcification.

Does physical activity and inflammation mediate the job stress-headache relationship? A sequential mediation analysis in the ELSA-Brasil study.

BACKGROUND: Evidence indicates that physical activity reduces stress and promote a myriad of health-enhancing effects through anti-inflammatory mechanisms. However, it is unknown whether these mechanisms interfere in the association between psychosocial job stress and headache disorders. OBJECTIVE: To test whether physical activity and its interplay with the systemic inflammation biomarkers high-sensitivity C-reactive protein (hs-CRP) and acute phase glycoproteins (GlycA) would mediate the associations between job stress and headache disorders. METHODS: We cross-sectionally evaluated the baseline data from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) regarding job stress (higher demand and lower control and support subscales), migraine and tension-type headache (ICHD-2 criteria), self-reported leisure-time physical activity, and plasma hs-CRP and GlycA levels. Conditional process analyses with a sequential mediation approach were employed to compute path coefficients and 95 % confidence intervals (CI) around the indirect effects of physical activity and biomarkers on the job stress-headache relationship. Separate models were adjusted for sex, age, and depression and anxiety. Further adjustments added BMI smoking status, and socioeconomic factors. RESULTS: In total, 7,644 people were included in the study. The 1-year prevalence of migraine and tension-type headache were 13.1 % and 49.4 %, respectively. In models adjusted for sex, age, anxiety, and depression, the association between job stress (lower job control) and migraine was mediated by physical activity [effect = -0.039 (95 %CI: -0.074, -0.010)] but not hs-CRP or GlycA. TTH was associated with higher job control and lower job demand, which was mediated by the inverse associations between physical activity and GlycA [Job Control: effect = 0.0005 (95 %CI: 0.0001, 0.0010); Job Demand: effect = 0.0003 (95 %CI: 0.0001, 0.0007]. Only the mediating effect of physical activity in the job stress-migraine link remained after further adjustments including socioeconomic factors, BMI, smoking, and the exclusion of major chronic diseases. CONCLUSION: In the ELSA-Brasil study, physical activity reversed the link between job stress and migraine independently of systemic inflammation, while the LTPA-mediated downregulation of GlycA was associated with lower job stress-related TTH.

Increased Expression of KNa1.2 Channel by MAPK Pathway Regulates Neuronal Activity Following Traumatic Brain Injury.

Recent studies have indicated that functional abnormalities in the KNa1.2 channel are linked to epileptic encephalopathies. However, the role of KNa1.2 channel in traumatic brain injury (TBI) remains limited. We collected brain tissue from the TBI mice and patients with post-traumatic epilepsy (PTE) to determine changes in KNa1.2 channel following TBI. We also investigated whether the MAPK pathway, which was activated by the released cytokines after injury, regulated KNa1.2 channel in in vitro. Finally, to elucidate the physiological significance of KNa1.2 channel in neuronal excitability, we utilized the null mutant-Kcnt2-/- mice and compared their behavior patterns, seizure susceptibility, and neuronal firing properties to wild type (WT) mice. TBI was induced in both Kcnt2-/- and WT mice to investigate any differences between the two groups under pathological condition. Our findings revealed that the expression of KNa1.2 channel was notably increased only during the acute phase following TBI, while no significant elevation was observed during the late phase. Furthermore, we identified the released cytokines and activated MAPK pathway in the neurons after TBI and confirmed that KNa1.2 channel was enhanced by the MAPK pathway via stimulation of TNF-α. Subsequently, compared to WT mice, neurons from Kcnt2-/- mice showed increased neuronal excitability and Kcnt2-/- mice displayed motor deficits and enhanced seizure susceptibility, which suggested that KNa1.2 channel may be neuroprotective. Therefore, this study suggests that enhanced KNa1.2 channel, facilitated by the inflammatory response, may exert a protective role in an acute phase of the TBI model.

Conservative treatment of Peyronie's disease: a guide.

PURPOSE: To review the literature on the topic, to suggest a common line of treatment applicable across a wide community of specialists, and to contribute in maintaining the high level of interest in this disease. METHODS: A comprehensive and exhaustive review of the literature was performed, identifying hundreds of articles on the topic. RESULTS: Peyronie's disease is a condition that has been recognized, studied, and treated for centuries; despite this, if one excludes surgery in cases in which the deformity is stable, no clear treatment (or line of treatment) is available for complete relief of signs and symptoms. Treatment options were divided into local, oral, and injection therapy, and a wide variety of drugs, remedies, and options were identified. CONCLUSIONS: Low-intensity extracorporeal shock wave therapy, vacuum therapy, penile traction therapy, phosphodiesterase type 5 inhibitors, hyaluronic acid, and collagenase of Clostridium histolyticum may be recommended only in specific contexts. Further studies on individual options or potential combinations are required.

Postoperative monitoring of ovariohysterectomy carried out with two types of surgical sutures in shelter cats and differences in selected blood parameters between shelter and pet cats.

BACKGROUND: Reduction of inflammation and early detection of complications after surgical procedures are important objectives for proper veterinary practice. This study aimed to evaluate the differences between shelter and pet female cats in selected acute-phase parameters scheduled to ovariohysterectomy. Postoperative monitoring after ovariohysterectomy with the same laboratory parameters was performed in shelter cats, in which two different types of surgical sutures were used for the entire procedure. The experimental group comprised 40 female cats from animal shelters ('shelter cats,' n = 40). These cats were divided into two subgroups: group A (n = 20) operated on with absorbable sutures and group NA (n = 20) operated on with non-absorbable sutures. In addition, the same parameters were evaluated in pet female cats (n = 19). Blood was collected from shelter cats immediately before surgery (term 0), at 24 and 72 h (terms 1 and 3, respectively), and at 7 and 14 days (terms 7 and 14, respectively) after ovariohysterectomy. Blood samples from the pet cat group were collected only once. RESULT: The mean haptoglobin concentration before ovariohysterectomy in pet cats was significantly lower than that in shelter cats. Fibrinogen concentration was significantly lower in pet cats than in cats from group A. Serum albumin, beta-1, beta-2, and gamma-globulin concentrations were significantly higher in the shelter cats than in the pet cats. Subcutaneous tissue thickening at the site of the postoperative wound was observed in five patients cats (25%) in group A, and two (10%) cats in the NA group. CONCLUSION: These results indicate that ovariohysterectomy leads to local and general inflammatory responses. The majority of cats from animal shelters suffered from subclinical inflammation.

Concentrations of selected immunological parameters in the serum and processing fluid of suckling piglets and the serum and colostrum of their mothers.

BACKGROUND: Blood sampling from neonatal piglets is related to multiple disadvantages. Therefore, a new, alternative matrix is required to assess piglets' early immune status efficiently. The present study aimed to assess the usefulness of processing fluid for determining selected piglets' immune parameters. 264 pigs - 31 sows, 146 male piglets, and 87 female piglets from commercial indoor farrow-to-finish pig herd were included in this study. 264 serum, 31 colostrum, and 146 processing fluid samples were collected. Serum was collected from all animals, colostrum was collected from sows, and processing fluid was collected from male piglets only. Using commercial ELISA tests, the concentration of various immunoglobulins, cytokines, and acute phase proteins was assessed in each matrix. Statistical analyses were employed to determine differences in the concentration of measured indices between piglets' serum and processing fluid and correlations in the concentration of tested indices between particular sets of matrices. RESULTS: Statistical analyses did not reveal significant differences in the IgG, IgA, IL-1ß, IL-4, IL-6, and IFN-γ concentration between piglets' serum and processing fluid (p > 0.05). A positive correlation (p < 0.05) regarding the concentration of some indices between processing fluid and samples collected from sows was also observed. CONCLUSIONS: Processing fluid can be considered a promising alternative to blood for assessing some immunological indices in piglets, such as IgG, IgA, IL-1ß, IL-4, IL-6, and IFN-γ, and, possibly, in the indirect assessment of some indices in lactating sows, including IgA, IL-1ß, IL-4, IL-6, IL-8, IFN-γ, or Pig-MAP.

Metabolic profile and gene expression pattern of cytokines and antioxidants markers during different physiological stages in Barki ewes.

BACKGROUND: In livestock, identifying the physiological and reproductive stages is valuable in guiding management decisions related to nutrition, veterinary procedures, and breeding programs. To achieve this goal, a cohort of Barki ewes in this research underwent observation across three pivotal physiological conditions: pre-pregnancy, late pregnancy, and early lactation. Blood samples were collected to investigate the changes in serum metabolic profile as well as gene expression pattern of cytokines and antioxidants markers during these stages. RESULTS: Our results showed that during late pregnancy, there was a significant (P < 0.05) increase in red blood cells (11.9 ± 0.5 1012/L), hemoglobin (10.8 ± 0.4 g/dl) and neutrophils count (7 ± 0.1 109/L) with significant decrease (P < 0.05) of total white blood cell count (9.1 ± 0.05 109/L). The packed cell volume (%) and monocyte count showed a significant (P < 0.05) decrease during both late pregnancy and early lactation stages. The serum concentrations of glucose, cholesterol, GSH, GPx, SOD and catalase displayed significant (P < 0.05) decrease during late pregnancy and early-lactation. Notably, during late pregnancy, there was a significant (P < 0.05) increase in the serum concentrations of albumin, globulin, urea, IGF-1, and malondialdehyde with significant decrease (P < 0.05) of total protein (4.9 ± 0.08 g/dl). Additionally, during early lactation, there was a significant (P < 0.05) increase in the serum levels of non-esterified fatty acids, triiodothyronine (T3), and thyroxin (T4). The gene expression profiles of cytokines (IL-4, IL-6, IL-8, and NFKB) were decreased in the ewes during late pregnancy compared to pre-pregnant and early lactation stages. In addition, the expression profile of antioxidant genes (SOD, CAT, GPX, and Nrf2) was significantly upsurged in the non-pregnant ewes compared to late pregnancy and early lactation ones. CONCLUSIONS: The results concluded that different physiological status significantly affects the blood metabolic profile and gene expression pattern in Barki sheep. Our findings can be helpful in monitoring animal health and applying in breeding programs of Barki sheep under harsh environmental conditions.

Identification of novel biomarkers of acute phase response in chickens challenged with Escherichia coli lipopolysaccharide endotoxin.

BACKGROUND: The chicken's inflammatory response is an essential part of the bird's response to infection. A single dose of Escherichia coli (E. coli) lipopolysaccharide (LPS) endotoxin can activate the acute phase response (APR) and lead to the production of acute phase proteins (APPs). In this study, the responses of established chicken APPs, Serum amyloid A (SAA) and Alpha-1-acid-glycoprotein (AGP), were compared to two novel APPs, Hemopexin (Hpx) and Extracellular fatty acid binding protein (Ex-FABP), in 15-day old broilers over a time course of 48 h post E.coli LPS challenge. We aimed to investigate and validate their role as biomarkers of an APR. Novel plant extracts, Citrus (CTS) and cucumber (CMB), were used as dietary supplements to investigate their ability to reduce the inflammatory response initiated by the endotoxin. RESULTS: A significant increase of established (SAA, AGP) and novel (Ex-FABP, Hpx) APPs was detected post E.coli LPS challenge. Extracellular fatty acid binding protein (Ex-FABP) showed a similar early response to SAA post LPS challenge by increasing ~ 20-fold at 12 h post challenge (P < 0.001). Hemopexin (Hpx) showed a later response by increasing ∼5-fold at 24 h post challenge (P < 0.001) with a similar trend to AGP. No differences in APP responses were identified between diets (CTS and CMB) using any of the established or novel biomarkers. CONCLUSIONS: Hpx and Ex-FABP were confirmed as potential biomarkers of APR in broilers when using an E. coli LPS model along with SAA and AGP. However, no clear advantage for using either of dietary supplements to modulate the APR was identified at the dosage used.

Interval-Based Secretomics Unravels Acute-Phase Response in Hepatocyte Model Systems.

Mass spectrometry-based secretomics approaches frequently utilize serum-free culture conditions to circumvent serum-induced interference and to increase analytical depth. However, this can negatively affect a wide range of cellular functions and cell viability. These effects become particularly apparent when investigating transcriptionally regulated secretion events and feedback-loops in response to perturbations that require 48 h or more to fully manifest. We present an "interval-based" secretomics workflow, which determines protein secretion rates in short serum-free time windows. Relative quantification using tandem mass tags enables precise monitoring of time-dependent changes. We applied this approach to determine temporal profiles of protein secretion in the hepatocyte model cell lines HepG2 and HepaRG after stimulation of the acute-phase response (APR) by the cytokines IL1b and IL6. While the popular hepatocarcinoma cell line HepG2 showed an incomplete APR, secretion patterns derived from differentiated HepaRG cells recapitulated the expected APR more comprehensively. For several APR response proteins, substantial secretion was only observed after 72 h, a time window at which cell fitness is substantially impaired under serum-free cell culture conditions. The interval-based secretomics approach enabled the first comprehensive analysis of time-dependent secretion of liver cell models in response to these proinflammatory cytokines. The extended time range facilitated the observation of distinct chronological phases and cytokine-dependent secretion phenotypes of the APR. IL1b directed the APR toward pathogen defense over three distinct phases-chemotaxis, effector, clearance-while IL6 directed the APR toward regeneration. Protein shedding on the cell surface was pronounced upon IL1b stimulation, and small molecule inhibition of ADAM and matrix metalloproteases identified induced as well as constitutive shedding events. Inhibition of ADAM proteases with TAPI-0 resulted in reduced shedding of the sorting receptor SORT1, and an attenuated cytokine response suggesting a direct link between cell surface shedding and cytokine secretion rates.

Association between COVID-19 severity and relatively high serum adiponectin levels at the time of admission.

At the beginning of 2020, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to epidemics worldwide. Obesity and visceral fat accumulation have been reported to be independent risk factors for severe COVID-19. Several reports have focused on the levels of adipocytokines/adipokines, including adiponectin (APN), which is exclusively secreted from adipocytes, although the importance of these factors in acute disease conditions remains unclear. Therefore, we investigated the relationship between serum adiponectin levels and COVID-19 severity. Patients with COVID-19 who were admitted to Sumitomo Hospital (Osaka, Japan) from May through October 2021 were included. A total of 107 patients were enrolled in this study. We obtained the anthropometric and clinical laboratory data of the patients at the time of admission and examined the associations between various parameters and COVID-19 severity. The mean period from onset to admission was 6.5 ± 2.8 days. We divided the patients into "non-severe" (mild, moderate-I and moderate-II) (n = 80) and "severe" (n = 27) groups. The "severe" patients were significantly older than "non-severe" patients. Additionally, no significant differences were observed in BMI, sex, or the period from onset to admission. The serum adiponectin levels of "severe" patients at the time of admission were significantly greater than those of "non-severe" patients even after adjusting for age, sex, and BMI. These results suggest that the serum APN levels at the time of admission can predict COVID-19 severity. However, further investigations on the changes in APN levels in acute diseases are needed.

Variability in sleep architecture and alterations in circadian rhythms in patients with acute cerebral infarction accompanied by sleep-disordered breathing.

PURPOSE: To continuously and dynamically monitor the sleep status of patients in the acute phase of cerebral infarction, and to investigate the characteristics of acute cerebral infarction(ACI)associated with sleep-disordered breathing (SDB), variations in sleep structure, and changes in sleep circadian rhythms. METHODS: Patients with ACI within 48 h of onset who were admitted to the Department of Neurology at Kailuan General Hospital from November 2020 to December 2022 were selected. Detailed baseline information such as age, gender, smoking history, drinking history, were recorded for the selected participants. From the beginning of their hospitalization, the selected participants were monitored for their sleep status continuously for 5 days using the Intelligent Mattress-based Sleep Monitoring Platform System(IMSMPS). Based on the heart rate data obtained from the monitoring, the interdaily stability (IS) and intradaily variability (IV) of the sleep circadian rhythm were calculated. RESULTS: 1,367 patients with ACI were selected. Monitoring results over 5 days indicated 147 cases (10.75%) without SDB, and 1,220 cases (89.25%) with SDB. Among the group with SDB, there were 248 cases (18.14%) with continuous mild SDB, 395 cases (28.90%) with moderate SDB, 295 cases (21.58%) with severe SDB, and 282 cases (20.63%) that fluctuated between different severity levels. Within this fluctuating group, 152 cases (53.90%) fluctuated between two severity levels, 120 cases (42.55%) between three levels, and 10 cases (3.55%) among all four levels. There were statistically significant differences (P < 0.05) in the sleep latency, sleep efficiency, non-rapid eye movement stages 1-2, rapid eye movement, proportion of non-rapid eye movement, proportion of rapid eye movement, wake after sleep onset, time out of bed, number of awakenings, respiratory variability index, and heart rate variability index among patients with ACI monitored from day 1 to 5. However, other monitored sleep structure parameters did not show statistically significant differences (P > 0.05). The coefficient of variation for all sleep monitoring parameters ranged between 14.54 and 36.57%. The IV in the SDB group was higher than in the group without SDB (P < 0.05), and the IS was lower than in the group without SDB (P < 0.05). CONCLUSION: Patients in the acute phase of cerebral infarction have a high probability of accompanying SDB. The sleep structure of these patients shows significant variability based on the onset time of the stroke, and some patients experience fluctuations among different severity levels of SDB. ACI accompanied by SDB can further reduce the IS of a patient's sleep circadian rhythm and increase its IV.

Systemic inflammation in early lactation and its relation to the cows' oxidative and metabolic status, productive and reproductive performance, and activity.

A dysregulated inflammatory response contributes to the occurrence of disorders in cows during the transition period from pregnancy to lactation. However, a detailed characterization of clinically healthy cows that exhibit an enhanced inflammatory response during this critical period remains incomplete. In this experiment, a total of 99 individual transition dairy cows and 109 observations (18 cows monitored in 2 consecutive lactations), submitted to similar transition management were involved to evaluate the relationship between elevated an inflammatory response and metabolic and oxidative status, as well as transition outcomes. Blood was taken at -7, 3, 6, 9, and 21 DIM, and concentrations of metabolic parameters (glucose, ß-hydroxybutyric acid, nonesterified fatty acids [NEFA], insulin, IGF-1, and fructosamine) were analyzed. Additionally, oxidative parameters (proportion of oxidized glutathione to total glutathione in red blood cells, the activity of glutathione peroxidase [GPx] and superoxide dismutase, concentrations of malondialdehyde, and oxygen radical absorbance capacity) and acute phase proteins (APP) including haptoglobin (Hp), serum amyloid A (SAA) and albumin-to-globulin ratio (A:G) were determined in the blood at 21 DIM. The 3 APP parameters were used to group clinically healthy cows into 2 categories through k-medoids clustering (i.e., a group showing an acute phase response, APR; n = 39) and a group not showing such a response (i.e., non-APR; n = 50). Diseased cases (n = 20) were handled in a separate group. Lower SAA and Hp concentrations as well as higher A:G were observed in the non-APR group, although for Hp, differences were observed from the APR group and not from the diseased group. Only 1 of the 5 oxidative parameters differed between the groups, with the non-APR group exhibiting lower GPx activity compared with the diseased group. The non-APR group showed the highest IGF-1 levels among the 3 groups and and lower NEFA concentrations compared with the diseased groups. Cows in the diseased group also showed reduced dry matter intake and milk yield compared with clinically healthy cows, regardless of their inflammatory status. Moreover, the APR group exhibited temporarily lower activity levels compared with the non-APR group. These findings highlight that cows with a lower inflammatory status after 21 DIM exhibited better metabolic health characteristics and productive performance, as well as activity levels. Nevertheless, the detrimental effects of a higher inflammatory status in the absence of clinical symptoms are still relatively limited.

Effects of prepartum concentrate feeding on reticular pH, plasma energy metabolites, acute phase proteins, and milk performance in grass silage-fed dairy cows.

We investigated how concentrate feeding during the last 21 d of pregnancy affects reticular pH, inflammatory response, dry matter (DM) intake, and production performance of dairy cows. We hypothesized that adding concentrates to dairy cows' diet before calving reduces the decrease in reticular pH postpartum and thus alleviates inflammatory response. We also hypothesized that prepartum concentrate feeding increases DM intake postpartum and consequently improves milk performance. Two feeding experiments were conducted using a randomized complete block design. In each experiment, 16 multiparous Finnish Ayrshire cows were paired based on parity, expected calving date, body weight, and milk yield of the previous lactation. Within the pairs, cows were randomly allocated on one of the 2 dietary treatments 21 d before expected calving. In experiment 1 (Exp1), diets were ad libitum feeding of grass silage as a sole feed or supplemented with increasing amounts of concentrate offered separately (increased to 4 kg/d by d -7). In experiment 2 (Exp2), diets were ad libitum feeding of a total mixed ration containing either grass silage, barley straw, and rapeseed meal (64%, 28%, and 8% on DM basis, respectively) or grass silage, barley straw, and cereal-based concentrate mixture (49%, 29%, and 30% on DM basis, respectively). Following calving, all the cows were fed similarly and observed until d 56 postpartum. Feed intake and milk yield were recorded daily, and reticular pH was monitored continuously by reticular pH bolus. Blood samples were collected at the beginning of the experiments, 7 d before the expected calving date, 1 d (in Exp1) or 5 d (in Exp2), 10 d, and 21 d postpartum. In Exp1, concentrate feeding increased metabolizable energy intake and tended to increase DM and crude protein intake prepartum. Moreover, prepartum concentrate feeding increased the concentrations of plasma ß-hydroxybutyrate and insulin, but differences in nonesterified fatty acids, glucose, or acute phase proteins were not observed. After calving, prepartum diet did not affect DM or nutrient intake, plasma energy metabolites, or milk production in Exp1. Although prepartum concentrate feeding increased reticular pH on the first day of lactation, it elevated plasma concentrations of serum amyloid-A and haptoglobin postpartum in the grass silage-based diet. In Exp2, adding concentrates to the diet based on a mixture of grass silage and straw did not affect prepartum DM intake or plasma concentrations of nonesterified fatty acids, glucose, or insulin. Adding concentrates to prepartum diet increased plasma concentration of ß-hydroxybutyrate before calving as in Exp1. After calving, prepartum concentrate feeding increased DM and nutrient intake during the second week of lactation in Exp2, but no effects were observed thereafter. In contrast to our hypothesis, prepartum concentrate feeding decreased reticular pH after calving in Exp2, but no differences in inflammatory markers were observed. Based on this study, close-up concentrate feeding in diets based on grass silage with or without straw does not alleviate the decrease in reticular pH or mitigate inflammatory response postpartum.